report 2005
Transcrição
Laboratório Associado para a Química Verde Tecnologias e Processos Limpos REPORT 2005 ii Executive Summary REQUIMTE (Rede de Química e Tecnologia) results from a long-standing collaboration between two university based research Centers - “CQFB: Centro de Química Fina e Biotecnologia da Universidade Nova de Lisboa“ and - CEQUP: Centro de Química da Universidade do Porto“. The association of the two centers was recognized as the Laboratório Associado para a Química Verde by the Portuguese Ministério de Ciência e do Ensino Superior in November of 2001, and was formerly chartered as a nonprofit scientific organization in January of 2003. The scientific expertise and complementary knowledge available, put together by CQFB and CEQUP, has been focused on the topic GREEN CHEMISTRY œ CLEAN TECHNOLOGIES AND PROCESSES with a wide range of tools and from different perspectives. In general terms, the existing competencies will be drawn from the areas identified as expertise fields within REQUIMTE: chemistry, (micro)biology, biochemistry and molecular biology, molecular modeling, bio(catalysis) and reaction mechanisms, (bio)conversion and bioremediation, transport phenomena, separation processes, sensor development, monitoring and control. The available expertise will be used to implement the use of cleaner products and cleaner technologies, preventing pollution at its source. By working with manufacturers, governmental agencies, consumers and general public new ideas and new attitudes can be implemented. REQUIMTE can act as a promoter and is available to answer questions and problems placed from outside. REQUIMTE will look forward to assist manufacturers in order to develop cooperative efforts to design and redesign products and processes that will reduce life cycles environmental impacts. Activities wil be implemented to provide reliable information on the environmental benefits, performances and economic feasibility of green chemistry and clean processes. The year 2005 was the fourth in which REQUIMTE was fully operational: frequent meetings of the board of directors (monthly), creation of formal incentives to strengthen the cooperation between researchers of the two Centers (seed money for joint projects) and the recruitment of six researchers (Investigadores Auxiliares) to carry out activities under the contract of Laboratório Associado. Laboratório Associado para a Química Verde iii Mission Statement a. REQUIMTE, which is recognized as the Laboratório Associado para a Química Verde by the Portuguese Ministério de Ciência e do Ensino Superior, is a voluntary association of two research Centers which have freely opted to collaborate in research and postgraduate activities, whilst still being fully committed to their respective Universities. b. REQUIMTE considers itself to have made a very important contribution to the way in which Chemistry, Biology and Chemical Engineering being carried out in Portugal is viewed internationally – as judged by the quality and quantity of its scientific publications and also by the number and quality of ongoing research projects. c. REQUIMTE will focus the activities of its reaserchers to implement the principles of Green Chemistry. d. REQUIMTE also aims to optimize internal collaboration and to identify the best international strategic partners. e. REQUIMTE, whilst wishing to preserve its well-founded and successful roots in traditional chemical, biological and engineering sciences, intends fully to strengthen its international research in innovative and novel topics and to become a pool of attraction for young and well established national and international scientists. f. REQUIMTE views its involvement in the postgraduate training of young people as a very important function. REQUIMTE policy is to select projects in such a way that students working within the organization will gain, in the course of their researches, the skills required by the employment market. g. REQUIMTE is presently the largest chemical network in Portugal, with approximately 380 researchers, of which more than 200 hold a Ph.D. degree. h. REQUIMTE provides an optimal environment to explore the synergies of the their expertise in answering the needs of the productive sector and providing specialized services and consulting. i. REQUIMTE is currently pursuing an environmentally driven reasearch in association with the chemical industry to develop closed loop industrial processes. j. REQUIMTE is working to foster public awareness of key chemical and biochemical concepts, to help understand the costs and benefits of technology in the modern world and to develop a balanced global appreciation of environmental issues. Laboratório Associado para a Química Verde iv History REQUIMTE stands for Rede de Química e Tecnologia and names the Chemistry and Technolology Network that has been formed by two Research Units, the Centro de Química da Universidade do Porto - CEQUP and the Centro de Química Fina e Biotecnologia da Universidade Nova de Lisboa – CQFB. Since November 2001 REQUIMTE became the “Laboratório Associado para a Química Verde – Tecnologias e Processos Limpos” of Fundação para a Ciência e a Tecnologia of MCTES, in which 374 researchers (208 holding a Ph.D. Degree) exploit the basic principles of Green Chemistry and aim to contribute to the practice of Sustainable Chemistry. The need of practicing a sustainable development in order to reach the social, economic and environmental objectives of the modern society is well accepted by governments, industrial sector and general public. Within this scope, Chemistry which is commonly associated with harmful products and not with materials absolutely essential for everyday life, must have a decisive role in the maintenance and improvement of living and environmental conditions. The awareness of contemporary society for the inevitability of the use of chemicals and chemical processes and the understanding of the concept of sustainability lead to a new way of thinking Chemistry. Having in mind the implementation of clean practices and clean industrial processes in which the amount of raw materials, energy, costs and risks are reduced a scientific movement, known as Green Chemistry, has emerged in the last quarter of the 20th century. Green Chemistry aims to redevelop laboratory and industrial processes in order to make them cleaner and economical viable. For that purpose scientists have to design new reactions and processes in which principles like an economy of atoms, use of renewable materials, use of nontoxic solvents and use of clean energy sources must be followed. The objectives of Laboratório Associado para a Química Verde – Tecnologias e Processos Limpos are: a) to encourage the use of clean products and technologies; b) to assist industry in the design and implementation of non-aggressive chemical processes; c) to train young researchers in interdisciplinary areas related with the practice of sustainable chemistry; d) to make public the principles of green chemistry and to alert society for the necessity of a sustainable practice in everyday life. Research is presently focused in the following thematic areas of: i) natural products, (ii) food quality and safety, (iii) clean production technologies and processes, (iv) environmental control and remediation and (v) catalysts, solvents and non-toxic compounds. The sharing of multidisciplinary scientific knowledge, technology and equipment between researchers of the two centers that formed the network has significantly contributed to the development of new projects in green chemistry and to the enrichment of training of graduate students by making easier the mobility of human resources. At present the network REQUIMTE can be described as a big Laboratory that has two operating sites, one at Universidade Nova de Lisboa and other at Universidade do Porto. Laboratório Associado para a Química Verde v REQUIMTE in 2005 2005 was the third year in which REQUIMTE operated as a —Laboratório Associado“, although the cooperation existed since 1996. The strategic plan was to focus expertise in Analytical, Biological, Inorganic, Organic, Physical and Theoretical Chemistry and in Chemical Engeeniring in a contemporary unifying concept œ that of Green Chemistry. 2005 was the second year when the funding contract with FCT-MCTES was operational and REQUIMTE hired six new researchers in order to carry out activities included in the contract with the Ministry. The profiles of the six new researchers are provided in Annex. The scientific production was also considered a key aspect of the activity of REQUIMTE, and the total number of articles in scientific journals, chapters in books and articles in proceedings has grown to 280, much to the dedication of its graduate students, 14 of which have completed their doctoral thesis and 14 their master thesis in 2005. Laboratório Associado para a Química Verde vi People at REQUIMTE Board of Directors Baltazar de Castro (UP) Isabel Moura (UNL) Manuel Nunes da Ponte (UNL) Co-ordinating Comittee of the Scientific Council Ana Lobo (UNL) Baltazar de Castro (UP) Isabel Moura (UNL) José Costa Lima (UP) José Moura (UNL) Manuel Nunes da Ponte (UNL) Maria de Lourdes Bastos (UP) Maria Rangel (UP) Advisory Comittee Prof. William B. Motherwell University College, Christopher Ingold Laboratories, 20 Gordon St., London WC 1 H OAJ, UK Organic synthesis; Reaction mechanisms in organic chemistry Prof. Luigi Marzilli Department of Chemistry, Louisiana State University, Baton Rouge, LA70803-1804, USA Spectroscopy; Inorganic synthesis; B12 chemistry; Bioinorganic chemistry; Metal based drugs Prof. Jean L. Rivail Université Henri Poincaré, Laboratoire de Chimie Théorique, B.P. 239, 54506 Vandoeuvre-les-Nancy, France Theoretical chemistry Prof. Marek Trojanowicz University of Warsaw, Faculty of Chemistry, Pasteura 1, PL-02-093,WARSAW, Poland Analytical Chemistry Professor James Clark Clean Technology Centre, Department of Chemistry, University of York, Heslington,York, YO10 5DD, UK Green Chemistry Prof.Harry Kuiper, Programme Leader and International Account Manager at the State Institute for Quality Control of Agricultural Products (RIKILT, UR Wageningen NL) Food Safety Laboratório Associado para a Química Verde vii http://www.requimte.pt LABORATÓRIO ASSOCIADO QUÍMICA VERDE – TECNOLOGIAS E PROCESSOS LIMPOS REQUIMTE, is the biggest network in Chemistry and Chemical Engineering established in Portugal and is recognized as the Laboratório Associado para a Química Verde by the Portuguese Ministério de Ciência e do Ensino Superior since November 2001. The scientific expertise and complementary knowledge available, put together by the two research centres that form the network (Centro de Química Fina e Biotecnologia-UNL and Centro de Química-UP), allowed us to deal with the topic GREEN CHEMISTRY - CLEAN TECHNOLOGIES AND PROCESSES with a wide range of tools and from different perspectives. REQUIMTE has the mission of corporate in a continuous form, in a competent and efficient way in the prosecution of the specific aims of the national scientific and technologic politics in the areas of: 1 NATURAL PRODUCTS: SCREENING AND SYNTHESIS Screening of biologically active compounds of traditional plant-based Medicine and support to certification on natural phamaceuticals. Synthesis of pharmacologically active compounds. Chromatographic purification of new compounds and spectroscopic studies of structure-function. 2 FOOD QUALITY AND SAFETY Food Quality and Safety regulations and inter-laboratory validation of analytical and certification methodologies. Screening of pharmaceutical residues and secondary metabolites Survey of critical points for microbiological control in food processing and development of microbiological methodologies for fast pathogen detection. 3 CLEAN PRODUCTION TECHNOLOGIES AND PROCESSES Implementation of clean separation processes – supercritical fluids, membranes, adsorption. Reaction/separation process integration. Monitoring, automation and control of bio/chemical processes. Scale-up. 4 ENVIRONMENTAL CONTROL AND (BIO) REMEDIATION Advanced analytical tools (AAS-EA, ICP-MS, GC-MS, HPLC-MS, AA and DNA sequencing, NMR, EPR, X-Ray Crystallography and MS) and implementation of good practices in chemical analysis. Development of control systems, probes, sensors and transducers (mainly oriented to environmental problems). Implementation of novel processes (including physical and biological) for treatment of water and industrial wastes, as well as soils. Energy recovery from waste and to recycljng of materials. 5 CATALYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS Green synthetic routes of chemicals and pharmaceuticals. Spectroscopic / computational techniques and molecular structure. Alternative solvents and catalysis. Enzymes in non-aqueous solvents. Laboratório Associado para a Química Verde viii Events The Day of Chemistry is organized every year bringing more than 600 students to the Campus of Caparica. In 2005 it occurred on the 8th of February. In the morning there is a session in the auditorium with live experiments and in the afternoon the students visit several laboratories in the Chemistry Deparpment. Every year the Faculty of Sciences and Technoly (Campus of Caparica) organizes an Open Day with visits to the laboratories of the Chemistry Department). In 2004 a Forum of Chemistry was organized by students and teachers of Chemistry. The VII Jornadas Tecnológicas de Engenharia Química took place on the... >>Igual a 2004:<< Program Ciência Viva In the week of the FCT Scientific Culture visits to several laboratories were organized. In the summer period two weeks courses, for students presently attending the secondary school, were organised at CEQUP and on the themes of Treatment of Laboratory Wastes and Toxicology. Monographic Courses (5 days) are ministrated in the themes: Determination of Molecular Structures by X-Ray Diffraction Methods Nuclear Magnetic Resonance: From Theory to Practice Liquid-liquid and gas-liquid chromatography Organization of Conferences , Symposiums and Workshops XIX Reunião Ibérica de Adsorção, July 2004 >>Retirei da info 2005:<< Participation in the organization of: - Acção de Formação de Professores “Histórias com Muita Ciência”, Lisboa, Nov 2005 Prof. P. Mata - Acção de Formação “Gastronomia Molecular: do Laboratório para a Cozinha. Azoto Líquido e Alginatos” , Lisboa, Nov 2005 Prof. P. Mata - Acção de Formação “Gastronomia Molecular: do Laboratório para a Cozinha. Gelatinas quentes e frias”, Lisboa, Mar 2005 Prof. P. Mata - Curso Satélite (trabalhos práticos do 12º ano) ao 4º DEDQ - Encontro Nacional da Divisão de Ensino e Divulgação da Química, Sociedade Portuguesa de Química, Out. 2005. Prof. M. Pereira - 4º DEDQ – Encontro Nacional da Divisão de Educação e Divulgação da Química da Sociedade Portuguesa de Química (http://www.spq.pt/congressos/4dedq/). P. Tavares - Curso Monográfico de Bioelectroquímica, 9-11 de Outubro 2005. J.Moura / M.G. Almeida Organização de eventos 2ª Jornada “Bebidas e Saúde do IBeSa” (Instituto de Bebidas e Saúde). 1st February 2005 in Faculdade de Farmácia including the presentation of the research work developed in 2003/2004 with grants from IBeSa and a conference intituled “Natural antioxidants, ageing and oxidative stress” presented by Prof. Vítor Costa from IBMC. The presented work researches were compiled in a book (ISBN 972-99931-0-6). Laboratório Associado para a Química Verde ix Report Organization The present report, after this introduction, is organized in Parts A and B and Annexes. Part A contains the achievements of the Associated Laboratory under its main themes. Part B contains the individual contributions of all the research groups from CQFB and CEQUP in a more detailed form. Annexes contain listings of the researchers, publications and on-going projects in 2005. Laboratório Associado para a Química Verde RESEARCH REPORT 2005 Part A 2 AREA 1 NATURAL PRODUCTS: SCREENING AND SYNTHESIS - A simple rearrangement of N-prenyl indole derivatives was uncovered and led to a simple entrancy into cancer useful natural molecules. - A new enantioselective inverse phase transfer reaction for the Markovinkov hydration of double bonds was disclosed, that makes use of cyclodextrins. - The synthesis of Ecstasy metabolites, produced by glutathione capture of the intermediate quinones, showed that the neurotoxicity paralels its redox potential. - Guinea-Bissau plant extracts revealed a library of cytotoxic phenolic compounds. - Chirality codes, previously developed, were applied to the prediction of enantiomeric excess in a combinatorial library of enantioselective reactions. - A new method was developed to represent chemical reactions with no specification of the reac tion center, from the difference of MOLMAP molecular descriptors of products and reactants. - The CIP rules to specify chirality in chemistry were revised in order to improve its logic, consis tency, scope and applicability. The work was adopted by IUPAC. Laboratório Associado para a Química Verde 3 AREA 2 FOOD QUALITY AND SAFETY - Participation in International Collaborative study ISO/CD 6885. - Membership of Technical Committees: elaboration of the industry code of Hygienic Practices and NP EN ISO documents. - Implementation of collaborative projects with producer associations: characterization of traditional food products and safety. - Quality Control of food products answering to industry questions and consumers concerns: GMOs and acrylamide. - Application of chemometric techniques (univariate, bivariate and multivariate statistics) to evaluate the relationships between different types of parameters in a given product, and to classify new observations in relation to predefined models. - Modelling and optimisation of food processing. - Measurement and prediction of physical and rheological properties. - Studies performed in poultry for human consumption to evaluate the occurrence of vancomycin resistant enterococci as well as in swines in Europe. - Patented voltammetric method for the determination of diacetyl directly in beer fermentation vessels. - In vitro evaluation of safety/toxicity/protection of food components and beverages for human consumption. Laboratório Associado para a Química Verde 4 AREA 3 CLEAN PRODUCTION TECHNOLOGIES AND PROCESSES - Membrane Processes were used for separation and/or reaction, in the recovery of aroma compounds, pre-enrichment for sample discrimination, extraction of amino acids and derivatives using ionic liquids, vapour separation . - Ion Exchange Membrane Bioreactos were used in the removal of polluting ions from drinking water streams. - Polysulfone membrane formation were prepared using a CO2-assisted phase inversion method. The Thermodynamics of ionic liquid-containing systems was studied. - Dynamic modelling of supercritical fractionation, nanofiltration and adsorption was carried out. The synthesis of new polyurethanes and PDLC, as well as the preparation of polymeric systems for drug controlled-release, were carried out in both conventional and scCO2 media. - Studies on the optimisation of pectin extraction from orange peels and the subsequent production of model edible films were carried out. - Process Integration of Supercritical Fluid Extraction and Membrane Separation continued to be studied. Laboratório Associado para a Química Verde 5 AREA 4 ENVIRONMENTAL CONTROL AND (BIO) REMEDIATION - Use of Ion Exchange Membrane Bioreactor (IEMB) for the removal of polluting ions (perchlorate and nitrate) from drinking water streams. - Biodegradation of organ-mercurial compound to metallic mercury, under aerobic conditions, by pure bacterial cultures in CSTR, fed with a real vaccine production wastewater. - Simultaneous removal of P and N and microbial population identification (FISH). - Pesticide Analysis (in food and environmental samples) - Laboratory Waste Management (waste separation/disposable strategies) - Soil Remediation (in-pulp solvent extraction procedures to remediate hydrocarbon contamination). - New trends on exploitation of different transducing schemes such us optical and electrochemical. - Studies on immobilization techniques for the monitoring low levels of drugs, food and pesticides (additionally studies for construction of flow-through voltammetry electrodes). - Development of continuous flow systems and dedicated equipment in automatic laboratorial determinations or in on-line control of industrial processes (multi-commutated flow methodologies and new concept of multi-pumping flow analysis). . Laboratório Associado para a Química Verde 6 AREA 5 CATALYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS - A new enantioselective inverse phase transfer catalysis reaction for the Markovnikov hydration of double bonds by an oxymercuration-demercuration reaction with cyclodextrins as catalysts was developed. - Sugars were used as sources for new chiral materials and microwave radiation was used in accelerated synthesis and/or in solvent free reactions. - Hydrogenation of limonene and the synthesis of new polyurethanes were performed in supercritical CO2. - Quantitative structure-property relationships between the melting point and density of ionic liquids was established, providing the design/synthesis of novel imidazolium and guanidinium based ionic liquids. A series of ionic liquids based on imidazolium and guanidinium cations were used as neoteric solvents in alkene asymmetric epoxidation. - Metals (Pd and Ni) and transition metal complexes (Mn, Cu and VO) immobilised in several supports (carbon materials, zeolites, clays and silica materials) and dispersed in PDMS matrix were tested in reductive and oxidative catalytic reactions. - Novel methods for the preparation of Pt and Pd nanoparticles with catalytic applications were developed, as well as their immobilisation on graphite. - Application of Monte Carlo methods to model the spontaneous formation of silica nanoparticles during the early stages of zeolite synthesis yielded results that are in close qualitative agreement with the experimental observations. - Design/optimisation of new leads for the treatment of cancer, malaria, AIDS and hypertension have been done by molecular modelling and computer simulation, as well as determination of inhibition mechanisms of RNR enzyme by anti cancer drugs. - Crystallographic and functional studies on several metalloproteins, on proteins involved in cellulose (polysaccharyde) recognition and degradation and on proteins from the glyoxalase pathway. Laboratório Associado para a Química Verde RESEARCH REPORT 2005 Part B 2 ANALYTICAL AND BIOORGANIC CHEMISTRY Head of Laboratory: Elvira Maria M. Gaspar, Assistant Professor Staff Members: Angela M.S. Relva Assistant Professor Marco Diogo R.G. da Silva Assistant Professor José Luiz Capelo-Martinez Assistant Researcher Post-Doct Fellows: Raquel Rial Otero Ph.D. Students: Laila H. Ribeiro Number of articles in scientific journals : 7 (1-7) Number of patents: 1 Number of Ph.D. Thesis: 1 Scope and description of on-going research During 2005 the group was mainly focused in some environmental problems. New sample treatments were applied to solve analytical problems. It was studied the hopanoid composition of sediments from lower Tagus basin. To decode the process of host tree selection by herbivore insects (a collaborative program with environmental scientists), namely the Mediterranean defoliator winter pine processionary moth, a profile of pine varieties cultivated in Portugal was achieved as an indicator for insect attack in the environment. New natural oligossacharides with potential pharmacological properties were isolated from an European Convolvulaceae plant. New chiral HPLC and enantioselective GC separation methods of an active natural compound (also present as a fragment group of complex molecules) has been developed as new separation techniques. Also new clean analytical methodologies for organometallic compounds speciation in food and environmental samples were used. Future Activities During 2006 the group will continue to be involved in environmental / human health problems. Human health will be focused by food analysis, monitoring the volatile profile to access the quality of important food matrixes and also by the analysis of human volatile compounds, potential bio-markers in cancer disease. Extraction of added value compounds from orange peel wastes and the potential of fruit stones to be used as adsorvents for the selective removal of organic compounds will be topics of interests. Clean analytical methodologies for organometallic compounds speciation in food and environmental samples. The creosote volatiles and other environmental specific problems will be studied by 1D GC-TOF-MS and also by GCxGC-TOF MS in a collaborative program. New analytical methodologies to control the quality of food samples, specially antibiotics and pesticides will be developed. Furthermore new methods for rapid protein identification based on MALDI-TOFMS will be assayed. Laboratório Associado para a Química Verde 3 PHOTOCHEMISTRY AND SUPRAMOLECULAR CHEMISTRY Head of Laboratory: Fernando Pina, Full Professor Staff Members: A. Jorge Parola Assistant Professor J. Carlos Lima Assistant Professor M. João Melo Assistant Professor Carlos Lodeiro Assistant Researcher Alexandra Bernardo Associate Professor Micaela de Sousa Invited Assistant Ana Claro Invited Assistant Post-Doct Fellows: Berta Covelo, Damián Fernández Ph.D. Students: Margarida Moncada, Sérgio Alves, Letícia Giestas,Carlos Pinheiro, André Vidal Pinheiro, Joana Ferreira Project Grantee: Bruno Pedras Number of articles in scientific journals: 10 (8-17) Chemosensors In the framework of the Project POCTI/47357/QUI/2002, “Chemosensors based on polyamine receptors containing fluorophoric units”, we have privileged our collaboration with the group of Prof. Enrique GarciaEspaña from the University of Valencia, Prof. António Bianchi e Andrea Bencini from the University of Florence, as well as Prof. Jaume Casabó e Lluis Escriche, from the University Autónoma de Barcelona, and the group of Prof. Rufina Bastida, from the University of Santiago de Compostela. In the case of the three first collaborations, the chemosensor is constituted by a polyamine receptor linked to a fluorophoric unit; in the case of the last two collaborations (papers 11, 16 and 17), the receptor is a macrocycle having oxygen or sulphur atoms in order to modify the coordinative properties and extend these chemosensors to other target metal ions or organic anions different from those achieved through the polyamine receptors. In the case of paper 1 the concept of chemosensor has been extended to a catalytic system. The ATP hydrolysis takes place in a system containing the chemosensor coordinated to Zn(II), and having non-coordinated Zn(II) in solution. In the framework of the Project POCI/55519/QUI/2004, “Towards New Supramolecular Devices: from new synthetic methods to multifunctional applications” we have been working in the developement of new chemosensors provided with nitrogen-sulphur and nitrogen-oxygen atoms in their structure for the detection of heavy metal ions, such us Hg(II), Cd(II) and Pb(II), as well as transition metal ions such as Zn(II), Ni(II), Cu(II) and Co(II). This subject was developed mainly in collaboration with the group Prof. Jaume Casabó and Lluis Escriche, from the University Autónoma de Barcelona, Spain (Papers 11 and 16), where a nice example of a Laboratório Associado para a Química Verde 4 square planar Ni(II) complex whas studied, whith colour tuning upon anion (Cl-, Br-, I-, F-, CN-) or neutral molecules (water, methanol, acetonitrile, pyridine, bipyridine) coordination (Figure 1). This behavior allows naked-eye detection of anions and could be used to develop new devices for detecting, for instance, iodide ions in mixed halide solutions (Paper 9). Upon synthetic modification (addition of an anthracene unit) the chemosensor was converted into an emissive system for Pd(II) and Cu(II) detection. (Paper 11). 4+ 4+ 4+ 4+ 2,2’-Bipy 4 4+ - F 4+ - Cl Py 4+ - H2O Br 4+ - CH3CN I S N Ni N S 2+ 4 Figure 1 – Color tuning upon coordination of neutral molecules of anions to a Ni(II) complex. In collaboration with the group of Prof. Rufina Bastida, from the University of Santiago de Compostela, an example of Energy Transfer from a macrocyclic to an Europium(IIII) or a Terbium(III) metal center was studied (Paper 10). With the group of Prof. Manuela Raposo, from the University of Braga, Portugal, we have started an intense study of new bisthiophene-phenanthroline systems and their Ru(II) complexes; part of this work was presented in the 8th National Conference on Photochemistry in Coimbra. With the aim of obtianing new “multifunctional applications”, interaction of new pyrene derived ligands with barbituric, parabanic and cianuric acids was carried out (work in press). The group was also involved in the study of the coordinative properties of several 1,10-phenanthroline derivatives in the solid state, following a crystal engineering approach (paper 8). This area was developed in collaboration with the group of Professor E. Vázquez-López from the University of Vigo, Spain. Into this collaboration one Postdoctoral student, Berta Covelo spent one year, since September 2004 and October 2005, in our group integrated as researcher in the project “Towards New Supramolecular Devices: from New Synthetic Methods to Multifunctional Applications”. Ionic liquids This work has been carried out in the framework of POCI/QUI/57735/2004, “Photochromism of Flavylium Compounds in Water/Ionic Liquid Biphasic Systems”.This project started on the 1st July 2005. Two synthetic approaches are being pursued: (i) synthesis of flavylium salts with chloride counter-ions in gram scale quantities as a source for ionic liquid synthesis; (ii) synthesis of flavylium salts with anionic substituents (e. g., sulfonate goups). Studies on the behaviour of flavylium salts in C8MIMPF6 and C10BMIMPF6 were also started. Flavylium chemistry This is a recurring subject in the research of our group. The study of the colour, and the possibility of changing it by pH, light or electric inputs have important applications in several fields from the art pigments to the food dyes. In particular, we are interested in the use of flavylium compounds to carry out cycles for write-readerase at the molecular level. A step further towards applications was done during the last year, through the incorporation of flavylium compounds in water permeable matrices (papers 12 and 13). Within our attempts to develop the concepts of multistate/multifunctional systems, we have reported an example of two connected flavylium networks operating through different algorithms, paper 14, see Figure 2. Laboratório Associado para a Química Verde 5 Figure 2 - Network of chemical reactions involving two flavylium compounds Anthocyanin blues NH2 4 In this project, we aim to explore the use of natural antioxidant compounds, the anthocyanins, as well as their pH jump pH jump pH=1.0 7.7 synthetic analogs, the substituted flavylium salts, as sources for blue, redCtH and violet colorantspH=Ctand pigments. + 4'-aminoflavylium 3 unlock O Read + pH=12.0 Ct- 5 Erase Enable auto-lock Write Unfortunately, the chemistry In modern world, color is ubiquitous, creating a bright and colourful environment. 5 1 hν Erase used to synthesize and to process colours is not always environment friendly. “Greener” colorants must hν, ∆ 4 auto-lock hν, ∆ hopefully be developed in order to guaranty a more sustainable development. 2 Write hν unlock pH= 0 pH= 1 AH+ pH= 7.7 3 + pH jump B/Cc Blue pigments based on zeolite inclusion complexes withAHanthocyanin or flavylium salts pH jump have been prepared Read and their photochemical and thermal stability will be studied and compared Maya 2 with those displayed by Enable hν 1 Blue, one of the most stable known blue inclusion complexes. pH jump pH=12.0 Cc- ∆ Read lock This project involves a feedback approach (experimental data Û theoretical calculations), that is beeing used 2 hν 2 3 these pH jumpstability in pH jump to gather a deep insight on the mechanisms that controlpH=0color and compounds. pH=1.6 AH+ AH+ 3 4 pH jump pH= 6.0 B/Cc It is a REQUIMTE funded project that joins the CQFB group and its knowledge of anthocyanin and flavylium unlock salts chemistry and the CEQUP group with its expertise in theoretical calculations and predictions. Write 1 The color in Art hν auto-lock Erase 5 ∆ 4 Write 1 + 5 pH jump i) the study of the colour of medieval Portuguese manuscript illuminations; the Book of Birds (O livro das Aves) and The Apocalypse of Lorvão (O Apocalipse do4'-acetamidoflavylium Lorvão), considered a national treasure were two of the Manuscripts that came to our Faculty for a more detailed study, namely with non invasive techniques, such as microXRF and micro fluorescence emission. The more relevant results obtained were submitted for publication. Laboratório Associado para a hν, ∆ hν CH3 Some of the more recent projects in the area of Conservation ofO the Cultural Heritage have acquired a nice C pH=1.6 pH= 6.0 Enable NH Ct momentum, in particular: Ct pH jump O pH=12.0 Cc- Química Verde pH=12.0 Ct- 6 ii) The study of the photochemistry / photophysics of ancient and historical dyes was also a success with the discovery of a new natural flavylium in Dragon’s blood resin from Dracaena Draco, and the full characterization of its network of pH dependent chemical reactions. This work was also submitted for publication. iii) Finally, the study of modern synthetic polymers used in Modern Art, in the framework of the PhD Project, Liaisons Dangereuses, where an interdisciplinary team was created, with Polymer Scientist Ana Ramos, Art Historian María Jesús de Àvila and Conservation Scientists Maria João Melo and Joana Ferreira for a better understanding and preservation of Portuguese Modern Art. The artists chosen for the case studies are Joaquim Rodrigo, Ângelo de Sousa and Lourdes Castro. The work obtained from the first analysis and characterization of the paintings by Joaquim Rodrigo will be presented in two international Meetings, at the MoMA, New York and at the Tate Modern, London; the work presented at the Tate will be the subject of a publication by the Getty Conservation Institute. Laboratório Associado para a Química Verde 7 ORGANIC SYNTHESIS AND CHEMISTRY OF NATURAL PRODUCTS Head of Laboratory: S. Prabhakar, Full Professor / Ana M. Lobo, Full Professor Staff Members: Pedro Abreu Assistant Professor Paulina Mata Assistant Professor Manuela Pereira Assistant Professor Ana M. Lourenço Assistant Professor Paula S. Branco Assistant Professor Luisa M. Ferreira Assistant Professor João Aires de Sousa Assistant Professor Maria Manuel Marques Assistant Researcher Post-Doct Fellows: Yuri Binev, Sunil Gupta, Qingyou Zhang, Yong Hong Liu, Susan Matthew, Mário Gomes, Ricardo Mendonça Ph.D. Students: Paulo Glória, Luís Pinto, Vasco Bonifácio, Rita Noronha, Susana Gaudêncio, Goncalo Carrera, Diogo Latino Valdemar Figueira Project Grantee: Marta Vilela, Artur Abreu, Carla Macedo, Catarina Soares Number of articles in scientific journals: 15 (18-32) Number of articles in books: 1 Number of M.Sc. Thesis: 1 ORGANIC SYNTHESIS AND CHEMISTRY OF NATURAL PRODUCTS Debromoflustramine B is a biological active alkaloid and its synthesis was recently reported by our group, starting with commercially available L-tryptofan. The method consisted in the stereoselective and chemoselective introduction of the 3,3-dimethylallyl chain at C3a of the known 1,2,3,8-tetrahydropyrrolo[2,3-b]indole skeleton with formation of two diasterioisomers with 36% ee. However, only the minor isomer could be converted into the final alkaloid. We envisaged starting from D-tryptofan and follow the same route. After performing several experiments, we observed the same behaviour and a mixture of two diasteriosomers were formed with 35% ee, in which the major isomer - (2R,3aS)-methyl-1-acetyl-1,2,3,3a-tetrahydro-3a-(3-methylbut-2-enyl)pyrrolo[2,3b]indole-2-carboxylate could be used for the total synthesis of the alkaloid. Novel rearrangements of the indole framework were studied. N-alkylation of N-acetyl L-tryptofan methyl ester with 3,3-dimethylallyl bromide gave (S)-methyl 2-acetamido-3-(1-(3-methylbut-2-enyl)-1H-indol-3-yl)propanoate with 78% yield. This product was treated with BF3OEt2. Several experiments were carried under diverse conditions and migration of the allylic chain to the C2 position was observed. This study enabled the synthesis of crucial intermediates that bear the skeleton of important biologically active alkaloids, as well as furnished information about the reaction mechanism. Theoretical computer calculations of the putative transition states point out towards a considerable ionic character of this rearrangement. Laboratório Associado para a Química Verde 8 A new enantioselective Inverse Phase Transfer Catalysis (IPTC) reaction for the Markovnikov hydration of double bounds by an oxymercuration-demercuration reaction with cyclodextrins as catalysts was disclosed. Moderate e.e. (up to 32%) and yields (14-60%) were obtained for allylic amines and protected allylic alcohols as starting materials. The alkenes reacted with different molar quantities of mercuric salts in a water/nhexane mixture (1:1) containing a phase transfer catalyst, resulting on the formation of a mercury species. The correspondent alcohol was obtained after demercuration with alkaline borohydrate. The best conditions determined for the allylic ether where further applied for the protected allylic amines. Although there were previously reported methods for enantioselective hydration of allylic alcohols, no equivalent method was up-to-date available for the direct enantioselective hydration of allylic alcohols. The present method was able to induce enantioselectiviy to the direct hydration of allylic amines. Future trends: The method will be applied to the semisyntesis of several quinolizidic alkaloids. Retrosynthetic analyses of halipeptins structure acknowledged a oxazoline tripeptide fragment, which included unnatural isoleucine-like residue, and a polysubstituted decanoic acid fragments as major building blocks to be synthesized. Synthetic strategies towards isoleucine-like residue took place along with its incorporation in the tripeptide structure in large scale. Building blocks’ coupling assembled the required carbon structure and, after some more chemical manipulations, halipeptin A was obtained along with some epimers. Related to benzopyran-derived bioactive molecules a new classes of compounds targeting the HIF-1 pathway were discovered and lead optimization took place in collaboration with Prof. Van Meir at Emory University (Atlanta, USA). Structure-activity relationship studies allowed molecules structural simplification and identified functionalities with strong impact in molecules activity. The Brazilian plant species of Solanum cernuum V. (Solanaceae) e Cissus sulciccaulis B. (Vitaceae) are currently used in popular medicine. Among other therapeutic activity S. cernuum exhibit potent antitumoral activity and C. sulciccaulis is important in the treatment of arthritis and beberi diseases. The dichloromethane and methanol extracts of these species proved to have low toxicity. The methanol extract (aerial parts) and dichlomethane (leaves) of S. cernuum were studied for their validation. The methanol extract has 10% of glicosyl alkaloids and peptides. The dichloromethane extract is composed of alkanes (C25-C34), steroids (were the most abundant is cycloeucalenone) and the xanthophyll lutein. The dichloromethane extract of C. sulciccaulis contains 1,76% of fatty acids (C14-C24), 1,71% of sitosterol, 0,14% of other terpenoid compounds and 0,12% of long chain alcohols. Future trends: The work will continue with further identification of steroid and terpenoid fractions of S. cernuum and C. sulciccaulis, together with the study of the methanol extract of C. sulciccaulis. The research activity has been focused on the search for bioactive natural products of plant origin. The evaluation of the anti-inflammatory and antioxidant activity of a Cuban medicinal plant extract (Pedilanthus tithymaloides) was carried out, and the identification of the corresponding active principlkes is in progress. From a medicinal plant (Ozoroa insignis) collected in Guinea-Bissau, a library of cytotoxic phenolic compounds has been characterized. The scientific collaboration with the University of Monastir (Tunisia) has been developed, leading to the isolation of new ceramides, and antioxidant phenolic glycosides from Tunisian plants. In collaboration with the Faculty of Pharmacy of Lisbon, new cytotoxic diterpenes with reversal effect on multidrug resistance, have been isolated from Euphorbia species of Portuguese origin. Architectural drawings on historical archives are often photoreproduced. When based in diazonium salts the discoloration problem is frequent. An interesting aspect of drawing collections from DGEMN is the presence of interleaving sheets of paper. This aspect gave us material to analyse the migrating coloured species Laboratório Associado para a Química Verde 9 without destruction of the museum objects. Organic extracts of the interleaving sheets were analyzed, with aim to study the migrating species, and the only compound isolated and characterized was resorcinol. HPLC studies of extracts of small samples of discoloured interleaving sheets and photoreproductions showed resorcinol as the only common compound. When a drawing in unable to promote the discoloration of adjacent interleaving sheets the resorcinol is absent. Future trends: After the confirmation of these observations in a larger number of cases, a method for the rapid identification of a ‘dangerous’ drawing by the use of HPLC can be applied to each new specimen before its introduction on the archive to assess its risk to degrade the rest of the collection. 3,4-Methylenedioxymethamphetamine (MDMA or “Ecstasy”) is a widely abused, psychoactive recreational drug. There are growing evidences that the MDMA neurotoxic profile may be highly dependent on its hepatic metabolism, leading to the production of reactive catechols, catechol thioethers, and quinones. In this study the electrochemical oxidation-reduction processes of MDMA main human metabolites, obtained by chemical synthesis, were evaluated by cyclic voltammetry. Parallely the neurotoxicity of á -methyldopamine (á-MeDA), N-methyl-a-methyldopamine (N-Me-á-MDMA) and 5-(glutathion-S-yl)- á -methyldopamine [5-(GSH)-á-MeDA] was also evaluated in cortical neuronal cultures2 and the results correlated with their oxidation-reduction potential. The lower oxidation potential observed for the catecholic thioethers of á -MeDA and N-Me- á -MeDA correlated with the higher neurotoxicity of these adducts. Future trends: Synthesis of the monomethoxylated cysteine and glutathione adducts of 4-hydroxy-3methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) will be conducted for neurotoxic evaluation. Quantitative structure-property relationships (QSPR) were established for the melting point and density of ionic liquids. These were applied to the design of new imidazolium and guanidinium ionic liquids that were synthesized. The chirality codes previously developed in the group were applied to the prediction of the enantiomeric excess in a combinatorial library of enantioselective catalytic reactions. The same codes were applied to train neural networks in order to automatically assign absolute configurations from 1D NMR data. A new method was developed to represent chemical reactions with no specification of the reaction center, from the difference of MOLMAP molecular descriptors of products and reactants. Future trends: Application of the MOLMAP representation of chemical reactions to the genome-scale classification of metabolic reactions. Development of QSAR models for the prediction of biological activity, biodegradability and toxicity. Further development of QSPR models for ionic liquids. Application of the SPINUS system for the simulation of 1H NMR spectra to the NMR-based classification of chemical reactions. Research activities concerning the specification of stereogenic units in organic chemistry, and particularly in the development of the Cahn-Ingold-Prelog System in order to improve its logic, consistency, scope and applicability were conducted. In this context a collaboration was established with IUPAC (International Union of Pure and Applied Chemistry) as a researcher of Requimte (Prof. Paulina Mata) belongs now to the Advisory Subcommittee of the Chemical Nomenclature and Structure Representation Division (VIII) http://www.iupac.org/ organ/members/m/mata.html. Laboratório Associado para a Química Verde 1 0 In 2005 this collaboration was very intensive, because of the ongoing revision of IUPAC recommendations on specification of molecular configuration to be included in the next IUPAC Book on Nomenclature of Organic Compounds. Molecular Gastronomy was also developed, through international connections with researchers in the area, particularly Dr. Hervé This (Lab. de Chimie des Interactions Moléculaires Collége de France et Lab. de Chimie Analytique - Institut National Agronomique Paris Grignon (INA P-G)), national collaborations (Prof. Maria da Conceição Loureiro Dias – Dep. de Botânica e Engenharia Biológica do Instituto Superior de Agronomia) and also the participation in the “Cours the Gastronomy Moléculaire 2004/2005 “ (INA P-G) . Work continued in the in science teaching and divulgation front which hopes to contribute to improve science teaching practices, to motivate students to continue their studies in science and to develop the general scientific culture in the public at large. This involves working with other educational levels (production of teaching aids and teacher training) and collaboration in research activities with other institutions, particularly Instituto Superior de Psicologia Aplicada. Concerning science divulgation activities special reference should be made to a) the collaboration with Ciência Viva and Pavilhão do Conhecimento by the participating in demonstrations and in international projects and b) the publication of science divulgation articles in the Portuguese press. Laboratório Associado para a Química Verde 1 1 SELECTIVE SYNTHESIS AND STRUCTURAL CHEMISTRY Head of Laboratory: Maria Teresa Barros, Associate Professor Staff Members: Maria Teresa Avilés Perea Assistant Professor António Gil de Oliveira Santos Assistant Professor Eurico José da Silva Cabrita Assistant Professor Marta Morais Saraiva de Andrade Assistant Researcher Ana Maria Madeira M. Faísca Phillips Principal Researcher Post-Doct Fellows: Krasimira Petrova, Snezhana Bakalova Ph.D. Students: Vitor João Salgueiro Rosa, Paula Correia da Silva, Maria João Morgado, Filipe José dos Santos Duarte, Paula Alexandra Carvalho Rodrigues Number of articles in scientific journals: 6 (33-38) The principles of Green Chemistry as applied to organic synthesis (economy and efficiency) needs advanced principles of organic synthesis applied to the efficient production of complex organic compounds from renewable natural (chiral) starting materials. We have continued to investigate the reactivity of saccharose and to apply our results to the study and application of sugar as a chiral auxiliary for asymmetric synthesis. Our final goal is to apply this chemistry to diverse problems under unusual but ecologically favourable reaction conditions. The selective derivatisation of carbohydrates with unsaturated moieties is one of the easiest routes for the preparation of polymers and for the construction of asymmetric molecules using carbohydrates as chiral auxiliaries. We have developed an efficient strategy to attach vinylic groups and dienes to the sugar nucleus (D-glucose, D-xylose and sucrose) by two different procedures (esterification and Wittig olefination), both under microwave irradiation. On the other way, Wittig reaction between O-formylated sugars and stabilised phosphoranes was performed in dry medium, with the formation of the isomer E as major product. By studying the application of microwave technology, we intend to develop cleaner and more efficient methods for synthesis, from a time and energy view point, in an attempt to contribute to a sustainable development. Water soluble dienes are also prepared by exposure of the hydroxyl groups of the sugar auxiliary. The chemistry of azides includes a useful synthetic applications in the preparation of 1,2,3-triazolines via 1,3 dipolar cycloaddition reactions. We have reported the preparation of several azides of low molecular weight, which have been useful for this work. We have been successful in the selective connection of acrylate to the sugar molecules (in some positions) and the copolymerisation with simple acrylates to form more or less dense sugar polymers. By attaching the sugar to a polymer it is possible to alter some of its properties. In collaboration with a research group in Biothecnology area we are preparing L-arabinose selectively marked in C2. The increasing interest in the production of ethanol from wood pulp (cellulose) has generated studies related to the metabolism of L- Arabinose (the second most abundant pentose in cellulose) by yeasts, which are excellent fermentative microorganisms. In order to elucidate the metabolic pathway of arabinose in selected yeasts. The technique to be used is in vivo NMR using C2-13C labelled L-arabinose. Laboratório Associado para a Química Verde 1 2 Asymmetric synthesis and homogeneous catalysis The syntheses of chiral bis(oxazolines) derived from (R,R)-(+)-tartaric acid and their application as ligands in the enantioselective Michael addition of diethylzinc to enones, already underway during 2004, was completed. The results were published. During this period, methodology for the syntheses of novel chiral diamines and sulfur-containing compounds derived from tartaric acid was also developed and continued. The possibility of using these compounds either as chiral organocatalysts or as ligands for metal-catalyzed processes, was investigated. A bis-Nmethylmethanamine derivative was found to be an organocatalyst for the diastereoselective and asymmetric Michael addition of cyclohexanone to b-nitrostyrene. The results were published in 2006. Stereoselective behavior of chiral a-haloacyl compounds in nucleophilic substitution reactions, under dynamic kinetic resolution (DKR) conditions. We studied, theoretically, the stereoselective behavior of nucleophilic substitution reactions of ?-haloacyl compounds, using a chiral auxiliary approach, based on ephedrine derived imidazolidinones, under DKR conditions. New chiral auxiliaries were proposed and some derivatives were synthesised and tested, confirming the theoretical previsions of expected high final diastereoisomeric excesses. Catalysts in asymmetric synthesis: A complexation study. The use of Lewis acids (LA) to enhance the reactivity and stereoselectivity of a number of reactions involving N-acyloxazolidinones and N-acylimidazolidinones is well known. For some transformations the stereoselectivity induced by the LA complexation can’t be entirely explained by the accepted models. We have undertaken a NMR and molecular modeling study of this type of complexation with different LA. Since the molecules in question have two key Lewis base sites, several solution complexes are possible, including chelated ones, which render the study very complex. In an attempt to simplify it, we have prepared several model compounds and studied their complexation with non-chelating (BF3.OEt2) and chelating (AlEtCl2, TiCl4 and Mg(ClO4)2) LAs. Only for Mg(ClO4)2 evidence for chelated forms in solution was found. This information can be of major importance in the revision of the accepted mechanisms for all reactions where N-acyloxazolidinones and Nacylimidazolidinones are used, prompting us to the proposal of new mechanistic pathways. Asymmetric additions to alkenes. Our interest in this subject has been mainly related with Diels-Alder reactions and the addition of electrophilic species (e.g amines) to unsaturated N-acyloxazolididinones and Nacylimidazolidinones. The accepted mechanism, proposed by Evans, more than 20 years ago is unable to explain many unexpected experimental observations made during these years. Based on our previous work on DKR reactions, we started the development of a full theoretical and experimental study (based on NMR techniques), with the aim of clarifying possible mechanisms and to propose structural variations, able of increasing the final diastereoisomeric excesses. Our studies brought us to a new proposal, which can explain the observed stereochemistry, both in Diels-Alder and in electrophilic additions. We also studied similar additions to alkenes connected to different chiral auxiliaries (as, for instance, Ethyl-S-lactyl acrylate) with more flexible structures, showing that even in these cases no chelated transition states are theoretically expected (results published in 2006). Intramolecular asymmetric aldol reactions. Based on our previous experimental results, we are currently studying the mechanistic aspects of asymmetric aldol reactions, using theoretical and experimental tools. Our aim is the understanding of the large amount of literature information on chiral and achiral systems. Until the moment, in spite of many reports describing the use of chiral catalysts and auxiliaries, there is no real understanding of the factors governing the mechanistic pathways of even the achiral processes. Our first studies indicate a strong dependence on electronic effects which, if confirmed, can reduce substantially the variety of structures able to work as starting materials. Application of NMR techniques to the study of intermolecular interactions. The complementary between the information obtained from NOE and relaxation studies with that from diffusion NMR was explored in the study of 1-buthyl-3-methylimidazolium (BMIM) ionic liquids. The determination of internuclear dis- Laboratório Associado para a Química Verde 1 3 tances, relaxation rates and relative strength of ion pair association have been interpreted in terms of rotational motion, molecular structure and molecular properties and allowed the proposal of a solvation model for [BMIM]PF6 dimethylsulfoxide solutions for a wide range of concentrations. The study of the molecular determinants of ligand specifity in family 11 Carbohydrate Binding Module (CBM11) is being conducted by NMR Saturation transfer in combination with ligand titrations of the protein. The protein was labelled with 13C and 15N and several experiments are being conducted in order to study the enzyme complexed with its carbohydrate ligands; In Organometallic chemistry, we have continued with the synthesis of cobalt (II) complexes of the general formula CoX2( a-diimine) where X= Cl, or I and the a-diimines are bis(aryl)acenaphthenequinonediimine) (ArBIAN) and 1,4-diaryl-2,3-dimethyl-1,4-diaza-1,3-butadiene (Ar-DAB) in order to study their structural and magnetic properties. The synthesised complexes are, [Co(Ph-DAB)Cl2], 1a; [Co(o,o’,p-Me3C6H2-DAB)Cl2], 1b; [Co(o,o’ –iPr2C6H3-DAB)Cl2], 1c; [Co(o,o’,p-Me3C6H2-DAB)I2], 1’b; [Co(o,o’-iPr2C6H3-DAB)I2], 1’c; [Co(o,o’,pMe3C6H2-BIAN)Cl2], 2b ; [Co(o,o’-iPr2C6H3-BIAN)Cl2], 2c; [Co(o,o’,p-Me3C6H2-BIAN)I2], 2’b; [Co(o,o’-iPr2C6H3BIAN)I2], 2’c. They were characterised by elemental analyses, mass spectrometry, IR spectrometry, and EPR, in some cases we were able to grow crystals suitable for X-ray structural determination. X-band EPR measurements in polycrystalline samples performed on all compounds indicate high-spin Co(II) ion (S=3/2) in an axially distorted environment. Single crystal experiments on compounds 1b and 1c allowed us to evaluate the orientation of the g-tensor in the molecular frame. This work is almost completed and it will be submitted soon for publication. 2006 activities to be developed Several polymers which we have recently synthesised and are based upon saccharose are being studied for their biodegradability. This work is being carried out in collaboration with another University. We are interested to use alternative energy source (microwaves) and alternative reaction conditions (use of ecologically friendly solvents such as water or no solvent). In a related project we pretend to study the use of concentrated saccharose solutions in accelerating stereoselective reactions. It is hoped that the structurally ordered sugar solution will both direct the reagents and accelerate the reaction in such a way that the product is obtained with high stereoselectivity and affords optically active products. Pericyclic reactions which can be carried out under aqueous conditions should be influenced by a chiral environment and organization of the reagents should occur. In another part of this project we hope to study the glycosylation of steroids. A number of important groups of drugs are glycosides. The sugars seem to play a key role in the interaction of the drugs with their receptors All the synthetic methodologies will be designed in order to develop cleaner technologies as a major emphasis in green chemistry. Among the several aspects, when is possible, the synthetic methodologies will be performed in solvent free conditions, or water-based medium, and/or under microwave radiation as alternative energy source. In another project, we intend to produce environment-friendly, ready-biodegradable complexones at a low cost, which may compete at industrial scale production with the well established and cheap synthetic NTA and EDTA compounds. For this purpose, the synthesis pathway will be based mainly in the life cycle; block units of stereo specific amino acids will be used for asymmetric synthesis of natural aminopolycarboxilates ligands (or derivates) potentially better biodegradability (i.e. they will be decomposed into non-toxic compounds, environmentally benign substances). In addition, the synthesis will be designed in order to be performed in a water-based medium, for reducing the use of organic solvents. Collaboration in a European project related to the synthesis and physical properties of azides will continue. Laboratório Associado para a Química Verde 1 4 Research aimed at the development of novel organocatalysts or ligands for metal-catalyzed asymmetric reactions, particularly using amino acids as chiral synthons, is currently underway. Concerning the Lewis acids - carbonyl compounds complexation studies, we plan to publish the results obtained in the last years and to continue to use the information obtained with these studies in the clarification of the mechanisms involved in asymmetric Diels-Alder reactions and other additions to double bonds. We expect also to continue our work in order to clarify several aspects dealing with intramolecular aldol reactions based on molecular modelling, NMR and synthetic work. Our aim is to understand the achiral systems and to concentrate efforts in understanding part of the literature information and, in special, the experimental results obtained in our research group during the last couple of years. We will continue to apply NMR techniques to the study of intermolecular interactions, especially ionpair strength in the BMIM family of ionic-liquids and also solute-solvent interactions. The study of the molecular determinants of ligand specifity in CBM11 will continue. The 2D 15N-HSQC will be assigned using double and triple resonance NMR experiments in order to identify the residues involved in ligand recognition. As before this work continues in strong collaboration with groups integrated in other research themes: Protein Crystallography and Biomimetic Systems and Simulation. We will continue with the synthesis of a-diimine cobalt complexes, trying to introduce ligands such as indenyl, cyclopentadienyl, alkyl, and hydride. New a-diimine ligands will be synthesised as well as new Schiff base ligands, The complexing properties of the newly synthesised ligands towards metal cations of environmental, analytical or catalytic interest, such as Co(II), Ni(II), Pd(II), Ag(I), Cu(I), Zn(II), Cd(II) and Hg(II), will be tested. Laboratório Associado para a Química Verde 1 5 PHYSICAL ORGANIC CHEMISTRY / RADICAL CHEMISTRY Head of Laboratory: Abel J. Vieira, Associate Professor Staff Members: João Paulo Noronha Assistant Professor Ph.D. Students: Pedro Manuel C. C. P. dos Santos, Mónica Pombinho de Matos, João Adalberto A. Lourenço Project Grantee: Patrique Nunes UndergraduateStudent: Marisa de Barros Sousa e Silva Number of articles in scientific journals: 3 (37, 39,40) Radical reactions of DNA bases and other biologically relevant compounds. Effect of antioxidants. Radical oxidation of caffeic acid, cinnamic acid and tetrahydrocannabinol. Identification of final products. Relative antioxidant properties of the above referred compounds. Synthesis of new precursors of alkyl radicals. Radical alkylation of xanthines. Evaluation of the in vitro antioxidant activity of non-steroidal anti-inflammatory drugs (NSAIDs) Chiral Quantitative Structure-Property Relationship / Counterpropagation Neural Networks study of enantiomeric separation on two HPLC columns Natural Products Research – Portuguese Autochthon Plants – Bioactivity Studies Arson analysis Laboratório Associado para a Química Verde 1 6 CHEMICAL ENGINEERING SCIENCE Head of Laboratory: Manuel Nunes da Ponte, Full Professor Staff Members: Pedro Brito Correia Invited Full Professor João Goulão Crespo Associate Professor Susana Barreiros Associate Professor Joaquim Vital Assistant Professor Maria Ascensão Reis Assistant Professor Isabel Ligeiro da Fonseca Assistant Professor Ana Maria Ramos Assistant Professor Pedro Simões Assistant Professor Henrique Guedes Assistant Professor Isabel Coelhoso Assistant Professor Ana Aguiar Ricardo Assistant Professor Madalena Dionísio Assistant Professor José Paulo Mota Assistant Professor Maria Margarida Cardoso Assistant Professor Rui Oliveira Assistant Professor Svetlozar Velizarov Assistant Researcher Post-Doct Fellows: Adrian Oehmen, Ewa Bogel-Lukasik, Filomena Feitas, Gilda Carvalho, Isabel Esteves, José Castanheiro, Luisa Serafim, Marta Eiroa, Paulo Lemos, Raquel Fortunato, Rui Ruivo, Svetlana Lyubchik , Teresa Casimiro, Vesna Najdanovic-Visak, Vitor Alves Ph.D. students: Ana Teixeira, Carla Brazinha, Carla Portugal, Carlota Veiga de Macedo, Cláudia Galinha, Cristina Matos, Graça Albuquerque, João Dias, João Fernandes, José Luís Santos, Liliana Guerreiro, Márcio Temtem, Maria Teresa Viciosa, Mariana Sousa e Costa, Mário Eusébio, Patrícia Oliveira, Pedro Vidinha, Rafal Bogel-Lukasik, Sofia Nunes Barata Project grantees: Ana Rita Brás, Angela Pereira Machado, Ricardo Couto Number of articles in scientific journals: 48 (5, 6, 38, 41-85) Number of patents: 5 Number of articles in books: 9 (2-10) Number of Ph.D thesis: 5 Laboratório Associado para a Química Verde 1 7 Scientific interests The scientific interests of the Chemical Engineering Science Area in 2005 spanned a wide range of subjects: - Adsorption processes - Biocatalysis in nonaqueous solvents - Bioprocess modelling, monitoring, optimisation and control - Bioremediation - Chemical reaction and supercritical carbon dioxide - Depolymerisation studies and characterisation of biopolymers - Dynamic modelling of supercritical fractionation processes - Drug controlled-release systems - Dynamical changes upon crystallization in a biopolymer - Extraction of biopolymers from industrial wastes for edible films applications - High-pressure NMR - Membrane Processing and Monitoring - Modelling of nanofiltration processes - Polymer processing and membrane formation in clean solvents - Polymeric Catalytic Membranes & Heterogeneous Catalysts - Polymer dispersed Liquid Crystal formation in both conventional and scCO2 media - Supercritical fluid extraction of liquid mixtures - Thermodynamics of Ionic Liquids - Viscous fluid mixing. Experiments and CFD The interplay of these multiple research interests resulted in a research effort leading, as an ultimate, strategic goal, to the study of Process Integration. The main areas of research can be grouped under three headings: - Clean Solvents and Clean Processes, - Process Simulation and - Polymeric Materials The above-described research activities led to collaborative efforts that joined different groups, in order to develop - Integration of Processes RESEARCH ACTIVITIES IN 2005 Clean Solvents and Clean Processes Adsorption processes 1- Activated carbon metal supported catalysts were tested on chlorobenzene hydrodechlorination. The types of materials used in this study were the following: synthetic activated carbon (SCN) and carbonaceous porous supports obtained from polystyrene-based organic salts pyrolysis. The introduction of the metal (palladium and nickel) on this support was performed using the method of reductive adsorption, discarding the need of subsequent reduction (in the case of palladium). These catalysts Laboratório Associado para a Química Verde 1 8 were successfully tested on the hydrodechlorination of chlorobenzene at room temperature and under H2 atmospheric pressure. 2- A novel porous material obtained from carbon-containing liquid and solid wastes has been used as adsorbents for 3d–transition metals removal from multi-components solutions. The activated carbon was prepared from the co-mingled natural organic wastes: sunflower husks of 25 %, petroleum waste of 50 % and low–grade bituminous coal of 25 %. The adsorption of the 3d-transition metals: copper (II), cobalt (III), nickel (II), iron (II) & (III), manganese (II) and chromium (III), on novel activated carbons were investigated from multi-components model and technological solutions. Emphasis has been done to the kinetics and thermodynamic studies of the simultaneous adsorption of the 3d–transition metals in a static mode. Polymeric Catalytic Membranes 1 - The limonene oxidation was performed in liquid phase, over cobalt acetylacetonate encapsulated in the supercages of zeolite Y and dispersed in a polydimethylsiloxane (PDMS) matrix. t-Butyl hydroperoxide was used as oxygen supplier. When the oxidation reaction was carried out over the PDMS embedded catalyst, the main product obtained was limonene oxide. On the other hand, when the Y-zeolite encapsulated cobalt Schiff base was directly used as catalyst, limonene oxide was also formed but the main product obtained was a polymer. The PDMS matrix seems to play an important role in tuning the reactants concentration near the catalyst active sites in such a way that polymerisation is inhibited. 2 - Poly(vinyl alcohol) (PVA) membranes were prepared using sulfosuccinic acid (SSA) by varying the amount of SSA (5-40 mol%) and were used for the esterification of acetic acid by isoamilic alcohol. The conversion of isoamilic alcohol increases when the amount of sulfosuccinic acid used in the polymer crosslinking is increased from 5% to 20%. 3 - The transesterification of soybean oil with methanol was studied using as catalysts two Nafion commercial membranes with different thickness and a poly(vinyl alcohol) membrane crosslinked with sulfossucinic acid (PVA-SO3H). The reactions were carried out at 60 ºC, in a batch reactor using the membrane cut in small pieces. The initial activities of the membranes are compared being observed an increase of initial activity with the amount of sulfonic acid groups. Membrane Processing and Monitoring 1 - Recovery of aroma compounds from diluted aqueous streams by pervaporation. was focused on the understanding of the molecular interactions between target solutes and the membrane material. Real-time monitoring of the permeating stream, by on-line mass spectrometry allowed the description of the process of solute permeation in steady-state and in transient operating conditions. New approaches for selective recovery of solutes from the permeating stream are under development. 2 - Development of integrated pre-enrichment techniques for sample discrimination by electronic sensorial systems (electronic nose) in pervaporation was integrated with electronic sensorial systems, in order to enhance discrimination of wine samples, in an automated mode. This approach allowed the improvement of sample discrimination using electronic nose, even when they present a high ethanol content. 3 - Design of clean membrane processes for recovery, separation and purification of high added-value products focused on the understanding and mathematical modelling of the transport process of the species involved across porous and non-porous membranes, with a special emphasis on water transport in liquid membranes, transport of solutes through ionic liquid supported liquid membranes, transport of hydrophobic molecules through non-porous membranes and transport of electrically charged species in ion-exchange membranes. 4 - Study of membrane processes for clean and selective recovery of biological products from dilute streams involved different membrane processes, namely liquid membrane extraction using selective carriers (chiral selectors), pervaporation for integrated reaction and recovery of solutes from dilute aqueous streams and from ionic liquids, ultrafiltration for fractionation of proteins with pharmaceutical interest, and nanofiltration for recovery and fractionation of antioxidant compounds from agroindustrial waste streams and for the reuse of catalysts in integrated reaction systems. Laboratório Associado para a Química Verde 1 9 5 - Extraction of amino acids and derivatives using ionic liquids was studied using different membrane configurations. The transport mechanism that regulates solute transport, diffusion through water clusters inside the ionic liquid, was compared for both configurations. 6 - Stability of supported ionic liquid membranes for vapour separation The use of a room-temperature ionic liquid (RTIL) immobilized in the pores of a supporting membrane is particularly interesting owing to the non-volatile character of RTIL´s which opens the possibility to obtain very stable liquid membranes. This new concept will allow the developpment of “taylor-made” membranes using an IL specifically designed to separate target solutes from vapour streams. 7 - Development of non-invasive, on-line monitoring techniques. used on-line mass spectrometry, Steadystate 2D-fluorescence, Fluorescence anisotropy, Time-decay fluorescence, Confocal Laser Scanning Microscopy and Raman Confocal Microscopy. Concentration gradients of solutes, in particular ionic liquids, within the membrane polymer was investigated by means of Raman Confocal Microscopy due to the lack of suitable molecular probes for this purpose. Bioremediation 1- Membrane Bioreactors Membrane bioreactors were used in the removal of polluting ions from drinking water streams, according to the Ion Exchange Membrane Bioreactor (IEMB) concept, for water contaminated with both perchlorate and nitrate. The process efficiency was compared using different types of membranes with a special emphasis on such having a comparatively low commercial price in order to promote the practical implementation of this technology. A study on the removal of other emerging water micropollutants (arsenate, ionic mercury etc.) was initiated. The arsenate separation potential of a number of anion-exchange membranes was evaluated. Batch tests under controlled conditions were performed in order to evaluate the ionic mercury reduction to metallic mercury by pure and mixed cultures. The AAS method for mercury determination was adapted and optimised for both liquid and gas phase measurements, which allowed for closing the corresponding mass balances. 2-Conventional biological processes. The simultaneous phosphorus and nitrogen removal was studied in two systems fed with propionate and acetate. The system adapted to propionate was more stable that the acetate one, that collapsed and lost the capacity of phosphorus removal. In the propionate reactor it was shown that the organisms able to remove both nutrients (DPAO, denitrifying polyphosphate accumulating organisms) are different from the classical organisms for phosphorus removal (PAO, polyphosphate accumulating organisms). Using Fluorescence in situ hybridisation (FISH) for the identification of phosphorus accumulating organisms, two different morphotypes were present in the propionate reactor, a dominant rod-shaped organism and a bacilli-shape one. Further research is being carried out for the identification of these two morphotypes The biodegradation organomercurial compound to metallic mercury, under aerobic conditions, by a pure culture of Pseudomonas putida spi3 strain was studied in a continuous stirred tank reactor (CSTR) fed with a real vaccine production wastewater. Chemical reaction in supercritical carbon dioxide In mixtures of high pressure carbon dioxide with liquids just below the critical pressure of the mixture, liquidvapour equilibrium shows a peculiar behaviour. While the gas phase remains essentially pure CO2, the liquid incorporates large amounts of carbon dioxide, which increase with pressure, dramatically changing its physical properties. Hydrogenation of limonene was performed in this pressure range, either in one phase or biphasic conditions, with surprising results inn terms of selectivity of products. The interpretation of the reactivity needed detailed measurements and correlation of phase equilibria. The hydrogen distribution ratios between the phases wee revealed to be extremely sensitive to pressure. Laboratório Associado para a Química Verde 2 0 Thermodynamics of Ionic Liquid-containing systems An extensive programme of measurement of phase equilibrium properties of ionic liquid mixtures with water, alcohols and high-pressure carbon dioxide continued. Biocatalysis in nonaqueous media Research efforts were directed towards the: (i) Generation of experimental data to test/upgrade the existing model for cutinase enantioselectivity (E), through studies on the impact of temperature and solvent on E of cutinase and of a lipase at temperatures down to -20 ºC (ii) Elucidation of the interactions between cutinase and sol-gel matrices prepared with different sol-gel precursors, by combining information obtained from experiments with sol-gel matrices containing additives, from fluorescence spectroscopy studies based on the single tryptophan residue of the enzyme, and from a calculation of the charge distribution at the surface of cutinase based on the existing model for the enzyme (iii) Implementation of a reaction/separation scheme, combining the chiral resolution of an alcohol in an ionic liquid catalysed by a lipase with extraction with supercritical CO2 (iv) Synthesis of geranyl acetate in a continuous packed-bed reactor via the esterification of geraniol with acetic acid catalyzed by a lipase in supercritical CO2 and in supercritical ethane, with an emphasis on water activity effects/control. Process Simulation and Modelling Dynamic modelling of supercritical fractionation processes A dynamic model of a supercritical fluid extraction plant was developed. The model incorporates a series of simulation models for the main pieces of equipment of the SFE plant, namely, the countercurrent structured packed column, the regeneration column, the heat exchanger for solvent warm-up and the make-up unit. The dynamic model of the heat exchanger takes into account the resistances to heat transfer in the gas as well as in the heating fluid (liquid water at ambient pressure) and across the stainless steel wall of the inner tube. Validation of the dynamic model was done by comparing predicted results with experimental data obtained for the fractionation of the squalene + methyl oleate with SC CO2. A good agreement was obtained between measured and predicted temperature and composition profile of the gas and liquid phases through the packed bed and extraction time. Modelling of biological processes 1-Metabolic model of Polyhydroxybutyrate production by mixed microbial cultures. The model is based on 7 metabolic reactions. Theoretical yields were evaluated. Experiments were performed with and without ammonia supply enabling the analysis of PHB formation independently of the cell growth process. The experimental yields partially confirmed the theoretical values. A full kinetic model was synthesized. The final model exhibited high accuracy in describing the process state of most experiments performed, thus opening good perspectives for model-based optimisation studies. 2- Modelling and optimisation of a recombinant BHK-21 cultures. A model-based optimisation study of fedbatch BHK-21 cultures expressing the human fusion glycoprotein IgG1-IL2 was performed. A hybrid mechanistic/ chemometric model identified the BHK metabolic fluxes. The optimisation studies showed that the glutamine and glucose concentrations should be maintained at low levels during the growth phase and then glutamine feeding should be increased. 3- Modelling and Control of Rotavirus Virus-Like Particle Production. Rotavirus-like particles were produced using the baculovirus-insect cells expression system. A mathematical model was developed to describe the intracellular dynamics of DNA, corresponding to the three structural genes of rotavirus, the respective mRNA concentration, single protein concentration (VP2, VP6 and VP7), and assembled VLPs concentration. Modelling of nanofiltration processes 1 - Modeling Rejection in Aqueous Nanofiltration – Solute Geometry and Orientation Effects Laboratório Associado para a Química Verde 2 1 A new approach to model the rejection of uncharged solutes by nanofiltration membranes was developed. A mathematical description of the solute size and geometry was developed, using the concept of geometric radius, in which the molecular geometry was represented by a prolate revolution ellipsoid. Additionally, the preferential orientation angle of the solute towards the membrane, and during permeation, was taken into consideration and, consequently, its effect on rejection was also introduced. 2 -Modelling the Transport in Solvent-resistant Nanofiltration The transport of solvents through a solvent resistant NF membrane was analysed by a hybrid modelling approach, integrating mechanistic and chemometric models that allowed for a better description of the experimental results. The permeation of water, n-butanol, iso-butanol and tert-butanol as single solvents as well as in their binary mixtures was studied and successfully modelled. 3- Application of CDF for Simulation and Optimization of the Flow Patterns CFD (computational fluid dynamics) was applied to characterize the flow in membrane modules, which allowed for studying velocity, pressure, concentration and pressure drop profiles inside the channels. The introduction of ladder-type spacers was found to disrupt the boundary layer near the membrane surface, thus decreasing its thickness, and therefore enhancing the mass transfer. Modelling of adsorption and adsorption processes Cyclic adsorption processes, including Pressure Swing Adsorption, Simulated Moving Bed and hybrid systems integrating adsorption and membrane permeation were modelled and optimized using state-of-the-art computational tools. A novel single-column mimetic alternative to multicolumn continuous counter-current chromatography has been developed. A novel approach for structural characterization of carbon nanotubes using molecular simulation and adsorption of probe molecules was developed and experimentally validated. Polymeric Materials Polymer processing in clean solvents New polyurethanes were synthesized in supercritical CO2, using as monomers the CO2 and aziridines. Aziridines have been reported to react with CO2 to give cyclic urethanes and polymers consisting of urethane and amine units. Substituted aziridines copolymerize with carbon dioxide to give copolymers of polyurethane in the presence or absence of catalysts. The use of supercritical CO2 simultaneously as reaction medium and reactant offers the opportunity to manipulate the outcome of these reactions, including the polymer structure. A new experimental apparatus was developed for carrying out reactions up to 120ºC and 34 MPa. The polymers obtained were analyzed by MALDI-TOF, NMR and DSC. Membrane formation in scCO2 Polysulfone (PS) membranes were prepared using a CO2-assisted phase inversion method. The effect of the solvent affinity and depressurization rate in the morphology of the membranes was investigated. These parameters had a significant effect upon the pore size of the membranes. Different casting solutions were prepared using the following solvents: methylpyrrolidone, dimethylformamide, dimethylacetamide, chloroform, dimethylsulfoxide and dimethylpropionamide. The morphology of the membranes obtained by fast depressurization (less than one minute) and slow (approximately one hour) were compared. Higher depressurization rates produce larger pores. Solvents with higher affinity to CO2 produce membranes with smaller pores and with narrow pore size distributions. Molecular size of the solvent and depressurization rate could be additional parameters used in CO2 – assisted phase inversion method to control membrane morphology. This technique was able to produce homogeneous membranes with a good interconnectivity between pores. Dynamical changes upon crystallization in a biopolymer Dielectric relaxation spectroscopy was used to monitor the development of crystallinity in poly(L-lactic acid) (PLLA) at 80ºC. Loss data was modeled considering the evolution of three relaxation processes: the aprocess of the bulk-like (non-restricted) amorphous phase, the a-process of the amorphous fraction influ- Laboratório Associado para a Química Verde 2 2 enced by the crystalline structure and the b-relaxation. It was found that the shape parameters and the position of the loss peaks are essentially constants during crystallisation, just with their dielectric strengths varying. This indicates that (i) the two segmental dynamics process develops independently and (ii) the dynamics features of the (slower) confined amorphous phase do not change during crystallization PDLC formation in both conventional and scCO2 media Polymer dispersed liquid crystals were produced by conventional thermally initiated polymerization of TrEGDMA+AIBN/E7 mixtures in different ratios, the 30/70 composition ratio revealing suitable electro-optical response. The formation process of the PDLC having the 40/60 composition as precursor was followed by dielectric relaxation spectroscopy in a pioneer study in literature. The degree of conversion of the monomers was evaluated by FTIR and its dependence on polymerization temperature, initiator concentration, E7 concentration, radiation power and radiation time were analysed. SEM was used to examine the morphology of the polymer dispersed liquid crystal (PDLC) produced. Differential Scanning Calorimetry (DSC) studies were made for not polymerized and partial polymerized samples to test the degree of conversion calculations. Dielectric Relaxation Spectroscopy (DRS) were used to compare the results obtained by this technique and FTIR in the same experimental conditions. In order to control the secondary thermal reactions in the photo-polymerization mechanism, a photoiniferter was used, p-xylylene bis(N,N-diethyl dithiocarbamate) (XDT), as a initiator in a living radical polymerization. Electro-optical studies of the PDLC film were made in order to evaluate the voltage needed to change the transmission characteristics of the film. These electro-optical characteristics are dependent on the polymer structure and thus the nature and the rate of polymerization mechanism, that means, size and number of the E7 microdroplets in the polymer matrix. High-pressure NMR High-pressure nuclear magnetic resonance, HP-NMR was applied to investigate the CO2-philicity of Krytox and the molecular interactions between different stabilizers, monomers and carbon dioxide. A new polyether ketone (PEEK) cell was built, with 10 mm o.d., suitable to record NMR spectra on a Brucker system. Pressure-dependent NMR spectra of CO2, CO2 + stabilizer and CO2 + stabilizer + monomer were obtained at 334 K and pressures from 15 MPa up to 22 MPa. Depolymerisation and monomer recovering The catalytic depolymerisation of polymethylmethacrylate (PMMA) was studied in order to obtain the pure monomer for further polymerisation, aiming at to maximize conversion at lower temperatures than those industrially used in simple pyrolysis. The ongoing studies involved the preparation and characterisation of catalysts based on La, Ce, Cs, Cu and Ni oxides inserted in the structure of mesoporous carbon xerogels, during the sol-gel synthesis of the polymer carbon precursor (a resorcinol-formaldehyde resin). These novel catalysts were tested in the depolymerisation of pure and waste PMMA, under previously optimised reaction conditions. The additives of the waste PPMA showed to play a synergetic catalytic effect on the reaction. Modelling of kinetic data was performed and a reaction scheme was proposed. Extraction of biopolymers from industrial wastes for edible films applications Studies on the optimisation of pectin extraction from orange peels (residue from juice manufacturing) by the flash pressure method (pressure from 2-5 bars) were carried out under a financed REQUIMTE project. Pectin extracts were chemically characterised (monosaccharide content, degree of esterification, galacturonic acid content, average molecular weights and polydispersity. The viscoelastic properties of pectin solutions were determined by the CEQUP group. The final purpose of this study is the preparation of edible films pectincarrageenan, being the carrageenan extracted from natural resources (seaweeds from the Portuguese coast –CEQUP group). The film preparation and the transport studies are carried out by another CQFB group. The thermophysical and mechanical characteristics of the films are performed by the CEQUP group. Three CQFB groups are involved in this project. Laboratório Associado para a Química Verde 2 3 Drug controlled-release systems Studies on polymeric formulations for drug controlled release have been performed. Microspheres containing anti-tumour drugs from the taxoid group and the antracicline group have been prepared using conventional methods, namely solvent evaporation method, and by an emulsion-membrane technique. Drug release studies were performed and mass transfer models were developed. Polymeric systems containing anti-inflammatory drugs were produced using supercritical fluid technology in different conditions: impregnation studies and SAS technology. The process efficiency and drug release behaviours were compared with the solvent evaporation method. Drug release formulations for target delivery of anti cancer drugs in the colon were developed using polymers pH dependent. Production of Biopolymers Strategies to obtain high cell concentrations of mixed microbial cultures without loosing the PHA storage capacity have been implemented. Experiments were carried out in Sequencing Batch Reactors (SBR). The effect of operating parameters such as substrate and ammonia concentrations, sludge age and reactor feeding strategies on the growth yield and specific growth rate were evaluated. Furthermore, microbial population was characterized by molecular biological techniques such Fluorescence in situ hybridisation (FISH) and the impact of the operating parameters on the microbial community structure was evaluated. A two-stage process including a fermentation step, in which the sugars of the molasses are converted into organic acids, CO2 and H2, and a PHA production step, in which the organic acids serve as the precursors to the formation of PHAs was implemented. The effect of different operating conditions on sugar to organic acids conversion yields and organic acids concentration profiles has been evaluated. The quality of the fermented molasses as a substrate for the production of PHAs was determined. Formulation and characterization of edible films for foods Model edible films based on commercial k-carrageenan and pectin from citrus fruit, were prepared, and their hygroscopic and mechanical properties, as well as their water vapour permeance, were determined. The model carrageenan-pectin films studied showed increased mechanical properties, lower glass transition temperatures, increased water permeability and hydrophilic properties with increased carrageenan content. A levelling off of the water permeance was observed at high carrageenan content and a maximum was seen in the mechanical properties. Film properties do not comply with simple mixing rules of carrageenan and pectin properties, suggesting a phase separation between pectin rich and carrageenan rich domains. Thermal-polymerization induced by 2,2’-azobisisobutyronitrile (AIBN) and photo-polymerization induced by 2,2-dimethoxy-2-phenylacetophenone (DMPA) of triethylene glycol dimethacrylate and their composites with and without the low molecular weight nematic liquid crystal E7 were studied. The degree of conversion of the monomers was evaluated by FTIR and its dependence on polymerization temperature, initiator concentration, E7 concentration, radiation power and radiation time were analysed. SEM was used to examine the morphology of the polymer dispersed liquid crystal (PDLC) produced. Differential Scanning Calorimetry (DSC) studies were made for not polymerized and partial polymerized samples to test the degree of conversion calculations. Dielectric Relaxation Spectroscopy (DRS) were used to compare the results obtained by this technique and FTIR in the same experimental conditions. In order to control the secondary thermal reactions in the photo-polymerization mechanism, a photoiniferter was used, p-xylylene bis(N,N-diethyl dithiocarbamate) (XDT), as a initiator in a living radical polymerization. Electro-optical studies of the PDLC film were made in order to evaluate the voltage needed to change the transmission characteristics of the film. These electro-optical characteristics are dependent on the polymer structure and thus the nature and the rate of polymerization mechanism, that means, size and number of the E7 microdroplets in the polymer matrix. Laboratório Associado para a Química Verde 2 4 Integration of Processes Process Integration of Supercritical Fluid Extraction and Membrane Separation The objective of this project is to explore the feasibility of combining two “green” separation technologies, based on different separation principles, for the separation of squalene from free fatty acids presented in the olive oil deodorized distillates (OODD): supercritical fluid extraction (SFE) and membrane technology. The first part of the project dealt with the determination of the vapor-liquid phase equilibria of the system squalene, oleic acid and carbon dioxide, to determine the best conditions for the subsequent step, the SFE/membrane extraction. A static mixer was used for this purpose. Different sets of pressure and temperature conditions were evaluated. Subsequently, a new apparatus for supercritical fluid extraction has been built where it incorporates the membrane module needed to the following project tasks. RESEARCH ACTIVITIES FOR 2006 Clean Solvents and Clean Processes Membrane Processing and Monitoring 1 - Membrane Design of New Selective Nafion Membranes Using Room Temperature Ionic Liquids This work aims the designing of new membranes with a high chemically, mechanically and thermal stability, exhibiting unique characteristics which are conferred by the incorporation of Room Temperature Ionic Liquids (RTILs) with distinct properties. These membranes may be used for different applications exhibiting a high selectivity, namely for gas and vapour permeation as well as for low resistance devices. Different characterization techniques will be used to evaluate if the cation is incorporated inside the membrane, namely Confocal Raman Spectroscopy, X-ray Photoelectron Spectroscopy and impedance measurements. 2 - Development of processes based on “smart” membranes This project will be focused on the development of membranes able to respond to environmental and/or external stimulus. This project will be directed to the conception of new membranes with the following (aimed) properties: self-cleaning, self-adjusted flux and rejection. This new materials will be applied for barrier packaging films, fractionation of biomolecules, catalytic degradation of pollutants. Bioremediation 1- Membrane Bioreactors Future work will be focused on integration of this biotransformation with the simultaneous transport of ionic mercury and arsenate through the pre-selected cation exchange membranes. Process optimisation will be performed with a real water and a different membrane configuration. 2-Conventional biological processes. The study on simultaneous phosphorus and nitrogen removal will proceed namely on the identification of the microbial species involved in the process. A new process concerning the biodegradation of xenobiotic compounds will be initiated Process Simulation and Modelling Modelling of biological processes PHA metabolic model. A metabolic model for PHA production from mixtures of volatile fatty acids by mixed microbial cultures will be developed. On-line monitoring of BHK-21 metabolic fluxes. Measurement techniques based on fluorescence spectra and automated sampling and analysis of glucose, glutamine, glutamate, lactate and ammonia (delivered by a bioprofiler device) will be integrated for on-line monitoring of BHK-21 cells’ metabolic fluxes. Laboratório Associado para a Química Verde 2 5 On-line optimising control of BHK-21 fed-batch cultures. Based on the previous information acquired on-line, the glutamine and glucose feeding strategies will be optimised on-line according to a model predictive control scheme. Genetic control in the baculovirus-isnsect cells expression system (BEVS). A mathematical model will be developed for gene expression in the BEVS taking into account the length of genes, strength of promoter and interaction with the host. Stochastic infection kinetics in the BEVS system. A model for the infection by three different recombinant baculovirus at low multiplicity of infection will be developed Culture screening based on hybrid modelling. A method for culture screening supported by hybrid models will be developed and applied to PHA producing bacteria. Membrane modelling. A predictive hybrid model for the flux of mixtures of solvents in nanofiltration membranes will be developed based on basic descriptors of solvents and membranes. Modelling of solute fractionation in vapour permeation / pervaporation will developed. Process Simulation and Modelling / Nanofiltration The planned activities will be focused on improving the application of the hybrid approach, and coupling the mechanistic with chemometric models for describing the solvent transport in nanofiltration in order to improve the predictive power and usefulness of this type of modelling. Furthermore, the CDF approach will be used to simulate and model the flow patterns. Spacers, of a different geometry and for a varying distance between two consecutive filaments, will be tested in a specially designed transparent cell with a rectangular channel under different flow conditions in order to identify the most efficient spacer and to validate the CDF simulations results. Polymeric Materials Drug controlled-release systems Polymers with functional groups will be produced using supercritical polymerization. Drug release formulations will be developed for target delivery of anti cancer drugs using polymers pH dependent as well as polymers with functional groups. Polymeric systems containing peptides for oral administration will produced. Design of biodegradable composite films for food packaging The objective of this work is to develop model composite films based on commercial pectin and carrageenan, and characterize them in terms of their hygroscopic and mechanical properties, and permeability to gases (oxygen and carbon dioxide) and water vapour. In order to design films with specific permeability properties, reactive compounds (ascorbic acid and/or calcium hydroxide to interact with oxygen and carbon dioxide, respectively) are added to the polymer matrix, while mica flakes are used as impermeable barriers. Ongoing work is focused on the characterization of the films in terms of permeability to gases (oxygen and carbon dioxide). The preliminary results obtained suggest that, these films are more permeable to water than to oxygen and carbon dioxide, and it is anticipated a decrease of the gas permeability with the inclusion of mica flakes. Moreover, the inclusion of ascorbic acid, which reacts with oxygen, leads to an improvement of the films selectivity (CO2/O2). Production of Biopolymers Research on the Production of PHA by mixed cultures from renewable sources will be continued. The analysis of the microbial population with a high PHA accumulation capacity will be followed by molecular probes. The process optimization based on on-line monitoring and control will be followed. Laboratório Associado para a Química Verde 2 6 Polymeric catalytic membranes The studies on limonene oxidation will be continued with the development of bifunctional catalysts able to not only epoxidise limonene to limonene oxide, but also to open the epoxide ring to the correspondent glycol and dehydrate this last to carveol. The optimisation of polymeric catalytic membranes and membrane reactors capable of minimise polymerisation and working under continuous feed of the oxidant, will be performed. The studies aiming the development of novel catalysts and polymeric catalytic membranes effective in the environmental friendly production of biodiesel, will be continued. Preparation of novel carbons from wastes and removal of organic compounds and heavy metals from wastewaters will be continued. The studies on the use of activated carbon as catalyst support for dioxins model compounds conversion will be continued. The studies on recovering of methylmethacrylate from waste polymethylmetacrylate will be continued, using as catalysts perovskites , pillared clays (argentine bentonites), exchanged with rare earth metals, and the same metals supported on mesoporous carbons. The characterisation of polyhydroxyalkanoates obtained by biosynthesis, in co-operation with other group of the same scientific area will go on. The characterisation of natural polysaccharides (concerning average molecular weights and polydispersity, by SEC) carrageenan extracted from seaweeds of the Portuguese coast, and pectins extracted from orange peels (waste of juice industries) will be performed, under cooperation projects involving three groups of CQFB, two of the same area, and a group from CEQUP/REQUIMTE. A novel method of pectin extraction, under pressure, will be studied and the operation conditions, pressure, temperature and time will be optimized in order to obtain the maximum pectin yield, keeping the desired pectin characteristics. Laboratório Associado para a Química Verde 2 7 BIOCHEMISTRY AND BIOPHYSICS OF PROTEINS and BIOINORGANIC CHEMISTRY AND PROTEIN ENGINEERING Head of Laboratories: Isabel Moura, Full Professor / José J. G. Moura, Full Professor Staff Members: Jorge Lampreia Assistant Professor Cristina Costa Assistant Professor Anjos Macedo Assistant Professor Jorge Caldeira Associate Professor Pedro Tavares Assistant Professor Alice Pereira Assistant Professor Carlos Salgueiro Assistant Professor Ricardo Franco Assistant Professor Stephane Besson Assistant Professor Gabriela Almeida Assistant Professor Sergey Bursakov Assistant Researcher Post-Doct Fellows: Rui Duarte, Patricia Sousa, Cristina Timóteo, Francoise Auchere, Ludwig Krippahl, Sofia Pauleta, Krisztina István Ph.D. Students: Patricia Raleiras, Cristina Cordas, Gabriela Rivas, Pablo Gonzales, Jorge Dias, Ana Martins, Filipe Folgosa, Carlos Martins, Joana Raimundo, Márcia Guilherme, António Nunes, Inês Isabel Fernandes Gomes. Project Grantee: Celina Santos , Américo Duarte, Andreia Barateiro, Miriam Sousa, Vanessa Cabral, Célia Silveira, Vanessa Cabral, Ricardo Alves, Sara Berguete, Ricardo Pais, Cristiano Mota, Duarte Alves , Ana Rita Fins , Andrea Mestre, Mª João Baleizão, Simmone dell´Acqua, Ana Teresa Lopes. Number of articles in scientific journals: 15 (86-100) Number of Ph.D Thesis: 1 DENITRIFICATION AND THE DINITROGEN BIOCYCLE Denitrification is a stepwise sequential pathway that transforms nitrate in dinitrogen, having nitrite, NO and N(2)O has intermediates. Further insights in nitrite and N(2)O reduction were obtained, as well as on ET proteins involved and Nitrate reductases (see section B.) Nitrous oxide Reductase Nitrous oxide reductase (N2OR) catalyzes the two-electron reduction of N2O to N2 and H2O in the last step of the bacterial denitrification process. It is a dimeric protein. The recently solved crystal structure of N2OR indicates that in each subunit there is a CuA center, which is the electron-transfer site, and a CuZ center, Laboratório Associado para a Química Verde 2 8 which is the catalytic site. The neighboring CuA and CuZ centers are from different subunits. The CuZ center has a strikingly new structural motif consisting of a m4-sulfide bridged tetranuclear cluster. The CuZ cluster is coordinated by seven His ligands with weakly bound water at the CuI/CuIV, which is the substrate access site. CuZ center in dithionite-reduced N2OR (the resting form) is a partially delocalized S=1/2, 1CuII/3CuI cluster. On the basis of the structural similarity with the CuA of COXs and the fact that mutant containing only this cluster showed no activity toward N2O, CuA is assumed to be an electron transfer center whereas CuZ is believed to be the active center of the enzyme. The activity of the Pseudomonas nautical N2OR was measured after periods of incubation of the enzyme in excess methyl viologen and dithionite solution. It was observed that the enzyme activity is dependent on the incubation time in the presence of reductant. It initially increases rapidly and then saturates slowly after 40 min, when highest activity is obtained. EPR measurements of the sample in the same reducing conditions were performed at periods of similar incubation times at which activities were measured to correlate with the activity assays. The characteristic EPR signal (gII=2.16) of the resting enzyme decreases with increasing incubation time. This cluster provides a mechanism for overcoming the reaction barrier of N2O reduction by a simultaneous two-electron reduction pathway to the substrate bound in a m-1,3-bridging mode. We demonstrate now that the redox active form of the CuZ cluster in enzymatic turnover is the all reduced 4CuI form by using a combination of activity determinations and EPR spectroscopy. This is the first demonstration that the S =1/ 2 form of CuZ can be further reduced. DFT calculations were performed to provide insight into the nature of N2O binding to and activation by this all-reduced 4CuI form relative to the 1CuII/3CuI resting form of the CuZ site. CuZ is a one-hole system (1CuII/3CuI) and the gradual decrease of the EPR signal on incubating with methyl viologen and dithionite indicates that the CuZ cluster is reduced to the 4CuI form. Nitric Oxide Reductase A Nitric Oxide Reductase (NOR) was purified from Pseudomonas nautica. It is a membrane bound enzyme, so the detergent dodecyl maltoside was used for solubilization. The purified enzyme consists of two subunits: NOR B which has a molecular weight of about 56 KDa and contains two b type hemes and one non-heme iron; and NOR C which has a molecular weight of about 17 KDa and contains one c type heme. In the native form of the enzyme, the main features of the EPR spectrum consist of a signal at gz=3.6 which belongs to a highly anisotropic heme c and a set of signals with a gz=2.99, which belong to a low spin heme b. Quantification of these signals gave a ratio of about 1:1. Since no other EPR signals are detected (apart from a small amount of a high-spin heme signal) it is thought that the non-heme iron is spin cupled to the other heme b originating a S=0. A typical signal of heme bound to NO with a spin quantification of only 0.2, appears after reduction and incubation with NO. Simulation of this signal revealed that it is a mixture of hexacoordinated and pentacoordinated heme. Copper containing Nitrite reductase The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO(2) (-). Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme. Pseudoazurin Pseudoazurin isolated from Paracoccus pantotrophus periplasm has a type I copper centre, coordinated by the N side chains of two His, S methione and S? cysteine residue, in a distorted tetrahedral geometry. This protein exhibits a spectrum with absorption bands around 450 nm, 590 nm and 760 nm in the oxidised form. Laboratório Associado para a Química Verde 2 9 The bands at 450 nm and 590 nm have a L-metal charge transfer character (Scys – Cu ion; 590 nm: CysS p ® Cu x2 – y2 and 450 nm: CysS s ® Cu x2 – y2). Pseudoazurin also presents a characteristic rhombic EPR spectrum with EPR lines split by a I=3/2 nucleus with a hyperfine constant of 70 gauss. We are carrying out studies in order to characterize two pH transitions. One was observed by visible spectroscopy, at low pH values; and the other by EPR at high pH values. Direct electrochemistry revealed two pKa values corresponding to these two transitions. In order to determine the mean g-values of the g-tensor of Cu site of pseudoazurin protein samples were crystallized as single and multiple crystal forms. EPR spectra of several crystals were recorded in the three mean axis. The protein was crystallized in the oxidized spectrum as a single crystal and the structure was determined at a resolution of 1.3 Å. The structure of pseudoazurin was determined in collaboration with the Prof. Maria João Romão group. STRUCTURE AND MECHANISMS IN MOLYBDENUM AND TUNGSTEN ENZYMES Membrane-bound nitrate reductase P. chlororaphis DSM 50135– initial step of Denitrification A nitrate reductase was solubilized with Triton X-100 from the membranes of Pseudomonas chlororaphis DSM 50135 grown microaerobically in the presence of nitrate. Like other membrane-bound nitrate reductases, it contains three subunits, of 129, 66 (64) and 24 kDa, referred to in the literature as alpha, beta and gamma, respectively. Electrocatalytic studies revealed that only the membrane-bound, not the solubilized form of the enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept electrons from methylviologen. The isolated enzyme possesses several iron-sulfur clusters and a molybdopterin guanine dinucleotide active center. The iron-sulfur clusters can be grouped in two classes according to their redox properties, the high-potential and low-potential clusters. In the as-isolated enzyme, two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy. The low-pH form shows a hyperfine splitting due to a proton, suggesting the presence of an -OHx ligand. Dithionite reduces the Mo(V) center to Mo(IV) and subsequent reoxidization with nitrate originates a new Mo(V) signal, identical to the oxidized low-pH form but lacking its characteristic hyperfine splitting. The isolated preparation also contains heme c (in a sub-stoichiometric amount) with the ability to relay electrons to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b usually found in this class of enzymes. EPR and kinetic studies on perilplasmic Nitrate reductase from D. desulfuricans ATCC 27774 Ammonification Periplasmic Nitrate Reductase from the sulphate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (Dd NapA) is an 80 KDa monomeric periplasmic enzyme having the bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) and a [4Fe-4S] cluster. EPR and enzymatic studies were performed to identify catalytic and non-catalytic relevant species. The sample in the as-prepared form presents a Mo(V) EPR signal that was assigned as non-catalytic relevant. The Dithionite reduced sample lacks Mo(V) ion EPR signal and presents a rhombic signal that is associated with the [4Fe-4S] cluster. The addition of nitrate to this sample yielded a Mo(V) rhombic signal with resonance lines split by a weakly interacting proton which is not solvent exchangeable. Addition of cyanide to the fully reduced sample yielded a new Mo(V) signal and decrease the g3 component of the Fe-S EPR signal. Kinetic studies show that Dd Nap has a complex enzymatic mechanism with more than one binding site for nitrate. Inhibition studies demonstrated that cyanide and azide inhibits nitrate depletion. Study of the spin-spin interactions between the metal centers of aldehyde oxidoreductase The aldehyde oxidoreductase from Desulfovibrio gigas belongs to the family of molybdenum hydroxylases. Besides a molybdenum cofactor which constitutes their active site, these enzymes contain two [2Fe-2S](2+,1+) clusters which are believed to transfer the electrons provided by the substrate to an acceptor which is either a FAD group or an electron-transferring protein. When the three metal centers of D. gigas AOR are simultaneously paramagnetic, splittings due to intercenter spin-spin interactions are visible when the EPR spectra Laboratório Associado para a Química Verde 3 0 are recorded at low temperatures. By studying quantitatively these interactions with a model based on the Xray crystal structure, which takes into consideration the interactions between the magnetic moments carried by all the metal sites of the system, it is possible to determine the location of the reducible sites of the [2Fe2S] clusters. When combined with the electron-transfer pathways proposed on the basis of the X-ray crystal structure, the results provide a detailed description of the electron-transfer system of D. gigas AOR. ANTAGONISTS Mo AND Cu Heterometallic clusters present on novel proteins Orange protein This protein contains a Mo centre containing other metals and had high sequence homology to proteins from iron-molybdenum cofactors biosynthesis family. Determination of the 3D structure of the apo-protein using NMR spectroscopy is being carried out. The coding sequence of the Orange protein (ORP) was obtain using Polymerase Chain Reaction (PCR) in which degenerated primers, based on the N-terminus and C-terminus, where applied. The DNA fragment of 350 base pairs (bp) obtained has revealed an open reading frame (ORF) that matched the known sequence of the ORP. In order to clarify the sequence of the protein, genome walker technique was applied, using a DNA library of Desulfovibrio gigas. The correct coding sequence was attained, revealing a length of 351 bp corresponding to a polypeptide chain of 117 amino acids. The ORP coding sequence was amplified with a new pair of non-degenerated primers. These primers contained in the 5’ end sequence for different restriction enzymes. The gene was inserted in the pET-21-d vector using NheI and HindIII, and the resultant vector was cloned in Escherichea coli DH5a ultra competent cells (Invitrogen). The resultant protein (rORP) differs from the native one in the N-terminus site, by the addition of 4 aminoacids. The new plasmid containing the ORP gene was transformed using Epicurian Coli BL21 (DE3) competent cells (Stratagene). The overexpression of the rORP was performed in different growth media and the induction of the target protein was made by the addiction of isopropyl â-D-thiogalactopyranoside (IPTG) when the bacterial culture was in the middle of the exponential phase. Sodium dodecyl sulphate-polycrylamide gel electrophoresis (SDS-PAGE) analysis of the total protein amount was performed to ensure the presence of the rORP. Optimal growth conditions were obtained (growth media and temperature, IPTG concentration) and large scale volumes (5 litres) of bacterial culture produced. The purification of rORP from the crude extract was accomplished in two chromatographic steps (1) gel filtration, using superdex 75 equilibrated with 300 mM Tris-HCl pH 7.6 and (2) an ion exchange diethylaminoethyl (DEAE) sepharose equilibrated with 10 mM Tris-HCl pH 7.6, using a linear gradient to a final concentration of 500 mM Tris-HCl pH 7.6. The purity of the protein after each step was followed by SDS-PAGE only as no colour was possible to observe in any of the fractions obtained. Toxicity of copper and molybdenum on a free living and in biofilm growing cells of D. gigas were studied to understand the chemistry and nature of copper, molybdenum and sulphur interactions. The toxic effect of copper was more emphasized in free than in biofilm cultures and biofilm cells were more competent to remove metals from solution. Blue Protein Blue Protein was purified from Desulfovibrio aminophilus. Cells were suspended and disruptured in a French press. After centrifugation and ultracentrifugation the supernatant was dialyzed. The soluble extract was subjected to a three step purification protocol that included: anion exchange chromatography (DEAE-52 cellulose), a Hydroxyapatite column and gel filtration chromatography (Superdex 200). The purity grade was determined by SDS-PAGE electrophoresis. Molecular mass determination. The molecular mass of the native protein was determined using gel filtration and a single symmetrical elution peak was detected. Molecular mass of the subunits was determined by mass spectrometryand SDS PAGE. Determination of the Free Laboratório Associado para a Química Verde 3 1 Sulfhydryl and Disulfide Bond Content. In order to determine free sulphydryl groups and disulphide bonds Desulfovibrio aminophilus, Blue protein was incubated with either iodoacetamide or with DTT and iodoacetamide and then the samples were then analysed by matrix-assisted laser-desorption ionisation time-of-flight mass spectroscopy. Aminoacid residues sequencing. Peptides were obtained by proteolytic digestion and separated by reverse phase chromatography. Aminoacid sequencing was performed on an automatic amino acid sequenator. The peptide sequences were aligned against Blue protein sequence from other sulphate reducer bacteria in order to find out conserved regions. DNA sequence determination. In a first attempt multiplex PCR was used in order to amplify a DNA fragment corresponding to Blue protein. Degenerated primers were designed based on reverse translation of aminoacid sequences from peptides generated by proteolytic digestion. PCR gaves a single which was isolated, cloned and sequenced. In a next step, inverse PCR protocol was used to amplify flanking sequences using genomic DNA as template. Specific primers pointing away from the known sequences were designed. PCR products were isolated and sequenced. Currently 169 amino acids of the protein sequence have been determined. CYTOCHROME c PEROXIDASE – CCP A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electrontransferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme. P. pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was reevaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase. Laboratório Associado para a Química Verde 3 2 Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase from Pseudomonas stutzeri Crystals of cytochrome c peroxidase from Pseudomonas stutzeri were obtained using sodium citrate and PEG 8000 as precipitants. A complete data set was collected to a resolution of 1.6 A under cryogenic conditions using synchrotron radiation at the ESRF. The crystals belong to space group P2(1), with unit-cell parameters a = 69.29, b = 143.31, c = 76.83 A, beta = 100.78 degrees. Four CCP molecules were found in the asymmetric unit, corresponding to a pair of dimers related by local dyads. The crystal packing in the structure shows that the functional dimers can dimerize, as suggested by previous biochemical studies. Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from P. stutzeri The production of cytochrome c peroxidase (CCP) from Pseudomonas ( Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus ( Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca(2+). The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca(2+)does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca(2+)binding site of low affinity.Two crystal forms of cytochrome c peroxidase from Pseudomonas nautica were obtained and reported last year. Study of the cytochrome c peroxidase (CCP) electron transfer complexes, specially the one between CCP and the cupredoxin – pseudoazurin.« Determination of the parameters of binding using different techniques; and structural characterization of the electron transfer complexes between these two proteins. Kinetic characterization of cytochrome c peroxidase using pseudoazurin as the electron donor. Identification of the important residues for the formation of the complex and/or for the electron transfer rate. In this study we have mutated some residues that are proposed to be important to drive the complex formation (residues belonging to the ring of lysines) or involved in the electron transfer pathway (residues belonging to the hydrophobic patch of cupredoxins). In the first stage, the mutant pseudoazurins are being biochemical characterized and then different parameters will be determined, such as the binding affinity, electron transfer rates and a structural characterization of the complexes will also be attempted. Structural and mechanistic studies of the effect of the calcium ion in the activation of the enzyme, using resonance Raman spectroscopy and also UV-visible spectroscopy. The electrochemical characterization of this enzyme is being done in collaboration with Prof. M.M. Correia dos Santos. Structural basis for the mechanism of Ca2+ activation of the di-heme CCP from P.nautica 617 Cloning and overexpression of P.nautica CCP is under way. Laboratório Associado para a Química Verde 3 3 STRUCTURAL STABILITY OF ADENYLATE KINASE Adenylate kinase was cloned and expressed in E. coli. Expression in E. coli was studied to understand the mechanisms of metal incorporation into adenylate kinase in vivo conditions. Bacteria were grown on the controlled media and essentiality and toxicity of metals were determined. Optimal conditions for the bacterial growth and recombinant protein production with different metals present were obtained. Cobalt, manganese and nickel used are the metals the most close by its characteristics to the zinc. Impacts of metals concentration on cell growth and on expressed enzyme activity were studied. Kinetic parameters and maximum rates of cell growth were determined. A cobalt-porphyrin(CoPf) containing protein was isolated from D. gigas and characterized. Molecular mass of the protein and porphyrin were established by application of MALDI-TOFF and ESI-MS spectrometry. The presence of one dissulfide bridge in the CoPf was demonstrated by molecule modification and following MS mass determination. Amino acid and nucleotide sequences of CoPf were obtained and used to identify this protein as a g-subunit of sulfite reductase of D. gigas. NANOSURFACES AND NANOPARTICLES The study of the interaction of biological molecules, namely proteins and nucleic acids, with noble metal surfaces including silver electrodes, nanostructured surfaces, and silver and gold nanoparticles. Our strategy is two-fold: (1) direct interaction of proteins with the metal surfaces and (2) coating the nanoparticles and nanostructured surfaces with self-assembled monolayers (SAM) of alkanethiols. This coating leads to stabilization and prevents uncontrolled growth and aggregation of the nanostructures. Colloidal gold is also being used to develop a new class of nano-biosensors that is able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. The aggregation of the nanoprobes is accompanied by a change of color from red to blue, providing the means for detection. Understanding the physical and chemical conditions that control the specific hybridization of gold nanoprobes to DNA and/or RNA targets will enable the design and preparation of probes to be used in the clinical environment. Transmission Electron Microscopy (TEM) is used to characterize the morphology and dimensions of the nanoprobes. Atomic Force Microscopy (AFM) imaging allows us to measure high resolution topographic images for characterization of the steps involved in the formation of the gold nanoprobe-DNA aggregates. New nanostructured surfaces functionalized with new ligands are being used to study heme-containing membrane proteins with high selectivity and sensitivity using Surface Enhanced Resonance Raman Spectroscopy (SERRS). New surfaces for SERRS are being generated including silver electrodes, nanostructured surfaces, and silver and gold nanoparticles with alkanethiols as capping agents. New modified alkanethiols for the formation of adequate Self-Assembled Monolayers (SAMs) for the immobilization of membrane proteins are being synthesized and used to cover the former surfaces. Two examples of the work in progress are the selective binding of an histidine-tagged protein to a SAM made of a Ni(II)-NTA-terminated ligand, and the utilization of a thiol-derivatised neoglycoconjugate of maltose to form a SAM at a electroactive silver surface. The former ligand is being synthesized by two different methods using the S-protected-mercaptocarboxylic acids: a) “Hell-Volhard-Zelinsky” type synthesis with N,N-bis(carboxymethyl)amine; b) coupling with Ná,Nábis(carboxymethyl)-L-lysine. The latter neoglycoconjugate mimics n-dodecyl-b-D-maltoside, a detergent widely used for membrane protein solubilization, and was utilized for adsorption and spectroelectrochemical studies of a tetraheme-containing membrane protein solubilized in that detergent. DEVELOPMENT OF ELECTROCHEMICAL BIOSENSORS In recent years, the recurrent anthropologic uses of nitrites have raised several public concerns. As a consequence, there is a growing demand to quantify these oxyanions in food and environmental samples. The development of analytical probes based on stable enzymes with high selectivity and specific activity to nitrite, such as the multihemic nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774, seems to be a good alternative to conventional protocols. However, previous attempts to design a ccNiR-based electro- Laboratório Associado para a Química Verde 3 4 chemical sensor were not fully successful from the stability and/or sensitivity viewpoints, due to an inefficient enzyme-electrode link. In the pursuit of better enzyme immobilization matrices, we constructed and characterized different bioelectrode configurations, using the following strategies: - entrappment of ccNiR in a ionomeric polymer (Nafion) loaded with methyl viologen as redox mediator; - attachment of biotinylated ccNiR molecules via avidin bridges to polypyrrole films bearing biotin groups; to establish the electronic communication between the enzyme redox centers and the solid electrode, the polypyrrole polymers were also functionalized with viologen groups. The kinetic behaviour of ccNiR was also investigated by homogeneous enzyme assays and direct electrochemistry. SIMPLE METAL SITES Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-ray The structure of Treponema pallidum (Tp) SOR was solved in collaboration with the X-Ray group at REQUIMTE (resp. Profª M.J.Romão). The structure determination of D.gidas SOR and Tp Rubredoxin are underway using NMR methodologies. VANADIUM(V)-COMPLEXES AND CALCIUM PUMP Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media. OXYGEN ACTIVATION Di-iron sites It is our research objectives the study of metal cofactors in biology, in particular the ones involved in molecular oxygen activation and detoxification mechanisms using fast kinetics coupled to spectroscopies such as Mössbauer spectroscopy. Is an integral part of these objectives the development of software tools that enable the necessary spectral data analysis. Currently, our interests are centered in two main projects. Laboratório Associado para a Química Verde 3 5 Although reactions of molecular oxygen with organic compounds are energetically favorable, they are kinetically stable (making aerobic life possible). To control dioxygen reactivity nature developed two different ironcontaining cofactors: heme and diiron clusters. While heme-containing enzymes are well-studied systems, only recently diiron cluster-containing proteins started to be studied in detail. Thus, understanding diiron cluster reactivity is understanding how nature overcomes the kinetic stability and controls oxygen reactivity, which in turn is of fundamental importance. It is our understanding that this type of study can only be successful if based in different techniques, such as spectroscopic, kinetic and electrochemical tools. Also, Mössbauer data analysis is becoming an emerging problem since the software used is incompatible with recent operating systems. This software is often developed by the user (becoming highly specific) or limited to basic functionalities, not open to third party plug-ins and usually expensive. We are currently developing a Mössbauer data analysis program that makes possible the analysis of spectra of biological samples (including kinetic data analysis). Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins Our research interests are centered in metalloenzymes involved in iron metabolism and cellular detoxification. Ferritin serves the dual functions of iron detoxification and storage; it catalyzes the oxidation of the toxic Fe2+ ions in the cells to the less toxic Fe3+ ions (ferroxidation) and stores the oxidized Fe3+ ions within its protein shell in a mineral form similar to ferrihydrite (mineralization). The rapid freeze-rapid quench technique in conjunction with Mössbauer and EPR spectroscopies has been used to study the mechanism of ferritin ferroxidation and mineralization. It is our purpose to further understand the kinetic and spectroscopic observations on the iron uptake mechanism of ferritins. Our short term objectives are to understand how ferritin binds Fe2+ ions in solution, catalyzes their oxidation, and directs the oxidized Fe3+ ions into the inner protein cavity to form the ferrihydrite mineral core. Since the structure of the putative ferroxidase site is highly homologous to those of the binuclear iron centers found in the R2 subunit of E. coli RNR and in MMOH, this study will also provide more general information concerning the chemistry of carboxylate-bridged diiron centers in proteins. MULTIHEME ENZYMES The Geobacteraceae, a family of dissimilatory Fe(III)- has been found to dominate the microbial communities present in a diversity of subsurface environments in which Fe(III) reduction is the terminal electron-accepting process. In the environment, Fe(III) is found mainly in the form of insoluble iron oxide particles and coatings, and many species of the family Geobacteraceae have evolved the ability to reduce a variety of electron acceptors that are either insoluble or too large to be transported into the cytoplasm. These include Fe(III) and Mn(IV) oxides. The members of the family Geobacteraceae are also capable of reducing many soluble metals, including the radionuclide U(VI). From the analysis of completed genome of G. sulfurreducens over 100 putative c-type cytochrome genes have been identified. Many of these cytochrome genes are more highly expressed during growth on Fe (III) than with fumarate as the electron acceptor and deletion of some of these cytochrome genes greatly reduces the capacity for Fe (III) reduction. The main goal of our research is to characterize thermodynamically Geobacter sulfurreducens multiheme cytochromes involved in such pathways using visible spectroscopy and NMR techniques to understand the functional mechanism of these electron transfer proteins. The present target proteins are: - Cytochorme c7 (PpcA) – trihaem cytochrome with 71 residues and with heme bis-His axial coordination which is involved in Fe(III) and UV(VI) reduction pathways. This protein shares high structural homology with a cytochrome c7 from D. acetoxidans and cytochromes c3 from Desulfovibrionacea family. - PpcA homologues in a total of four that were revealed from the analysis of the G. sulfurreducens genome. Laboratório Associado para a Química Verde 3 6 - Polymers of cytochrome c7-type domains. The polymer under study consists of four c7-type domains with a total of 12. Interestingly, the individual domains of this polymer contain a heme which is coordinated by a methionine and a histidine residue (having the other two hemes bis-His axial coordination), and thus representative of a new type of cytochromes. PROTEIN-PROTEIN INTERACTIONS We developed a new algorithm for (BIGGER/CHEMERA) for macromolecular interactions. Synechocystis ferredoxin/ferredoxin-NADP(+)-reductase/NADP+ complex: Structural model obtained by NMR-restrained docking Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes. Laboratório Associado para a Química Verde 3 7 PROTEIN CRYSTALLOGRAPHY Head of Laboratory: Maria João Romão, Associate Professor Staff Members: Ana Luisa Carvalho Assistant Researcher Cecília Bonifácio Laboratory Technician Pedro Assunção Computer System Administrator Post-Doct Fellows: José Trincão, Shabir Najmudin, Roeland Boer Ph.D. Students: Teresa Santos Silva Research Students: Catarina Coelho, Filipe Freire, Aldino Viegas, Lídia Barata Number of articles in scientific journals: 5 (89, 101-104) We have focused our work on structural and functional studies of different systems grouped into (A) Metalloproteins; (B) Proteins involved in cellulose (polysaccharyde) recognition and degradation; (C) Proteins from the glyoxalase pathway. An important focus of our research have been metalloenzymes, in particular those dependent on the molybdopterin cofactor. In addition, we have pursued, together with the Faculty of Veterinary Medicine of Lisbon, structural and functional studies on several new components of the Cellulosome assembly, a complex machinery responsible for plant cell wall degradation. More recently we started, in collaboration with the Faculty of Sciences of Lisbon, a new project on enzymes from the pathogenic organism Leishmania infantum. METALLOPROTEINS Isoquinoline 1-oxidoreductase from Brevundimonas diminuta Isoquinoline 1-oxidoreductase from B. diminuta catalyzes the first step of isoquinoline degradation to isoquinoline-1-one. It consists of two subunits, one of 80 kDa, containing the Mo active center, and another of 16 kDa, containing two [2Fe-2S] clusters. Its primary sequence and behavior, specifically in its substrate specificity and lipophilicity, differ from other members in the family. A crystal structure of the enzyme is expected to provide us with an explanation for these differences. We have optimized a previously described purification procedure and crystallized the enzyme. A structure Mo site [2Fe-2S] solution was obtained and model building and refinement to 2.5 Å resolution are under way. [2Fe-2S] Periplasmic nitrate reductases of Ralstonia eutropha and Desulfovibrio desulfuricans Aiming at studying the amino acid residues important for its enzymatic specificity, we used nitrate reductase from R. eutropha (R.e. NAP) as a model system for mutational studies of the active site. As a first step we have been trying to develop an efficient Laboratório Associado para a Química Verde 3 8 expression system in order to produce adequate amounts of pure protein. Aiming at clarifying the reaction mechanism for this class of enzymes we have performed several crystallographic studies on reduced, inhibited and substrate-bound forms of the enzyme from D.desulfuricans. With this purpose, most of the experiments were carried out in an anaerobic chamber. Crystals were obtained in anaerobic conditions using protein reduced with either methyl viologen or dithionite. This protein was also co-crystallized in the presence of substrate, substrate analogues and inhibitors. Comparing the crystal structures of oxidized and reduced Cytochrome c Peroxidase from P. stutzeri We have solved the P. stutzeri Cytochrome c Peroxidase crystal structure in the oxidized as well as in the reduced form. The latter crystals were grown in an anaerobic chamber, under an argon atmosphere. Crystals were cryo-cooled in liquid nitrogen inside the glove box. A complete dataset was collected in-house and was used to solve the structure. The structural differences between the two redox states of the enzyme contribute to clarify mechanistic aspects for CCPs. The non-heme Superoxide Reductase from Treponema pallidum Superoxide reductase is responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined to 1.55 Å resolution. Unlike class I SOR, in TpSOR the desulforedoxin-type structural Fe centre is absent but the N-terminal domain is maintained and exhibits a desulforedoxin-like fold. The C-terminal domain harbors the Fe(His)4(Cys) active site. The participation of a glutamate as the sixth ligand of the iron centre in P. furiosus SOR was not observed in TpSOR. As suggested for PfSOR, X-ray photoreduction could be affecting the oxidized form of the TpSOR centre, preventing hexacoordination of the iron atom. The CELLULOSOME Structural basis for the specificity of Carbohydrate Binding Modules (CBM 30 and CBM 44) The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential. Enzyme systems that attack the plant cell wall contain non-catalytic carbohydrate-binding modules (CBMs), that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. The C-terminal domain of the modular Clostridium thermocellum enzyme ctCel9D-Cel44A comprises a novel CBM44 which binds to cellulose and xyloglucan with equal affinity. The crystal structures of CBM44 and another CBM (CBM30) show how celluloligosaccharides can be accomodated. CBM30 Mutagenesis studies confirm that the cleft comprises the ligand binding site and that a hydrophobic platform plays a pivotal role in ligand recognition. CBM 44 Laboratório Associado para a Química Verde 3 9 Insights into the Structural Determinants of Cohesin—Dockerin Specificity Cellulosomes assemble via the Type I dockerins on the catalytic subunits binding to the reiterated Type I cohesins in the molecular scaffold, while Type II dockerin-cohesin interactions anchor the complex onto the bacterial cell surface. Type I and Type II cohesin-dockerin pairs show no cross-specificity. The structure of CohII is very similar to Type I cohesins, and the dockerin binding site is likely to be conserved in the two proteins. Subtle differences in the topology of the binding sites and a lack of sequence identity in the betastrands that comprise the core of the dockerin binding site explain why Type I and Type II cohesins display such distinct specificities for their target dockerins. PROTEINS FROM THE GLYOXALASE PATHWAY Crystallographic studies on the glyoxalase II from Leishmania infantum One of the most remarkable features of trypanosomatids is the functional replacement of glutathione by N1N8bisglutathionilspermidine (tripanothione). Cloned glyoxalase II from Leishmania infantum was overexpressed in E. coli, purified and crystallized. The structure was solved using the human glyoxalase II structure as a model. These results, together with future detailed characterization using other substrate analogues will contribute to the understanding of why trypanosomatids evolved to make use of trypanothione instead of glutathione. Planned research activities for 2006 Metalloproteins Multi-heme nitrite reductase from D. desulfuricans: Crystallization of the membrane hetero-oligomeric complex of NIR; Crystal structures of inhibitor and substrate-analogue bound complexes as well as structures of different redox states of the enzyme. Nitrate reductase from Ralstonia eutropha – Optimization of a suitable expression system. Mutagenesis studies and purification of wild-type and mutants. Crystallographic studies of the purified proteins. Co/Zn containing adenylate kinase – Crystallization of Co-containing enzyme and structure solution. Superoxide reductase from T. pallidum – Mutagenesis studies and crystal structures of the mutants. Plant cell wall degrading enzymes Novel studies on the structure and mechanisms that determine the molecular recognition of carbohydrates towards different CBM modules. It will involve mutagenesis and structural studies. The latter will include a combined approach of crystallographic, NMR and computational chemistry methods (in collaboration with other researchers from Requimte). Proteins from the Glyoxalase Pathway Structural and mutagenesis studies of L. infantum glyoxalase II in order to clarify the reaction mechanism; Crystallographic studies on other enzymes from the glyoxalase pathway Laboratório Associado para a Química Verde 4 0 BIOLOGICAL TRANSPORT Head of Laboratory: Teresa Maria Fonseca de Moura, Associate Professor Staff Members: Hugo Gil Ferreira Invited Full Professor Karin Tonnies Gil Ferreira Associate Professor Isabel Borges Coutinho de Medeiro Assistant Professor Maria da Graça Soveral Rodrigues Assistant Professor Ana Isabel Dias Bicho dos Santos Assistant Researcher Project Grantee: João Filipe da Custódia Dias, Rui Manuel Lucas Gameiro Domingues Tostões Membrane Transport 1. Detection and role of aquaporins in different Strains of S. cerevisiae (in collaboration with Prof. Conceição Dias from Instituto superior de Agronomia) The functional expression of aquaporins was investigated S. cerevisiae strains differing in the expression level of AQY1 and AQY2: double mutant, parental and overexpressing AQY1 or AQY2. The functionality of these proteins was accessed in vivo by measuring the activation energy of water fluxes in protoplasts. The behavior of the different strains when submitted to osmotic challenges revealed to be different and dependent on the type of aquaporin expressed, giving values of the osmotic permeability coefficient and activation energy that reflect the preferred pathway for water transport in each strain. 2. Inhibition of the mitochondrial pyruvate carrier by valproate and its metabolites (in collaboration with Prof. Margarida Silva from Faculdade de Farmácia de Lisboa) Pyruvate driven oxygen consumption and ATP synthesis is severely inhibited by the antiepileptic Valproic acid (VPA). To test whether this effect results from an inhibition of the mitochondrial pyruvate carrier by VPA or by its â-oxidation metabolites 3-keto-VPA and 4-Delta-VPA we used inverted submitochondrial vesicles (ISMV) prepared from rat liver cells. We found a marked inhibition on pyruvate transport with the metabolite 4Delta-VPA at physiological concentrations (0.5 mM and 1 mM). The inhibition mechanism and the kinetic parameters are under study. 3. Functional characterization of a P-ATPase from Nicotiana tabacum: Patch Clamp studies (in collaboration with the Plant Development group of Prof. José Feijó from Institute Gulbenkian for Science) The proton P-ATPase from Nicotiana tabacum is responsible for proton fluxes observed during pollen tube growth (4µm/sec, one of the fastest polarized cellular growth in nature). The electrophysiological patch-clamp technique can be applied to access the proton currents through this ATPase and to access the mechanisms by which pump activity is regulated. cDNAs for the wilt-type of the protein and a construct with GFP (green fluorescence protein) on the C-terminal were subcloned into pcDNA with the CMV promoter for expression in mammalian cells. The conctruct cDNA has been used to transiently transfect mammalian HEK293 cells for patch-clamp characterization. Expression of the protein in transfected cells has been accessed by means of a laser light (with 473 nm wavelength). So far, the detected fluorescence levels (24-48hrs after transfection) are low. A control of the transfection protocol was made with GFP alone with almost 60% of the cells showing fluorescence after 48hrs. Preliminary results obtained in the whole-cell configuration show an increase of the cell current upon application of 40µM fusicoccin, a fungal toxin known to enhance proton pumping by this pump. Laboratório Associado para a Química Verde 4 1 4. Electrophysiological studies on membrane transporters (in collaboration with Prof. Constanta Ganea of the Biophysics Department, “Carol Davila” University of Medicine and Pharmaceutics, Romania; our lab. received the visit of Anca Popescu, Ph.D. student, Out-Dez 2005, for this joint project) This line or research consists on studying the effects of various physico-chemical factors of general interest, as heavy metals (Ni, Cd, etc.), food supplements, pollutants and/or drugs (quercetin, hypericin, galantamine, etc) on the electrical properties of different membrane transporters and ion channels, by means of the sensitive patch-clamp (CQFB/FCT/UNL) and BLM, black lipid membranes (“Carol Davila U.M.P.). The results will provide better understanding of molecular mechanisms underlying membrane transport and of the influence of external factors on these mechanisms. To start this project a culture of TE671LH cells which express the nicotinic acetylcholine receptor was established and maintained. A fast perfusion system that allows accurate temporal control of application of solutions and drugs to the cells (on the millisecond range) was built. The whole-cell configuration of the patchclamp technique was used to examine the effect of quercetin (an antioxidant of plant origin used as food supplement) on the acetylcholine induced transient currents by the neuronal receptor. Patch-clamp results show a possible decrease of the currents after exposure to quercetine and alteration of the receptor kinetics. 5. Patch clamp characterization of ionic currents of HEK293 cells HEK293 cells are commonly used for transfection of heterologous membrane proteins (channels and transporters). It is therefore important to know which endogenous membrane transporters these cells have. Several types of potassium and anionic channels were characterized. However, an integrative study of HEK293 cells is lacking to understand the magnitude of these endogenous currents under experimental conditions, and the relative contribution of sodium, chloride and potassium to the total current of a cell. Ion substitution patch-clamp experiments have shown that the total ionic current by a cell does not account for the summation of the individual currents of these three ions. Mathematical models of biological systems Mathematical models for cells and epithelial systems are being developed. Numerical simulations are implemented in the Berkeley Madonna package (http://www.kagi.com/authors/madonna). 1. Tubular organ - thick ascending limb of the Henle´s loop - TAL The flat sheet cTAL model, developed for the behaviour of the thick ascending limb of the Henle´s loop of mammalian kidney, was extended to a tubular geometry in order to simulate the behavior of cTAL in in vitro and in vivo conditions as referred in the last report. The model is now able to fit experimental reported results and predict behaviors where data is not yet available. In particular the model: i) simulates reported output values for the ion concentrations and predicts their steady state profiles along the lumen; ii) simulates the increase of the trans-epithelial electrical potential difference, in contrast with the potential difference across the basolateral barrier that remains unchanged, when perfusing at high rates; iii) predicts the same value for the static head independently of the NaCl load at the entrance of the tubule. 2. Calcium homeostasis - A minimal model (col. with Dr. J.F. Raposo and Prof. L. Sobrinho from Instituto Português de Oncologia Lisboa, and Dr. A. Pires from the Hospital Amadora-Sintra) In mammals the calcium concentration in the extracellular compartment (ECC) is finely regulated (Calcium Homeostasis) and controls a large number of physiological processes: blood coagulation, the excitability of nerve and muscle cells, the epithelial secretion, the secretion of parathormone (PTH), the functional state of many ionic channels, etc. As a result of the development of reliable and easy techniques of measurement of the main variables of this control system (Calcium, PTH and Calcitriol) it is now possible to characterize with some detail many of the system’s disturbances which are frequently seen by the endocrinologists. Although there is now published information on the behaviour of a number of the components of the calcium control Laboratório Associado para a Química Verde 4 2 system we lack tools that may help us to analyze the behaviour of the system as a whole. This is an attempt to develop such a tool. A first version of the model covering acute situations was published (J. Clin. End. Met., 2002, 87, 4930-40). Most of 2005 was dedicated to data gathering from several hospitals (Instituto Portugues de Oncologia, Hospital de Santa Cruz, Hospital de Setúbal) and to its statistical analysis. Studies on Food Components 1. Analysis of the mineral and trace elements composition of human milk (in collaboration with Prof. Matilde Castro and Dr. Lino Mendes from Faculdade de Farmácia de Lisboa) As stated in the report of 2004, the mineral and trace elements composition of human milk was characterized. The screening of potentially toxic mineral elements in breastfeeding mothers was performed and correlated with socio-demografic data and dietary patterns through a food frequency questionnaire. The great variability of the values determined in the different samples of maternal milk (n= 140) has to be emphasized. It was possible to detect the presence of major minerals and trace minerals in the human milk, especially Mn, Si, B, Ba, Sr among others. Hg was detected in 15 samples, showing a correlation between fish consumption and its presence. All these studies are being performed using the ICP (Induced Coupled Plasma) technique. 2. Behaviour of natural pigments in solution and in organized media NMR 1D and 2D data was obtained for aqueous solutions of anthocyanin isolated from the peel of Passiflora edulis (Sims) fruit. Preliminary results showed it to be a monoglucoside derivative of a non-substituted aglycone. UV-Vis spectroscopy absorption and emission data were obtained for several fluorescein derivatives of the protein bovine serum albumine (BSA), namely Erythrosine (food color E 127), Eosin and Rose Bengal in water and in aqueous solutions. For all systems, phosphorescence was readily observed at room temperature in well deareated solutions both with and without protein. Complementary studies using the triplet-emitter bromonaphtalene in deareated water-BSA systems also showed phosphorescence at room temperature Laboratório Associado para a Química Verde 4 3 DEVELOPMENT OF TRANSDUCERS Head of Laboratory: José Luís Costa Lima, Full Professor Staff Members: Alberto Nova Araújo Associate Professor Rui Alexandre Santos Lapa Associate Professor Mª Conceição B. S. M. Montenegro Full Professor Maria Beatriz V. N. Q. G. Junqueiro Associate Professor Agostinho Almiro Almeida Assistant Professor João Luís Machado Santos Assistant Professor Maria Lúcia M. F. Sousa Saraiva Assistant Professor João Pedro Almeida Lopes Assistant Reseracher Rita Isabel Lemos Catarino Assistant Professor Paula Cristina Gerónimo Assistant Professor Ivone Valente Oliveira Assistant Professor Cristina Maria C. Morais Couto Assistant Professor Adriana Martins Pimenta Assistant Professor Maria Isabel Almeida Cardoso Technical Staff João Rodrigo da Silva Santos Laboratory Technician Cristina Maria Delerue-Matos Professor Coordenador Maria do Carmo V. F.Vaz Professor Coordenador Abel José Assunção Duarte Assistant Ph.D. Students: Maria Luísa Soares Silva, Sofia Alexandra Lopes da Piedade Gomes, Emília Maria Gonçalves Santos, Célia Maria Pinto Gomes Amorim M.Sc. Students: Branca Sofia Teixeira, Ricardo Nuno Mendes Jorge Pascoa Number of articles in scientific journals: 7 (105-111) Number of Ph.D thesis: 1 Number of M.Sc thesis: 1 New trends were investigated based on exploitation of different transducing schemes such us optical and electrochemical. Studies on immobilization techniques were implemented for the monitoring low levels of drugs, food and pesticides. Additionally studies were developed concerning the construction of tubular and wall-jet electrodes dedicated to the implementation of manifolds with FIA multi-side detection or multicommutated flow systems. In more detail it can be referred the following results: Laboratório Associado para a Química Verde 4 4 - A new nitrate-selective electrode was been constructed based on the analytical potentialities of the oxaazamacrocycles as potentiometric ionophores with improved selectivity characteristics when compared with commercial available unities. The working characteristics of this new electrode constructed without inner reference solution, made possible its application to the determination of nitrate in different types of vegetables and bottled mineral waters without the use of a support electrolyte for elimination of possible interferences. - An inexpensive and easy to construct miniaturized biosensor for the determination of uric acid in biological fluids was been developed. The amperometric biosensor was prepared by using a carbon paste electrode prepared with uricase from Arthrobacter globiforms and tetracyanoquinodimethane as electron transfer mediator. - A tubular amperometric detector coupled to a multicommutated flow system was been constructed and applied in the determination of uric acid in urine. The exploitation of the analytical potentialities of the multicommutated flow manifold allowed the implementation of the on-line sample dilution based on the zone sampling approach. The dilution capability exhibited by the developed methodology allowed a direct insertion of the samples in the flow system without any pre-treatment. - Tin (IV) porphyrins were used as ionophores for phthalate selective electrodes preparation. The influence on the electrode response of the ionophore structure and the membrane composition, namely the amount of the incorporated ionic sites was been studied. The analytical usefulness of the electrodes was been assessed by potentiometric determination of phthalate in water and industrial products. - The reagent generation by ultrasonic irradiation to produce chloride and hypochloride for analytical purposes derived from the residues of organochlorines of laboratory effluents was been studied. The dependence between the production of chloride obtained from the sonication of the effluents containing organochlorines and the irradiation time was evaluated for CCl4, CHCl3, CH2Cl2. It was observed that under saturation conditions the production of chloride was highest for CHCl3. The extent of sonochemical generation of hypochlorite was also evaluated in the presence of several NaOH concentrations. - An optical biosensor based on immobilised carbonic anhydrase and its application to the determination of the anti-glaucoma agent acetazolamide by enzyme inhibition measurement, was been evaluated. The enzyme and a pH indicator dye, cresol red, were physically immobilised in overlapped sol-gel thin films, in a dual-layer format. The sensor was integrated in a flow cell and coupled to a continuous flow system operating on a multicommutation and binary sampling approach. - Two types of ion selective electrodes (with and without inner reference solution) have been developed using selective membranes based on iron oxyhydroxide embedded on silica gel mixed with ultra pure graphite at a 2/29 (w/w) ratio. The working characteristics of the electrodes and the effect of interfering ions was investigated. The accuracy and the precision of the determinations with the constructed ESI’s have been tested on arsenicfree drinking water spiked with known amounts of arsenic and groundwater samples containing high levels of arsenic. Fiber-optic Fluorescence optodes Sensors regarding an optical transduction are also known as optodes and constitute an increasing area in green chemistry. They are suitable for chemical recognition and quantification, allowing selective readings at low levels and with low sample volumes. Within this context, fiber-optic fluorescent optodes are currently being developed for the determination of ionic species in aqueous solutions. Optimization of the integration of the different components among optodes is carried out. Chemometric techniques to process sensor signals under steady state or lifetime fluorescence are also under development. Laboratório Associado para a Química Verde 4 5 AUTOMATION AND INSTRUMENTATION Head of Laboratory: José Luís Costa Lima, Full Professor Staff Members: Alberto Nova Araújo Associate Professor Rui Alexandre Santos Lapa Associate Professor Mª Conceição B. S. M. Montenegro Full Professor Maria Beatriz V. N. Q. G. Junqueiro Associate Professor Agostinho Almiro Almeida Assistant Professor João Luís Machado Santos Assistant Professor Maria Lúcia M. F. Sousa Saraiva Assistant Professor Marcela Alves Segundo Assistant Researcher Adriano Fachini Assistant Researcher IIvone Valente Oliveira Assistant Professor Cristina Maria C. Morais Couto Assistant Professor Adriana Martins Pimenta Assistant Professor João Alexandre Velho Prior Assistant João Rodrigo da Silva Santos Laboratory Technician Ph.D. Students: Paula Cristina into, Cristina Vieira, Pedro Maia, Marta Filipa Ribeiro, Karine Marques, Luís Miguel Magalhães, Eunice Rodrigues, Hugo Oliveira, Marieta Passos, André Araújo M.Sc. Students: Daniel Ribeiro, Ana Isabel de Castro, Cristina Silvestre Number of articles in scientific journals: 22 (112-133) Number of Ph.D thesis: 1 Number of M.Sc. Thesis: 1 Continuous flow systems and dedicated equipment were developed and applied in automatic laboratorial determinations or in on-line control of industrial processes. Special attention was been dedicated to procedures based on the multicommutated flow methodologies and sequential injection analysis. Additionally new flow analysis concepts and methodologies was been present and evaluated, namely multi-pumping flow analysis and single reaction interface flow analysis. In this theme the following activities can be stressed: - The development of a new separation method based on a novel reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations. The chromatographic resolution between peak compounds was lower than 5.0 and analysis time was less than 9 minutes under the optimal conditions. The method was found to be applicable for rotine analysis of the active compounds in several pharmaceutical preparations. Laboratório Associado para a Química Verde 4 6 - The development of a sequential injection analysis system based on the sensitized effect of cationic surfactants on the reaction between metal ions and chelating dyes. The quality of the results obtained with the developed systems for 18 water samples were evaluated by comparation with conventional procedures, with no statistically significant differences for a 95% confidence level. - A critical review on flow-injection systems with multi-side detection was been published. The paper stress the improvements in system simplicity, ruggedness and performance of the multi-side detection and emphasize situations where relocating the detector results in significant advantages over traditional FIA systems. - Two review on sequential injection analysis (SIA) was also published. One of the papers stressed that SIA with electrochemical detection is an important tool for the automation of chemical procedures and presents a overview of the principles of operation and applications and the other referred the application of this methodology in food analysis. - The new concept of single interface in flow analysis was been present and discussed. The dual or multiple interface concept, commonly associated to the distinct flow techniques, was replaced by a single interface concept, which do not rely on the utilisation of a well-defined and compelling sample volume but only on mutual penetration of sample and reagent zones at a single reaction interface where both sample and reagent met together prior to detection. The detector is positioned at the core of the manifold (not in the conventional terminal position) and repetitive flow reversal enable interface manipulations, including multi-detection of the entire reaction interface or the monitoring of the evolution of a pre-selected interface zone by using reversal cycle times. - A fast and sensitive flow-based procedure for chemiluminometric determination of carvedilol was been proposed. The developed methodology takes advantage of the antioxidant capacity of carvedilol to inhibit the chemiluminescence response resulting from the oxidation of luminol by hypochlorite, by acting as a hypochlorite scavenger. - A sensitive sequential injection analysis methodology for the fluorimetric determination of naproxen is proposed. The developed automatic analytical procedure is based on the complexation of naproxen with cyclodextrin yielding an enhanced fluorimetric signal and applied to the determination of naproxen in pharmaceutical preparations. The results obtained with the flow system were compared with those furnished by the reference procedure and the relative deviations were lower than 3.6%. - A flow injection multisite detection system was developed for correction of the sample blank in a colorimetric determination. By using detector relocation, a single spectrophotometer is used for the sequential reading of the sample blank and the coloured product; the sample crosses the flow cell to measure intrinsic absorption, the colour reagent is subsequently added, and the the flow cell is relocated to provide the reading of the resulting plug. - A fully automated analytical methodology for chemilluminometric determination of propranolol hydrochloride in pharmaceutical preparations was been proposed. The developed procedure was based on the oxidation of propranolol by potassium permanganate in acidic medium and was implemented in a multicommutated flow system. - Restricted acess material (RAM) column containing 25µm C18 alkyl-diol support was integrated into the sequential injection analysis (SIA) manifold and the SIA-RAM system was tested for direct determination of furosemide in serum. The integration of RAM material into SIA enabled creation of a comprehensive on-line sample clean-up technique combined with fluorescence determination of the analyte. - A multi-pumping flow system for the chemiluminometric determination of the hypoglycaemic drug metformin was implemented. The developed methodology was based on the metfotmin-induced inhibition (metformin acts as Cu(II) scavenger) of the catalytic effect of Cu(II) ions on the chemiluminescent reaction between luminal and hydrogen peroxide. - A computer-controlled multi-syringe flow injection system was proposed to perform the spectrophotometric determination of avaible iron and boron in soil extracts. The methodologies were based on the formation of ferroin complex (determination of iron) and azomethine-H reaction (determination of boron). Both determinations Laboratório Associado para a Química Verde 4 7 were performed in manifolds with similar configurations by changing the reagents present in the different syringes. - A multi-syringe flow injection system for the determination of total phenolics based on the 4-amino-antipyrine reaction was been proposed. In line preconcentration using solid-phase extraction was implemented, offering an environmentally friend alternative to organic solvent extraction performed in batch procedure and in previous described flow methodologies. - A new flow methodology exploiting the multi-pumping approach was developed for the spectrophotometric determination of ambroxol hydrochloride in pharmaceutical preparations. The flow manifold was implemented by using exclusively multiple solenoid actuated micro-pumps which acted simultaneously as sample insertion, solutions propelling and reagents commutation units. - The development of a pulsed flow sequential injection analysis (SIA) system based on the utilisation of pulse generating solenoid micro-pumps, wich replace the conventional solutions propeplling units commonly used in SIA systems. The influence on sample/reagent zones interpenetration and reaction zone formation of the reproducible pulsed flow produced by micro-pumps operation, which is characterised by a chaotic solutions movement, was comparatively evaluated with the typical laminar flow conditions of conventional SIA systems. - Four configurations for sample introduction (volume or time based) were tested in order to establish if the different strategies affect the analytical signal in multi-syringe (MSFIA) systems. The influence of the best sampling approaches were also evaluated in MSFIA systems for the spectrophotometric determination of phenolic compounds and the potentiometric determination of chloride. - A potentiometric flow system has been developed for the direct determination of arsenic (V) in water samples after on-line preconcentration in a minicolumn packed with a new adsorbent material selective to As(V). The flow system has been successfully applied to the determination of inorganic arsenic species in groundwater samples collected in areas affected by arsenic contamination. - An enzymatic flow analysis approach, based on the Griess-Ilosvay reaction, was developed for the evaluation of nitrite and nitrate aiming an environmental benign alternative reduction method using nitrate reductase and the development of on-line dilution procedure for the analysis of water samples, with a wide range of nitrate concentrations. - An automatic system that performs two analytical procedures allowing the evaluation of the alternative antioxidant capacity of wine samples, was evaluated. Automation was carried out using sequential analysis system that allowed the development of the two methodologies that were evaluated using trolox as standard and subsequently using other antioxidant substances which are abundant in wine and whose antioxidant activities were compared to that shown by trolox. - An automatic methodology was been proposed based on multi-syringe flow injection analysis for the determination of avaible phosphorous in soil extracts. This fully computerized flow technique allowed the development of a flow network where sample and reagents were intercalated and sent further towards detection system. - A novel sampling approach in flow analysis was proposed and applied to the spectrophotometric determination of glucose and fructose in invert sugar syrups. The samples are collected in gelatine capsules and brought to the laboratory. The capsules are placed directly into a dissolution chamber of a flow-batch system, therefore, the manual sample dissolution step is not required. Laboratório Associado para a Química Verde 4 8 QUALITY CONTROL AND AUTHENTICITY OF FOOD PRODUCTS Head of laboratory: Rosa Seabra, Full Professor / Beatriz Oliveira, Assistant Professor Staff Members: Isabel Maria P. L. V. O. Ferreira Assistant Professor Paula Andrade Assistant Professor José Fernandes Assistant Professor Patrícia Valentão Assistant Professor Susana Casal Assistant Professor Rui Alves Professor Coordenador Olívia Pinho Associate Professor Isabel Mafra Assistant Researcher Luísa Maria S. Vieira Peixe Assistant Professor Helena Maria Neto Ferreira Assistant Professor Miguel F. de Albuquerque Cabral Assistant Professor Cristina Réu Technician Cristina Maria Delerue-Matos Professor Coordenador Maria do Carmo V. F. Vaz Professor Coordenador Maria Goreti Ferreira Sales Assistant Maria de Fátima S. Barroso Assistant Post-Doct Fellows: Sónia Dopico Ph.D. Students: Sara Cunha, Miguel Faria, Joana Amaral, Bárbara Ribeiro, Sandra Maria Basílio Quinteira, Carla Alexandra Novais Oliveira e Silva, Patrícia Sofia Carneiro Antunes, Elisabete Maria Pereira Machado, Sandra Quinteira, Ana Raquel Freitas M.Sc. Students: Ana Filipa Gomes, Áurea Roxo, Carla Barbosa, Lúcia Maia, Sílvia Marisa da Cruz Barros, Sónia Mendes, Alexandra Soares, Cristina Soares, Aida Reis, Filipa Grosso. Number of articles in scientific journals: 27 (134-160) Number of articles in books: 2 Number of Ph.D. Thesis: 2 Number of M.Sc. Thesis: 4 The leading research lines of our group are quality control, authenticity and safety of foodstuffs, with emphasis on macronutrients (fatty acids, proteins, triglycerides), micronutrients (vitamin E, phytosterols, carotenoids, organic acids) residues and contaminants (biogenic amines, pesticides, acrylamide, heterocyclic amines, Laboratório Associado para a Química Verde 4 9 polycyclic aromatic hydrocarbons) of conventional and traditional food products, with “Protected Designation of Origin”, PDO. In this context, the development of analytical procedures is a constant challenge and the goal of our research team. The principal activities under development in 2005 were: Molecular biology techniques for the evaluation of food safety and food authenticity The research includes the use of DNA based techniques (PCR Polymerase Chain Reaction) applied to food products. The identification of species of milk producers in dairy products, namely cheeses PDO (Protected Designation of Origin), has been carried out for the detection of fraudulent procedures. Particularly, the “Queijo de Cabra Transmontano” is a product with PDO, whose manufacture should include only milk of one caprine breed, the “Serrana Transmontana”. A duplex PCR technique for the simultaneous detection of bovine and caprine milks in cheeses has been developed. By means of a linear normalized curve between the log of the ratio of the bovine band intensity and the sum of bovine and caprine intensities vs. the log of cow’s milk percentage, it was possible to quantify adulterations with bovine milk in the range of 1-50%. The method was successfully applied to “blind” and commercial caprine cheeses. This work has resulted in an M.Sc, thesis to be presented next year. Vitis vinifera clonal identification is of great importance as the choice of the best clones can contribute for wine quality and authenticity. By means of an optimized detection method of clones identification, it was possible to obtain 14% discrimination within Touriga Nacional (TN) clones, identifying two different groups. The polyclonal origin of TN clones was previously verified with eight microsatellite markers. This research line resulted in a PhD thesis presented in December 2005. Next year we intend to develop Molecular PCR-based techniques for Olive Oil authenticity evaluation. In the international scene there are particular signs that olive oil has been adulterated with chemically similar oils, like hazelnut oil (Corylus avellana L.), and in some cases with other crops. To develop highly specific PCR methods one needs to obtain (1) highly pure DNA extracts using alternate extraction techniques and (2) PCR primers throughout the analysis of DNA sequences publicly available like the ones of NCBI (GenBank) and EMBL. This last goal can be achieved only by knowing how to analyse and retrieve data from these huge databases using tools frequently used by Bioinformatics. This work programme will be a basis of the Postdoctoral Grant concerning the safety and authenticity of foodstuffs by biological molecular procedures. The detection of genetically modified (GM) soybean RR (Roundup Ready) with herbicide tolerance (glifosato) has been carried out by PCR techniques. The method under development included the design of specific primers for the lectin gene and the 3’ NOS/plant junction specific of RR soybean. Those primers were successfully used to detect the RR soybean in foodstuffs by conventional PCR and to quantify it by real-time PCR with SYBR Green as fluorescence dye. The planned research for the future includes the comparison of several DNA extraction methods from foodstuffs, the detection of Bt maize in raw materials and foodstuffs and the development of more specific real-time PCR techniques with hydrolysis probes (such as TaqMan). Chemical Markers of Food Authenticity Characterization of conventional and traditional foodstuffs is a custom, being examples of the developed work: Physico-chemical characterization of PDO cheeses, that includes lipolytic and proteolytic products characterization, and correlation between volatile components (determined by SPME/ GC/MS) and sensory characteristics. Proteolysis and authenticity criteria based in lactoglobulins HPLC analyses for identification of homologous proteins in milk mixtures from different species were also evaluated. This work resulted in a M. Sc. Thesis. (Isabel Ferreira, Beatriz Veiros, Olívia Pinho) Some coffee constituents were determined in both green and roasted coffee and their behaviour with roasting evaluated and discussed. Actually was under evaluation the presence of heterocyclic amines correlated with beneficial effects in neurological system. The evaluation of Harman and norharman, two beta-carbolines that might be involved in the protection for Parkinson disease, was accomplished with 17 samples analysed in the Laboratório Associado para a Química Verde 5 0 raw state and after three different roasting degrees. It was verified that the amounts are proportional to the roasting intensity and that robusta coffee amounts are always higher. These conclusions are important for the consumption of “expresso” coffee, where high degrees of roast and blends with robusta are a must for a good final quality. This research has resulted in an M.Sc, thesis to be presented next year as well as a Grant from IBeSa. Several quality parameters are determined in varietal olive oils in order to characterize some cultivars of Olea europaea (fatty acids, sterols, tocopherols, triglycerides) and were applied to PDO olive oils “Azeite de Trásos-Montes”. This research line concerns to the PhD Grant about safety and authenticity of olives and olive oils in preparation. An HPLC methodology to characterize the carotenoid profile (lutein and ??carotene) of olive oils was implemented. This methodology was developed, validated and applied to several PDO Portuguese olive oils from different regions (Trás-Os-Montes and Alentejo). This subject will be described in a M.Sc. thesis in preparation. The chemical composition (including fatty acid, sterols, tocopherols and tocotrienols and triacylglycerol profiles) of different cultivars of hazelnuts (Corylus avellana L.) and walnuts (Juglans regia L.), grown in different geographical areas was evaluated as well as the importance of these parameters in the discrimination of the cultivars in study by chemometric methods. The experimental work is concluded and a PhD thesis is finished and presented to evaluation. Sheep milks of two autochthonous Portuguese breeds were evaluated for their conjugated linoleic acid (CLA) levels. The results suggest that this milk can be considered an interesting source of CLA. In prosecution of this line a similar study with Spanish breeds was performed in collaboration with a Spanish Faculty aiming the detection of interesting differences for genetic improvement of the breeds. The conjugated linoleic acid levels (CLA), total fatty acid profile and total fat were determined in the adipocite depots of suckling lambs from 4 different breeds in a total of 122 animals. The same parameters were also evaluated on the fat from their ingested milk. The variability of the results seems to be correlated with the “ganaderia”, lamb age, breed and the mother’s milk production per day. This research line continues with more milk samples from another breeding as well as with traditional Portuguese sausages aiming protected geographical indication (PGI) as a form to obtain added value from the regional products of underprivileged regions. Measurement of flavour characters in food by sensory and instrumental techniques is other goal in development. Optimization of SPME/GC/MS methods for separation and identification of volatile compounds in foodstuffs and beverages like, cheese, honey, beer and coffee for extraction of a wide variety of compounds belonging to very heterogeneous groups, such as, esters, alcohols, acids, aldehydes, ketones, hydrocarbons, eters, sulphur compounds, alicyclic compounds, aromatic compounds, heterocyclic compounds, etc. will be performed. Train of sensory panels to establish Quantitative Descriptive Analysis of those products is also part of this research line. Within the scope of a research project regarding the chemical characterization and the evaluation of the antioxidant potential of tronchuda cabbage (Brassica oleracea L. var. costata DC). seventeen phenolic compounds from the internal leaves of this cabbage were characterized and quantified by reversed-phase HPLC-DAD-ESI-MSn and HPLC/DAD, respectively: quercetin 3-O-sophoroside-7-O-glucoside, 3-pcoumaroylquinic acid, kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-(caffeoyl)-sophoroside-7O-glucoside, sinapoyl glucoside acid, kaempferol 3-O-(sinapoyl)-sophoroside-7-O-glucoside, kaempferol 3O-(feruloyl)-sophoroside-7-O-glucoside, kaempferol 3-O-(p-coumaroyl)-sophoroside-7-O-glucoside, 4-pcoumaroylquinic acid, sinapic acid, kaempferol 3-O-sophoroside, 3 isomeric forms of 1,2-disinapoylgentiobiose, 1-sinapoyl-2-feruloylgentiobiose, 1,2,2-trisinapoylgentiobiose and 1,2’-disinapoyl-2-feruloylgentiobiose. A phytochemical study was also undertaken on in vivo material cultivated under conventional and organic practices and collected at different times. Six organic acids (aconitic, citric, ascorbic, malic, shikimic and fumaric acids) were identified and quantified by HPLC-UV. Qualitative and quantitative differences were noticed between internal and external leaves. Analysis of the internal leaves phenolics was achieved by HPLC-DAD, and the phenolic profile obtained revealed to be distinct from that of the external leaves previously determined. Laboratório Associado para a Química Verde 5 1 By this means it were identified and quantified eleven compounds: 3-p-coumaroylquinic acid, kaempferol 3-Osophoroside-7-O-glucoside, kaempferol 3-O-(caffeoyl)-sophoroside-7-O-glucoside, kaempferol 3-O-(sinapoyl)sophoroside-7-O-glucoside, kaempferol 3-O-(feruloyl)-sophoroside-7-O-glucoside, kaempferol 3-O-sophoroside, two isomeric forms of 1,2-disinapoylgentiobiose, 1-sinapoyl-2-feruloylgentiobiose, 1,2,2’-trisinapoylgentiobiose and 1,2’-disinapoyl-2-feruloylgentiobiose. In general, internal leaves exhibited more constant chemical profiles. Tronchuda cabbage seeds were also studied for their chemical composition. Thirteen phenolic compounds were characterized and quantified by reversed-phase HPLC-DAD-MS/MS-ESI and HPLC-DAD, respectively: two sinapoylgentiobiose isomers, three sinapoylglucose isomers, kaempferol-3-(sinapoyl)sophorotrioside-7glucoside, sinapoylcholine, kaempferol-3,7-diglucoside-4’-(sinapoyl)glucoside, three disinapoylgentiobiose isomers, 1,2,2’-trisinapoylgentiobiose and 1,2-disinapoylglucose. 1,2-Disinapoylgentiobiose was the compound present in highest amounts, followed by kaempferol-3-(sinapoyl)sophorotrioside-7-glucoside and 1sinapoylglucose, being 1,2-disinapoylgentiobiose isomer the minor compound. Seven organic acids (aconitic, citric, ascorbic, malic, quinic, shikimic and fumaric acids) were also identified and quantified by HPLC-UV. Ascorbic acid was the main compound, followed by citric acid. Studies regarding the influence of the white cabbage butterfly Pieris brassicae on tronchuda cabbage chemical composition are in course. Within the scope of a Ph.D. project the phenolic compounds and the organic acids composition of Edible mushroom species, the edible beefsteak fungus Fistulina hepatica was determined by HPLC/DAD and HPLC/ UV, respectively. The results showed a profile composed by five phenolic compounds (caffeic, p-coumaric and ellagic acids, hyperoside and quercetin) and six organic acids (oxalic, aconitic, citric, malic, ascorbic and fumaric acids). The quantification of the identified compounds revealed that ellagic acid and malic acid are the main compounds in this species. In a general way the phenolic profile revealed to be more constant than the organic acids one and could be more useful for the quality control of the species. Studies with other edible mushroom species are in course. Within the scope of a post-Doctoral project the analytical methodology for the chemical characterization of white “Vinho Verde” grapes, by their most important phenolic compounds and organic acids, was achieved by the sequential application of the experimental design that allows a large number of factors to be screened simultaneously, to determine which of them has a significant effect on the analytical signal instead of a traditional strategy of optimizing each variable separately. Plackett-Burman design was used as a screening method in order to select the variables that have influence on the system and subsequently a statistical model of central composite design was used to calculate the optimum value for each variable. The developed method combines solid-liquid extraction of both phenolic compounds and organic acids from grapes and a subsequent clean-up step using solid-phase extraction (SPE). Phenolic compounds and organic acids were determined by HPLC/DAD and HPLC/UV, respectively. The identified and quantified phenolic compounds were: quercetin-3-o-glucose, kaempferol-3-o-rutinoside, isohamnetin 3 glucose, quercetin, kaempferol and epicatequin. The determined organic acids were oxalic, citric, tartaric, malic, shykimic and fumaric acids. The methodology will be applied to the determination of these compounds in white “Vinho Verde” grapes from several varieties. In another work the anthocyanins, organic acids and voltatile phenols composition of red wine obtained from Touriga Nacional grapes growing in Dão region (Portugal) was determined by HPLC/DAD, HPLC/UV and GC/ FID, respectively. By these means nine anthocianic compounds (malvidin-3,5-O-diglucoside, cyanidin-3-Ogalactoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin, cyanidin, pelargonidin and malvidin), six organic acids (ketoglutaric, tartaric, malic, quinic, lactic and shikimic acids) and two volatile phenols (4-ethylguaiacol and 4-ethylphenol) were identified and quantified. Malvidin-3-Oglucoside, the pair lactic plus shikimic acids and 4-ethylguaiacol were the main anthocyanin, organic acids and volatile phenol, respectively. The effect of nine different Dekkera bruxellensis strains on these chemical parameters was also evaluated. The results obtained indicate that some strains of D. bruxellensis yeast are able to deteriorate red wine from Dão region during its maturation by the production of volatile phenols, namely 4-ethylphenol. Laboratório Associado para a Química Verde 5 2 Ballota nigra (Lamiaceae), known as black horehound, is a perennial herb which is commonly distributed in most areas of the world that have mild climate. It is a small herb, which flowered aerial parts are gathered, dried and used for medicinal purposes. A phytochemical study was undertaken in this species, and seven phenolic compounds (chlorogenic, caffeic and caffeoylmalic acids, ballotetroside, forsythoside B, verbascoside and allysonoside) and eight organic acids (oxalic, aconitic, citric, ascorbic, malic, quinic, shikimic and fumaric acids) were identified and quantified by HPLC/DAD and HPLC/UV, respectively. Forsythoside B and quinic acid revealed to be the main compounds in its infusion. The phytochemical characterization of Linaria vulgaris (Scrophulariaceae), known as yellow toadflax, is in course. Food Safety In the field of food contaminants, the project (approved and financed by FCT) aiming the assessment of the dietary exposure of Portuguese consumers to acrylamide proceeds. With this goal an isotope labelled GCMS analytical methodology was developed and allowed the accurate quantification of the compound in distinct kind of food matrixes. The referred method was applied to the determination of the levels of the compound in some starchy food products that include chips, potato crisps, breakfast cereals, biscuits and crisp bread. Furthermore, we started to study the role of some raw material properties in acrylamide formation during food processing, namely in cheap frying. A special attention has been devoted to study the presence of acrylamide in coffee and coffee derivatives, probably one of the most important sources of acrylamide for Portuguese consumers. A specific SPE/GC-MS analytical methodology was optimised and used for screening the presence of the compound in this kind of products. Furthermore, studies to explain the formation of the compound during the processing of coffee rawmaterials are in execution. An MSc thesis will be presented next year concerning this subject. Also pesticides residues are a real concern in food chain and, used without knowledge, can contribute to the expansion of serious environmental and public health effects. Either in their original chemical structure or in metabolic or degradation products, it is therefore common to find trace amounts in agricultural and agroalimentary products. Pesticides remain the first line of defence in pest control when crop injuries and losses become economic. They are the only answer to a severe pest outbreak or emergency and are typically dosed close to or post harvest. Even though some times unexpected, another problem arising at pesticide control is the presence of metabolites or conversion products of the analyte. Depending of its pathway at the environment, different chemical structures can be found within the several matrices. Additionally, the concentration levels expected here are, in most cases, much lower than those of the original pesticide but much more toxic. This is the case of Fenthion (I) an organosphophorus insecticide, widely used in the control of olive fruit fly (Bactrocera oleae Gml.) pest. It is rapidly converted to fenthion sulfoxide (II) and subsequently by oxidation gives origin to fenthion sulfone (III). Another oxidative process due to the plant enzymatic action can involve the other sulphur atom in the fenthion moiety, thus leading to the formation of another three metabolites (fenoxon (IV), fenoxon sulfoxide (V) and fenoxon sulfone (VI). All the metabolites show higher toxicity than the parent compound: the letal dose (LD50) is 220 mg/kg for I, 125 mg/kg for II-IV, 50 mg/kg for V and 30mg/kg for VI. Due to the possibility of the presence of these compounds olives and olive oils a matrix solid-phase dispersion (MSPD) methodology has been developed to extract the fenthion and its 5 metabolites in olives being the method compared with a conventional liquid-liquid extraction. The extracted compounds were analyzed by gas chromatography using a nitrogen phosphorus detector for quantification and mass spectrometry detection for identification. The developed methodology MSPD provide alternative method to liquid–liquid extraction and offers additional benefits due to its high sensitivity, simplicity and small size of sample required. Two papers in this field are in preparation. In the same context and similar approach Fhosmete were also determined in olives after the development of specific techniques for it extraction as well as its metabolites. The results obtained are in evaluation and a paper will be submitted to publication. Laboratório Associado para a Química Verde 5 3 Dimethoate are another pesticide in study but it determination and metabolites will be performed in the Microbial Biophysics and Residue Chemistry Research Unit - Eastern Regional Research Center (USA) with supervision of Prof. Steven J. Lehotay. Olive trees are attacked by a variety of insects and other pests, which cause a reduction in the quality and quantity of the olives and oil produced. Many of these are controlled by organophosphorus pesticides, endosulfan and several synthetic pyrethroid pesticides, which are registered for use in olive groves in some UE members (Greece). The compounds are lipophilic, with high n-octanol-water partition coefficients ranging from 4.7 to 7.0 (log values), which suggests that residues will concentrate in the oil during extraction from olive oils. Portugal is a olive oil producer and it will be important to control the presence or not of these products or their metabolites in order to answer to health concerns and simultaneously to prevent unethical operating and marketing practices and implement the value of regional or traditional references. To answer these questions a GC/NPD methodology was implemented for the multiresidual determination and quantification of organophosphorus pesticides being applied to olives and virgin olive oils from biological and conventional agriculture. Samples harvested in different regions of Portugal and submitted to phytossanitary treatments with the referred product are in evaluation. The presence of biogenic amines in foodstuffs constitutes an important food safety problem, due to its implication in phenomena of food intolerance and intoxication. Traditional foods have been an important part of the Portuguese diet and it was observed that a high intake of harmful amounts of biogenic amines from traditional Portuguese fermented foods is possible, since the few data available for Portuguese traditional cured sausages and cheeses indicate the presence of high contents of putrescine, cadaverine, tyramine and in some cases histamine. Methodologies previously validated for characterization of biogenic amines of Protected Denomination of Origin (DOP) Portuguese meat products and cheeses were applied to extend the current limited database on this topic, and contribute to evaluation of exposure of the Portuguese consumer to biogenic amines and in some cases propose modifications in traditional manufacture to reduce biogenic amines. Oils and fats are rich sources of essential fatty acids and other nutrients but, when used over prolonged periods, their biological value decreases and several hazard substances are formed by oxidation, hydrolysis, and polymerization. The quality of the frying oil is a critical factor to preserve high quality and produce safe fried products. The used oil rejection should be easily determined. The polar components (TPC) comprise a multitude of different artefacts and their content is a suited indicator of fat quality, being the basis of legislation in many countries for the control of used frying fats. Nevertheless, the official methodology is time consuming, and unsuitable for on-line assessment. Several quick tests measuring colour and dielectric constant have been developed and are commercially available. An MSc. student developed this subject including the study: 1. test the efficiency of several quick tests while comparing with official methodologies; and 2. evaluate several frying operations in a restoration unit. Different foods and frying times were evaluated until the usual rejection point in that unit, based in visual inspection. The quick tests evaluated were simple to use, relatively inexpensive, “green”, and mostly able to stand up to the rigors of frying and handling by workers with reduced analytical knowledge. The samples collected in the restoration unit clearly demonstrate that the oils are precociously rejected comparatively with the legislation limits, being therefore on no health concern. With the diffusion of these quick tests the oils will probably be used for longer periods, still within legislation, with advantages for both industry and environment. The higher concerns are indeed the small restaurants were the oils are intermittently used, for uncontrollable periods, and were no quality control is implemented. A MSc. thesis is practically concluded. In this context another MSc. student will develop an HPLC methodology to determine polymerized triacylglycerol contents in the previously referred oils correlating these contents with TPC and safety of the frying products. Also in this subject some studies to evaluate the performance of several oils and fats to be used in industrial frying (catering and industry) are being performed by request of an oil producer. The catering frying procedures are simulated in laboratory and the quality of the frying oils evaluated along the frying time. Trans fatty acids are naturally present in animal fats (ruminants) and formed in vegetable oils and fats by hydrogenation. Some health concerns (e.g. increase blood cholesterol levels) about its presence in foodstuffs Laboratório Associado para a Química Verde 5 4 conducted to the government’s decisions to limit its contents of certain fatty foodstuffs, the agreement between manufacturers in Europe to this effect and USA’s ruling that this content must be declared. As a result of the decisions mentioned above, the edible oils and fats industry is required to lower and control the trans content of its products while maintaining a fatty acid composition acceptable in terms of health nutrition. To know the scenery in Portuguese products, namely cookies / biscuits, a project accepted and supported by UP rectorate are in execution with the collaboration of GRAC. The objective is the implementation of environmental friendly procedures to fat extraction (microwave). The project includes the evaluation of 100 brands of cookies marketed and available to the consumers. Polycyclic aromatic hydrocarbons (PAHs) are a diversified family of more than 100 lipophilic organic contaminants composed by two or more fused aromatic rings. These compounds are mostly formed by incomplete combustion of organic matter as a consequence of a series of natural and anthropogenic processes. Due to their multiple potential sources of contamination, PAHs are ubiquitously distributed in nature. Therefore, human exposition is virtually unavoidable, which raises an important public health concern due to their recognized carcinogenic activity. Exposition to PAHs was epidemiologicly associated with an increased risk of skin and lung cancer. Sixteen PAHs are actually classified as priority pollutants by Environmental Protection Agency (EPA) on the basis of their occurrence and carcinogenicity being six with 4-6 rings classified as heavy PHAs the potential toxic in food chain. Diet is the major non-occupational source of PAHs for non-smokers. Due to their lipophilic nature, PAHs may contaminate fats and oils, which represent, along with cereal products, the principal food sources but fish and smoked foodstuffs are also subject of concern. In order to determine the dietary exposure of Portuguese consumers to PHAs a work program was drew up and will be developed along the next year. Beneficial compounds of foodstuffs It is important to emphasize that the major chemical markers of authenticity referred above are also beneficial compounds of foodstuffs but will not referred in this item. Separation and characterization of bioactive peptides from whey hydrolysis to be applied in food and pharmaceutical industries: Whey is a by-product in the manufacture of cheese. Although whey protein content is relatively low (0.6%), finding alternative uses for whey solutions is a concern for the dairy industry. Hydrolysis of whey proteins by enzymes immobilized on an inert support can either change or evidence functional properties of the produced peptides, thereby increasing their applications as food ingredients. A process for separation of bioactive peptides based on affinity chromatography and using continuous systems will be developed for ALPMHIR, the most potent antihypertensive peptide obtained from trypsin hydrolysis of blactoglobulin. Similar process will be developed for other bioactive peptides with antimicrobiane action. This process will enable recovery of bioactive peptides with minimal destruction thus enabling utilization by returning these active peptides to functional cheese. Other peptides that can be used as ingradientes in infant formula industry will also be recovered.Bioactive peptide profile of cheeses rich in probiotic microorganisms with low fat content and appreciated sensory characteristics will be compared with functional cheeses enriched with selected peptides from whey. In order to obtain data on the nutritional value of some dishes largely consumed in Portugal, traditional Portuguese dishes and largely consumed fast food meals were evaluated, indicating that our feeding style is already far from the original and health claimed “Mediterranean diet”. The nutritional composition of the traditional Portuguese sausage “alheira” was determined and evaluated the modifications that occur with different cooking processes. In this field were also evaluated the nutritional compositions of confectionery cakes. (Susana Casal, Eulália Mendes, Rita Carneiro) “Expresso” coffee is being studied for its chemical and sensorial attributes in order to provide a safer product by reducing the levels of unhealthy substances and increasing those with known health benefits. Different blends and degrees of roast will be studied. This subject will be developed by a PhD student that obtained a Grant from FCT. Laboratório Associado para a Química Verde 5 5 Garlic (Allium sativum) has the reported benefits of lowering cholesterol levels, reduction hypertension, and acting as an antimicrobial agent in living systems. The active ingredients in garlic are thought to be the sulphur containing compounds, or thiosulfinates, present in the volatile oil. Allin, which converts to allicin (Sallyl-2-propenthiosulfinate) in the presence of the enzyme allinase, is present at about 1% in garlic cloves. About 60 accessions belonging to the Portuguese garlic germplasm collection held at Banco Português de Germoplasma Vegetal, Braga, are currently analyzed for the allicin content using an RP-HPLC-UV method. Preliminary results indicate a high degree of variation in the allicin content which can be very valuable for garlic breeding and improvement of Portuguese varieties. This line research will be included in a MSc. thesis in execution in collaboration with CIBIO-CEQUP. To evaluate the influence of the diet in the composition of serum, plasma and red cells lipids, their fatty acid profiles will be evaluated in some restricted populations, including weight loss or pathologies like diabetes. Beer composition and beer foam stability Comparison between composition of two types of beer: with alcohol and without alcohol. Study of beer foam stability, by characterization of protein profiles using reversed phase and size exclusion –HPLC. Contribution of iso-alpha acids, organic acids, sugars, amino acids and others to foam stability. Development of an HPLC method using a “light scattering” detector was developed for separation and quantification of sugars in food matrices (beer, honey, dairy products). Chemometric techniques Given the great number of analyses and techniques applied to the same foodstuffs, chemometric techniques were used in order to evaluate the relationships between different types of parameters, to determine the best parameters to characterize a given product, and to classify new observations in relation to predefined models. Univariate, bivariate and multivariate statistics were applied, mainly principal components, canonical variate and discriminant analysis, canonical correlation, procrusts and generalized procrusts analysis, as well as 3way Tucker analysis. Complete algorithms were written in order to developed three main lines of work: (i) producing detailed analysis for pedagogic purposes, (ii) enhancing the use of statistics in the department through the application of predictive biplots for data description and analysis, and through interpolative biplots for routine laboratory classification purposes, (iii) generalizing and evaluating the advantages and disadvantages of biplots in current industrial and investigational practices. This work resulted in a PhD thesis that will be presented next year. Simultaneously a book in this subject is in preparation by orders from the editor. Microbiology The rapid emergence and spread of several antibiotic resistant bacteria in both clinical and community settings during the last years have been facilitated by the use of antimicrobial agents in hospitals and in human husbandry. In an attempt to control the problem of antibiotic resistance the European Union banned the use of antibiotics that are analogues to those used in human medicine for purposes of growth enhancement in food-producing animals. Portugal is one of the EU countries with the highest antibiotic use in veterinary medicine and with the highest rates of vancomycin resistance in enterococci. Thus, studies on diversity of both resistant bacterial strains and their genetic elements involved in antimicrobial resistance are essential for the control of antimicrobial resistance dissemination. Following, the achievements obtained in this field were: High occurrence of antibiotic resistant enterococci suggests maintenance of selective pressure by the use of antibiotics/other substances in the Portuguese poultry production. Persistence of a number of widespread PFGE types containing different resistance genes might reflect environmental/host adapted enterococcal strains that might contribute to the maintenance of antibiotic resistance, thus constituting a resistance reservoir that is non-sensitive to banning interventions. Laboratório Associado para a Química Verde 5 6 The simultaneous occurrence of a vancomycin-resistant E. faecium (VREF) strain among swine in four distant European countries for at least a 4 year period and the presence of a common Tn1546 type D indicate stability of strains and vancomycin genetic elements in swine production. The finding of a group of genetically closely related strains, including both vancomycin-susceptible E. faecium and VREF isolates, harbouring a particular purK allele previously associated to E. faecium swine strains, might mirror wide dissemination of a host specific clone more prone than others to acquire and spread different antibiotic resistance as reported for human clinical E. faecium isolates. The study with Portuguese Salmonella isolates indicates that antibiotic resistance mediated by integrons is common among diverse serotypes. The presence of several categories of these genetic elements shared among human and food isolates suggest that resistance determinants among animals may contribute to the problem of multi-drug resistant salmonella in humans. The newly described sul3 gene which confers resistance to sulphonamides has now been identified in 14 Salmonella isolates from three serotypes collected from human and nonhuman sources in Portugal, being mainly observed in isolates from swine food products. The consumption of sulphonamides for veterinary use is generally widespread in Portugal, particularly for swine production. So, the appearance of a newly described gene and the simultaneous presence of several sul genes may reflect its high usage in food-producing animals. The persistence of several sulphonamide resistance genes may be the result of the successive pressure exerted by sulphonamides and other antimicrobial agents that are also commonly used and may be mitigated by the fact that not all sulphonamide-resistant determinants exert a fitness cost, as described for the sul2encoding plasmid. Quantification of food aditives Never before has the range and choice of foods been so wide either in supermarkets, specialist food shops or when eating out. Food additives play a key role in maintaining the food qualities and characteristics that consumers demand, keeping food safe and protected from many environmental adverse conditions, such as temperature changes, oxidation and exposure to microbes, which can change their original composition. All food additives must have a demonstrated useful purpose and undergo a rigorous scientific safety evaluation before they can be approved for use. Each commercialized product must also meet all legal requirements, which includes controlling most levels of food additives. This creates the necessity of performing food analytical controls in a routine fashion. Thus, simple, quick and selective methods producing low levels of effluents of small toxicity and with reduced reagent consumption are required to address main environmental and economical aspects of today’s society. Electrochemical techniques are explored for this purpose, and applied to the determination of acidity regulators and flavour enhancers in foods of different sources. Determinations of glutamate and maltol, two flavour enhancers, are being developed by means of voltammetric procedures with gold and mercury electrodes. Both analytes are electrochemically reduced. The inherent mechanism was studied after cyclic voltammetric experiments. Main analytical features such as detection limit, quantification limit, slope, reproducibility, and selectivity were evaluated. Developed methods were meant for analysis of soups, sauces and cakes. Microwave assisted procedures were also carried out to assist preparation of complex food samples Regarding acidity regulators, tartaric acid was determined by means of spectrophotometric procedures, carried out in flow injection analysis set-ups. Solid-phase reactors are also in preparation for this purpose. Main flow parameters are optimized and the optimum conditions are selected for analytical application of the method. Laboratório Associado para a Química Verde 5 7 ENVIRONMENTAL CONTROL AND REMEDIATION Head of laboratory: Cristina Delerue Matos, Professor Coordenador / Helena Soares, Assistant Professor Staff Members: Maria do Carmo Vaz Professor Coordenador Simone Barreira Morais Assistant Maria Goreti Ferreira Sales Assistant Sónia Adriana Figueiredo Assistant Maria Isabel Branco Alves Martins Professor Adjunto Maria Teresa de Oliva Teles Moreira Professor Adjunto Abel José Assunção Duarte Assistant Florinda Figueiredo Martins Assistant Hendrikus Petrus Antonius Nouws Assistant Maria de Fátima de Sá Barroso Assistant Maria João Dantas Ramalhosa Ferreira Assistant Maria Manuela Barbosa Correia Assistant Olga Manuela Matos de Freitas Assistant Valentina Maria Fernandes Domingues Assistant Salomé Sousa Teixeira Assistant Maria Isabel Limpo de Serra Assistant José Tomás Soares de Albergaria Technician Maria Aurora Soares da Silva Technician Sérgio Alberto Monteiro de Morais Technician Paula Celeste Baptista Paíga Technician Ph.D. Students: Carina Machado, Cristina Maria Rodrigues Ferreira Alves, Oriza Paula Guedes Tavares Number of articles in scientific journals: 10 (161-170) Number of Ph.D Thesis: 2 Pesticide Analysis Determination of pesticide residues in food and environmental samples has received much attention in the last few decades. Following the need to develop faster, cleaner and more reliable analytical methodologies new chromatographic and electroanalytical methods have been developed. Concerning chromatographic procedures such as LC-DAD, LC-MS, GC-ECD or MS, they have been applied for the analysis of tomato samples, wine, must, grape and water. Microwave-assisted solvent extraction, liquid-liquid extraction (LLE), and solid-phase extraction (SPE) and microextraction (SPME) have been used to extract the analytes under study for chromatographic analysis. Laboratório Associado para a Química Verde 5 8 Voltammetric procedures for the determination of azinphos-methyl, parathion-methyl and diuron in soil samples using gold ultramicroelectrodes were developed. Regarding soil pretreatment, microwave-assisted solvent extraction was performed. This strategy is effective when compared to traditional extraction techniques, providing reduced extraction times, reduced solvent consumption and increased sample throughput. Other electroanalytical techniques were used to analyse natural waters and commercial phytopharmaceuticals. Voltammetric, amperometric and potentiometric based procedures were used for this purpose. These were applied to the determination of metam and molinate. Experiments were adapted to flow conditions, allowing reduced time, cost and operator intervention. These electroanalytical approaches generated also effluents of small toxicity. Laboratory waste management “Greening” of chemistry has clearly brought many advantages. Still, wastes from chemical analytical laboratories are typically hazardous and present a high diversity. The fact that only small quantities are generated has justified negligence of their existence or has induced laboratories to hire specific companies to perform this task. Thus, a waste management program was created and currently implemented for supporting all laboratory activities of students in a Chemical Engineering degree. This program is responsible for waste separation/ disposal strategy, collection, and characterisation of all inorganic liquid toxic wastes produced. Once entering the research laboratory, a proper chemical treatment is investigated and implemented. Resulting solids are isolated, purified, and evaluated in terms of purity and contaminants. When possible, solids are redirected as reagents towards another laboratory experiment. This feature provides a sustainable perspective to the waste management program. Before discard in the public sewage system, all liquid effluents emanating from suitable treatment are chemically controlled. Still, fulfilling chemical regulations does not ensure lack of adverse effects in living organisms when aquatic environment is chosen as final destiny for liquid treated wastes. Thus, chlorella vulgaris has been exposed to these wastes in order estimate inherent toxicity effects. As comparison, untreated wastes have also been subject of ecotoxicity trials. Soils remediation Soil contamination is closely related with inadequate practices of some industrial activities, disposal of industrial and municipal wastes or environmental accidents. Minute amounts of an organic compound are able to contaminate soil and groundwater, which grants serious risks for environment if toxic or carcinogenic compounds are concerned. Several technologies, like soil vapour extraction (SVE), in-pulp solvent extraction and bioremediation, are suitable to enable remediation at low cost. SVE is simple, relatively cheap and very efficient to remove from soils volatile organic compounds, usually correlated to underground fuel reservoirs. This strategy has been applied to remediation of soils of sand, organic matter and clay contaminated with benzene, toluene, ethylbenzene, xylene, trichloroethylene and tetrachloroethylene. SVE experiments are performed in a pilot installation with vacuum pumps to produce airflow. This is meant to enable estimation of remediation time, a feature that a conventional SVE procedure does not preview. In-pulp solvent extraction procedures were used to remediate soils with petroleum hydrocarbons. Ternary systems composed by solvents of small environmental concern and able to establish a single phase mixture were used for this purpose. Contaminants used in this study were 2,4-trimethylpenthene, xylene, naphthalene and hexadecane. Main analytical features for an improved efficiency were established. Effect of soil parameters and possibility of solvent regeneration were also appraised. Bioremediation by means of dual bio-augmentation is a novel approach that enhances degradation of organic pollutants regarding the use of microorganisms resistant to toxic metals. Four robust strains (Methylobacterium strains PM1, Mi1 and F5.4 and Methylophilus str. EHg7) are currently used to improve degradation of trichloroethylene (TCE) and methyl tert-butyl ether (MTBE) by soil microbial communities. Ability of natural microflora to degrade organic compounds in soils is under study prior to its enrichment with Methylosinus trichosporium str. OB3b as active degrader. It is proposed to set up microcosm experiments in the presence Laboratório Associado para a Química Verde 5 9 or absence of inocula of the 4 bacterial cultures in the presence and absence of heavy metals (Cd, Cr or Pb) and to study the effect of the addition of the selected organisms. Gas chromatographic methodologies are developed for the quantification of pollutants and process monitoring. Removal of toxic compounds by means of adsorption strategies Adsorption plays a key role in modern industries, especially in the field of environmental protection engineering, with the increasing environmental awareness of people all over the world. Many companies are looking at tertiary treatment processes to remove contaminants from their effluents. Several studies are in development with (inexpensive) natural adsorbents: cork, algae and wastes from corn production. One of them explores the adsorption capacity of cork (Quercus suber L.) to remove pyrethroids from waters, in order to attenuate the impact of this compound in aquatic life. An ecotoxicological evaluation showed that synthetic pyrethroids are amongst the most toxic pesticides for aquatic organisms. Other study aims to explore the ability of Portuguese macro algae species (Ascophyllum nodosum, Sargassum muticum, Fucus Spiralis and Pelvetia canaliculata) for the removal of toxic metals (Cd(II), Zn(II), Ni(II), Cr(III), Cu(II) and Pb(II)) from aqueous solutions is currently under development. This work is divided in the following parts: (i) the effect of temperature, pH and initial concentration of Pb (II) and Cu(II) in sorption process onto marine macro algae Ascophyllum nodosum was studied by using a Box-Behnken factorial design method, which gives a mathematical model that shows the influence of each variable and their interactions; (ii) various types of reagents, mineral acids (HCl and H2SO4) and aqueous solutions of ethylenediaminetetracetic acid (EDTA) disodium salt, calcium chloride and sodium chloride were investigated for their efficiency to recover Cu adsorbed by Ascophyllum nodosum; (iii) and finally equilibrium studies were conducted with mono and bicomponent metal solutions using batch adsorption experiments. Finally, wastes (Zea mais) from corn production have been tested successfully as adsorbents for textile dyestuffs. Batch equilibrium and Kinetic studies were performed. Kinetic studies were conducted using batch adsorption experiments and fixed-bed columns and the experimental results were fitted to models. Interaction between pH buffers and metal ions In this project (POCTI/39950/QUI/2001), the complexation properties of several M-(buffer)x-OHy systems involving six metal ions (Cd2+, Co2+, Cu2+, Pb2+, Ni2+, and Zn2+) and seven pH buffers (CAPS, CAPSO, CHES, TAPSO, TAPS and AMPSO) were characterized. No complexation was detected between three buffers (CAPS, CAPSO and CHES) and four metal ions (Cd2+, Cu2+, Pb2+, and Zn2+) tested. For the other systems, results indicated complexation and the final models of thirteen systems were fully defined. Biorremediation of wastewaters using microorganisms The main aim of this project, which started in 2005, is to develop a clean technique, using yeast brewer’s cells, for the treatment of wastewaters containing heavy metals The evaluation of yeasts flocculation capacity in the presence of heavy metals, as well as the optimization of metal ions accumulation conditions by the biomass was studied (financial support: POCTI/CTA/47875/2002 and UP/CGD /2005). New environment-friendly chelating agents for industrial and domestic applications This is a multidisciplinary project, financed by REQUIMTE and FCT (POCI/QUI/57891/2004), which involves researchers from the two centers of REQUIMTE. The project started in 2005 with the synthesis of the new ligands. Then, biodegradation and metal complexation studies will be performed to check the environmental impact of these ligands and their strength as metal chelators, respectively. Laboratório Associado para a Química Verde 6 0 ANALYTICAL METHODOLOGIES Head of laboratory: Aquiles de Barros, Associate Professor Research Team: José António Maia Rodrigues Assistant Professor Paulo Joaquim Ferreira de Almeida Assistant Professor Luís Guilherme de Lima Ferreira Guido Assistant Maria Isabel Afonso Rocha Assessor Maria Fernanda Rocha M. L. O. Cabral Associate Researcher Cristina Maria F. Delerue Alvim de Matos Professor Coordenador Simone Barreira Morais Assistant Maria do Carmo Vaz Professor Coordenador Maria Goreti Ferreira Sales Assistant Simone Barreira Morais Assistant Maria de Fátima Sá Barroso Assistant Hendricus Petrus Antonius Nouws Assistant Paula Celeste Baptista Paíga Assistant Post-Doct Students: Pedro Miguel Gonçalves Rodrigues Ph.D. Students: Andreia Filipa da Silva Curto M.Sc. Students: Marta Sofia Roma Pires, Maria Fernanda Andrade Resende, Sérgio José Pinto Teixeira Number of articles in scientific journals: 7 (171-177) Number of M.Sc. Thesis: 1 Activity in 2005 The strong lines of the project announced for the triennium 2003-2005 are the consolidation of the growing investigation related with beer and the diversification of the analytical techniques used. In this context a special reference is made to the research that is being devoted to the studies related with the problem of beer ageing, no doubt the main particular topic considered in the development of the project. Anyway, it is important to note that voltammetric analysis still represents an important field of investigation in the context of the project, as a continuation of the research produced before 2003. The main activities under development during 2005 have been: The cooperation with Unicer, Bebidas de Portugal SGPS, S A - Strengthening of the good relationship that already exists with the main Portuguese brewery. As result of this cooperation, several papers and communications in congresses were produced and the three years joint project Beervolt, approved by “Agência de Inovação” in 2004, was continued. This project, initiated in April 2004, is based on a patent meanwhile Laboratório Associado para a Química Verde 6 1 submitted (already approved in Portugal and in phase of EC approval). It consists in the development of a equipment for the voltammetric determination of diacetyl directly in the fermentation vessels and involves two other companies: Carlsberg S/A and CAI. The cooperation with Carlsberg S/A, Denmark - The joint project previously referred involving this important beer company will allow the intensification of a collaboration that exists since the year 2000. The cooperation with Institut Français de la Brasserie et de la Malterie, I. F. B. M., Nancy, France In the context of a collaboration involving Unicer and I. F. B. M., the Ph. D. student of this group Andreia Curto was involved in a post-graduate training of 6 months at I. F. B. M., in the context of a Ph. D. “mix grant” that she obtained from FCT. The main objective of this joint research was the study of the impact of several technological factors of the malting and brewing processes on the organoleptic stability of beer. The cooperation with the Instituto Superior de Engenharia do Porto (ISEP) – This collaboration consists essentially in the co-supervision of the Ph. D. work of Henri Nouws, lecturer at the ISEP. The work is cosupervised by Aquiles Barros and Cristina Delerue-Matos. A HMDE flow cell developed in our group is being used in the investigation, and some results were already presented in scientific meetings and published in scientific journals. Another branch of the research worthy of mention is: The cooperation with the Department of Civil Engineering of the Faculty of Engineering of Porto – It is a collaboration essentially devoted to studies on the accelerated degradation of geosynthetics; the work involves the investigation of the behaviour of these materials after being submitted to the effect of some chemical agents, under especially intense degradation conditions. Plan for 2006 For 2006, the objective of the group is to continue the activity of 2005, with special focus on: — consolidation of the work conducting to the construction of an equipment for the determination of diacetyl on line (in collaboration with the companies Unicer, Carlsberg and CAI); also, it is expected that there are some news about the European Patent that is pending on this subject; — continuation of the study of the impact of several technological factors of the malting and brewing processes on the organoleptic stability of beer (also involving IFBM and Unicer); — continuation of the studies on the accelerated degradation of geosynthetics, in collaboration with the Department of Civil Engineering of the Faculty of Engineering of Porto, involving a Ph. D. student. — opening of a new line of investigation focused on analytical, industrial and biochemical studies of some phenolic compounds present in beer and in hop. — opening of a new line of investigation focused on the use of membrane based extraction techniques with application in the development of analytical methodologies in flow for food analysis. Electroanalysis Electrochemistry is often required in the analytical chemistry field because of its selectivity, sensitivity, and generation of effluents of low toxicity. It may also provide information concerning oxidative/reductive mechanisms. Different voltammetry-based methods were developed, exploiting oxidation/reduction of the analyte as well as its adsorption, using conventional electrodes like glassy carbon and hanging mercury drop electrodes or ultramicroelectrodes. Development of flow methods was done by coupling amperometric and stripping voltammetric detectors to flow-injection analysis set-ups. Different working electrodes were used here, including glassy carbon, carbon paste, chemically modified and hanging mercury drop electrodes. This strategy was applied to the analysis of different compounds within pharmaceutical and environmental fields. Laboratório Associado para a Química Verde 6 2 Ion-selective electrodes are also constructed for presenting the attractive feature of selectivity. Selective membrane composition and configuration were regarded. Preparation of polymeric sensors was selected after evaluating the effect of ionophore and plasticizer natures. Devices of cylindrical configuration were prepared for flow set-ups. This arrangement ensured a laminar flow, decreasing dead volumes and the response time of the detector. Laboratório Associado para a Química Verde 6 3 PHYSICOCHEMICAL CHARACTERIZATION OF FOOD PRODUCTS Head of laboratory: Maria do Pilar Gonçalves, Associate Professor / Alberto Sereno, Associate Professor Staff Members: Loïc Hilliou Assistant Researcher Post-Doct Fellows: Marta Isabel de Glória V. M. Silva Ph.D. Students: Luis Mayor Lopez, Wancheng Sittikijyothin, Fabio Donato Soares Larrotonda Number of articles in scientific journals: 3 (178-180) Number of M.Sc Thesis: 1 Rheological behaviour and morphology of protein-polysaccharide mixed systems Studies were conducted on the effect of shear on b-lactoglobulin gels, pure or mixed with galactomannans using dynamic oscillatory measurements. Different shear rates, time of shear and temperature conditions were tested. The results showed that the rheological behaviour of the pure and mixed gels was very sensitive to shear treatment. In all cases studied, the time needed to reach the gel point decreased and the gel strength increased with increasing shear rate. The strain at fracture for mixed gels subjected to shear was higher than for unsheared gels; whereas the contrary was observed for pure gels. The mechanical spectra were analyzed quantitatively by fitting a Cole-Cole model to the storage and loss compliance versus frequency data. The fits matched reasonably well the data in the high frequency region for the mixed gels. The work is in its final stage. In 2006, some articles will be submitted to international journals and a PhD dissertation will be also submitted by Wancheng Sittikijyothin in the Faculty of Engineering of the University of Porto (FEUP). Cold gelation of globular proteins a) The denaturation conditions, namely temperature and holding time, and the protein concentration were systematically varied in order to screen their impact on the resulting heat denatured whey protein isolate (HDWPI) solution viscosity and gel elasticity. Results show that the former variables can be reconciled in a single parameter - the aggregates concentration - which controls the rheological properties of the HD-WPI solutions and the texture characteristics of the corresponding cold-set gels. b) In the frame of a scientific collaboration with a Brazilian working group, a study about the rheological behavior of ‘Polpa de Açaí (Euterpe oleaceae)’ was initiated. c) In the frame of a CAPES/GRICES project an investigation about biodegradable superabsorbent polymers (SAP) has been developed. The swelling ratio, the degree of swelling, the water sorption isotherms and the texture profile of the new materials were determined. The rheological behavior of the SAPs is also under investigation. Work plan for 2006 The studies mentioned in points b) and c) of the above paragraphs will be concluded at the beginnings of 2006. In the frame of our study about cold-set gelation of WPI, it will be developed the necessary hardware (a transparent measurement cell and an optical train) to make structural studies during the gelation process. Laboratório Associado para a Química Verde 6 4 Technology for edible biodegradable films and coatings for foods In this FCT project (POCTI/EQU/45595/2002), phycocolloids were extracted from Portuguese seaweeds. The research activity was continued by mixing the isolated polysaccharides (k-i hybrid carrageenans) with starch. Films were obtained from these mixtures by casting. These films were characterized about their physical and chemical properties. The results of this characterization together with a comparison with model films prepared from commercial k-carrageenan and starch were reported at the ENPROMER 2005 and the CIBIA 2005 meetings. Workplan for 2006 Efforts in film formulation will primarily aim at lowering film hydrophilic properties by incorporating hydrophobic chemicals. Finally, film formulation will be extended to pectins as planned in the work plan of a separated project (see REQUIMTE project below). COMFOOD: Controlling the microstructure of food products by rheo-optical methods. This FCT funded project started in October. Workplan for 2006 Building of an optical train and preliminary rheo-optical characterization of model food systems. Physical characterization of PHA biopolymers This FCT funded project started in September. Workplan for 2006 PHA samples, produced by our partners at CQFB, will be characterized by Differential Scanning Calorimetry (DSC) and rheology. Characterization of biopolymer mixtures for food applications. This GRICES/DAAD cooperation aims at studying commercial k- and i-carrageenan gels as well as k-i hybrid carrageenans extracted from Mastocarpus stellatus seaweeds (see project POCTI/EQU/45595/2002) by means of Fourier transform rheology (FTR) and new Magnetic Resonance Imaging (MRI) techniques. In a preliminary study, a steady shear applied on cooling carrageenans solutions was shown to impede the gelation mechanism. After shear cessation, gels can set with an elasticity that depends on the applied shear. The FTR signatures of i- and k-i hybrid carrageenans gels are virtually similar, thus indicating resembling structures. The FTR behavior of -carrageenans gels is more complex as it appears to be a combination of hard gel-like behavior at low strains and of soft gel-like behavior at larger strains. Workplan for 2006 Results reported above will be presented at the ISFRS 2006 and AERC 2006 meetings and 2 articles will be prepared right after additional structural characterization (optical microscopy and/or cryoSEM imaging). Then, polysaccharide mixtures such as gelatin-carrageenan blends should be studied with the same set of techniques. Extraction of added value compounds from orange peel wastes: application to mixed biodegradable films. This project is funded by REQUIMTE. Films containing commercial pectins have been first characterized. The rheological characterization of pectins produced by our CQFB partners has been initiated. Laboratório Associado para a Química Verde 6 5 Work plan for 2006 Characterization of newly extracted pectins by rheology and DSC. These new products will then be used in films whose properties will be compared to commercial pectin-based films. Studies in Food Structure The last year studies about changes in food structure during processing were performed and some mathematical models were applied in order to establish relations quality-macrostructure-microstructure of processed food materials. This work focused on the study of structural changes during osmotic dehydration of pumpkin fruits. The work is in its final stage, and in the next year some articles will be submitted to international journals in the area of Food Science and Technology. A Ph Degree dissertation, with the title “Characterization and modelling of structural changes in fruits and vegetable tissue submitted to dehydration processes”, will be also submitted by Luis Mayor López in the Faculty of Engineering of the University of Porto (FEUP). Laboratório Associado para a Química Verde 6 6 TOXICITY AND PROTECTION STUDIES Head of Laboratory: Maria de Lourdes Bastos, Full Professor Staff Members: Rosa Seabra Full Professor Paula Andrade Assistant Professor Felix Carvalho Assistant Professor Fernando Remião Assistant Professor Helena Carmo Assistant Maria Elisa Soares Assessor Principal Eulália Mendes Assessor Principal Carla Rodrigues Technician Maria João Marques Researcher Ph.D. Students: João Paulo Soares Capela, Ricardo Jorge Dinis Oliveira , Helena Pontes, Vera Costa, Miguel Lopes, Ema Alves, Márcia Carvalho M.Sc. Students: Luis Correia, Renata Silva Number of articles in scientific journals: 14 (181-194) Biological activity of plant extracts and beverages The hot water extracts of tronchuda cabbage internal and external leaves and of the seeds were investigated for their capacity to act as scavengers of DPPH· radical and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid), exhibiting antioxidant capacity in a concentration dependent manner against all radicals. Despite the antioxidant capacity exhibited by tronchuda cabbage internal leaves, in general terms they exhibited lower antioxidant potential than external leaves. This can be ascribed to the higher content of both phenolics and organic acids in the external leaves. According to the results obtained in all assays, and in comparison with data from both tronchuda cabbage internal and external leaves, it could be observed that, in general terms, tronchuda cabbage seeds exhibit higher antioxidant potential than its leaves. This is not surprising once seeds often contain the highest concentration of lipids of any plant tissue, with high levels of polyunsaturated fatty acids. The occurrence of high amounts of phenolic compounds, particularly of hydroxycinnamic derivatives, and organic acids, namely ascorbic acid, in tronchuda cabbage seeds suggest that these compounds protect storage lipids from oxidation. Studies regarding the antioxidant capacity of tronchuda cabbage against reactive nitrogen species (nitric oxide and peroxynitrite) are in course. Beefsteak fungus Fistulina hepatica was investigated for its capacity to act as a scavenger of DPPH· radical and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid). Good results were obtained against DPPH and superoxide radicals and hypochlorous acid but a prooxidant effect was observed for hydroxyl radical. The study of the antioxidant potential of other edible mushroom species is in course. Laboratório Associado para a Química Verde 6 7 The antioxidant ability of the medicinal species Ballota nigra (Lamiaceae) infusion against DPPH radical, reactive oxygen species (superoxide radical, hydroxyl radical, hypochlorous acid) and nitric oxide was investigated. The tested infusion mainly exhibited a potent scavenging effect on DPPH, nitric oxide and superoxide radicals. In hydroxyl radical assay a potent pro-oxidant activity was noticed. No effect was found against hypochlorous acid. Studies regarding the antioxidative capacity of Linaria vulgaris (Scrophulariaceae) are in course. Due to the high content of antioxidant compounds, some foods could be able to delay or even revert the neurodegenerative events related to the increased oxidative stress. The central nervous system is particularly vulnerable to this phenomenon, a manifold process that plays a pivotal role during aging, after chronic ethanol consumption and in some neurodegenerative diseases. This vulnerability is related to the generation of harmful reactive oxygen species that lead easily to a depletion of antioxidant defences. Polyphenols present powerful in vivo antioxidant properties and are particularly abundant in red wine and green tea. Thus, we thought of interest to test the antioxidant efficacy of red wine and green tea after prolonged consumption in the hippocampus and in the cerebellum of the adult rat. Using biochemical methods we intended to quantify the activity of antioxidant enzymes, lipid and protein oxidation, glutathione levels and transcriptional activity changes in these brain areas. These studies are in progress. Studies of mechanisms of toxicity using in vitro and in vivo models An increasing body of data has being demonstrating that mammalian cells have elaborate networks of molecular signalling in counteracting heat shock and in developing adaptation to oxidative stress to avoid cell death. However, the precise mechanisms linking heat shock, oxidative stress and cell survival/cell death mechanisms are not yet clearly understood. The purpose of this study was thus to determine the usefulness of the freshly isolated mice hepatocytes model for the study of the time course of hyperthermia-induced oxidative stress and cellular signalling. Freshly isolated mouse hepatocytes obtained from Charles River CD1 male adult mice were incubated under normothermia and hyperthermia. The toxic effects were evaluated by measuring cellular viability and oxidative stress. Hyperthermia-induced cell signalling was evaluated through the measurement of HSF1 activation and HSP70 expression. The results accomplished in this work demonstrated that mild continuous hyperthermia leads to oxidative stress and loss of cellular viability in a time-dependent manner. Additionally, it was also found that hyperthermia was capable of inducing a heat shock response reflected the activation of HSF1, which emerged before the formation of HSP70 levels. Continuing our mechanistic toxicity studies of amphetamine derivatives, a new in vitro model was implemented in our lab in order to extend our research to the brain, an important target organ of the toxicity of these compounds. The widely abused psychoactive recreational drug 3,4-Methylenedioxymethamphetamine (MDMA or “Ecstasy”) and MDMA-metabolites-induced were evaluated for their neurotoxicity in neuronal cultures from rat cortex under normal (36.5ºC) and hyperthermic conditions (40ºC). Our studies showed that MDMA and MDMA-metabolites produce a neurotoxicity that is potentiated under Hyperthermia. Also that MDMAinduced neurotoxicity is dependent on its 5-HT2A-receptor agonism and on the generation of reactive oxygen species (ROS)/reactive nitrogen species (RNS). These studies bring new insights on the mechanisms by which MDMA and its metabolites elicit neurotoxicity. Amphetamine and it’s alike are substances with the ability of inducing pleasant sensations in those who use them, being classified as psychostimulant drugs of abuse. The consumption of amphetamine analogous compounds, in particular MDMA, increased in an expressive way in the last years. The clinical and experimental evidences of the amphetamine and it’s alike toxicity, allied to the consumption in the younger population, specially in the reproductive period, imposes the analysis of the situation. It’s our purpose to contribute to the characterization of the behaviour, and in a long term, of the exposure to MDMA, using adolescent Wistar rats. It’s being used a dosage of MDMA known to induce neurotoxicity. With the purpose of identifying the biochemical and cellular modifications responsible for the behaviour changes, are being executed a set of biochemical Laboratório Associado para a Química Verde 6 8 quantifications. These studies are being accomplished in brain areas known to be specially damaged by MDMA, namely hippocampus, striatum, accumbens, rafe and pre-frontal cortex. All these studies will be equally executed to determine the simultaneous consumption of MDMA and ethanol gives the high frequency with which they are consumed in the younger population. Since ethanol and MDMA are usually consumed simultaneously, it is very relevant to evaluate the effect of the association of these two substances on the hepatic cells as they are involved in the metabolism of both drugs. Additionally, hyperthermia has been recognized as a life-threatening effect associated with MDMA exposure. Therefore, a comparative in vitro study is being performed in isolated mouse hepatocytes to evaluate the hepatotoxicity of MDMA, ethanol, and their association under normothermic and hyperthermic conditions. These studies are in course. The pesticide paraquat is responsible for several acute fatal intoxications being the lung the main target organ of toxicity. However, no antidote or effective treatment was developed until now to decrease the paraquat accumulation in the lung or to disrupt its toxicity. In vivo studies, particularly those directed to decrease paraquat lung accumulation and those to interfere with the subjacent signaling pathways, which lead to edema and subsequent fibrosis, have been performed. All these toxicity studies are being developed by the PhD students and consistent results are expected in the next year. Some validated in vitro models, namely the microcrustaceous Daphnia magna and Artemia parthenogenica, as well as the microalgae Tetraselmis chuii and the mosquitofish Gambusia holbrooki were used for the evaluation of environmental contamination by acethylcholinesterase inhibitors and pharmaceutical drugs. Biotransformation of compounds: in vivo and in vitro studies In this research line important biomarkers of exposure (metabolites) and biomarkers of effect are being identified and validated and we hope they will be of great value to establish toxicity mechanisms involving the biotransformation of xenobiotics. Sustained high levels of circulating catecholamines can lead to cardiotoxicity and neurotoxicity. We have previously shown that the catecholamines oxidation products can further conjugate with GSH in isolated rat cardiomyocytes and hepatocytes leading to the formation of potentially more toxic species. The next points were studied during 2005: 1) An isocratic reverse-phase HPLC with photodiode array and/or coulochem detection methodology was developed in order to analyse biogenic catecholamines-GSH adducts. 2) A sample treatment procedure for catecholamines-GSH adducts analysis in biological samples was also developed and applied to human serum. The obtained recoveries were between 60 and 70% and the chromatograms were clean. 3) (Glutathion-S-yl)-catecholamines were characterized by their UV and mass spectrometry (MS). 4) The development of this methodology allows a direct analysis of catecholamines-GSH adducts in blood, enabling the study of the catecholamines oxidation process. 5) To establish the possible toxicity of cathecolamines in the presence or absence of a system capable of generating oxygen free radicals (Xanthine and Xanthine Oxidase) on calcium tolerant cardiomyocytes isolated from adults rats. Results are being analysed and it is expected to be published in current year. The influence of genetic polymorphism of the main metabolizing enzymes of the designer drugs of abuse 4MTA, 2C-B, and MDMA in their toxic effects was evaluated. By using cell lines that express the genetic variants of human metabolizing enzymes (CYP2D6, CYP3A4) we are able to identify the enzymes that are mostly involved in the detoxification of these drugs and also the relationship between polymorphic Laboratório Associado para a Química Verde 6 9 metabolism and toxicity. Subsequent use of human liver microsomal fractions profiled for the enzymes of interest and incubation with susceptible cell lines will be performed in order to determine if genetic polymorphism is in fact related with the overexpression of the toxic effects of these designer drugs of abuse. With this aim we have developed the following tasks: 1. Evaluation of the influence of polymorphic metabolism in the cytotoxic effects of MDMA and 4-MTA. Studies with V79 cell lines genetically engineered for the expression of polymorphic variants of human CYP2D6 enzyme. 2. Development and validation of an analytical technique for the quantification of the oxidative metabolite of MDMA, N-methyl-á-methyldopamine. 3. Characterization of the oxidative metabolism of MDMA catalyzed by CYP2D6 in V79 cell lines genetically engineered for the expression of polymorphic variants of human CYP2D6 enzyme. Identification and quantification of metabolites. 4. Evaluation of the cytotoxicity of N-methyl-á-methyldopamine towards the addopted in vitro experimental model. Comparison with the parent compound MDMA Important results were obtained and were already submitted for publication and other studies under this subject are in course Laboratório Associado para a Química Verde 7 0 BIOMIMETIC SYSTEMS AND SIMULATION Head of Laboratory: Maria João Ramos, Associate Professor Staff Members: Alexandre Magalhães Assistant Professor André Melo Assistant Professor José Augusto Pereira Assistant Professor Pedro Fernandes Invited Assistant Professor Agostinho Antunes Pereira Assistant Researcher Nelson Brito Technician Ph.D. Students: Akapong Suwattanamala, Rute Fonseca, Susana Pereira, Nuno Cerqueira, Fátima Lucas, Vineet Pande, Sérgio Sousa, Ricardo Branco, Alexandra Carvalho, Irina Moreira, Zenaida Mourão, Alexandre Carvalho, Marco Preto, Daniel Dourado, José Rui Marques, Alexandra Marques, Bruno Tamames, Juan Tamames. M.Sc. Students: Eva Cunha, Daniel Osório Project Grantee: Natércia Brás, Sónia Patrício Number of articles in scientific journals: 23 (195-217) Number of articles in books: 1 Number of M.Sc Thesis: 2 Just as in the past few years, we have been generally interested in the molecular modelling of biological and chemical systems. To further these studies, we have been using both molecular simulations and quantum mechanics techniques, as well as quantum mechanics/molecular mechanics (QM/MM) or quantum mechanics/ quantum mechanics (QM/QM) hybrid methods. Last year we have added to our present, general interest in proteomics, a new line of computational genomics. Our idea is to eventually establish a close link between both areas, which we have already started doing. A more detailed description concerning our main present interests, which have derived from year 2004 and will continue in 2006, follows: I. Disease Related Research Cancer (i)The study of the catalytic and inhibition mechanisms of enzymes P450 1A2 1,2 (an enzyme involved in the metabolic pathway of carcinogenesis) RNR 3-5 (ribonuclease reductase, an enzyme thought to be involved in cancer), and other enzymes 6-11, resorting to both quantum mechanics, QM/QM and QM/MM methods, has advanced very much. These studies have been financed by the National Foundation for Cancer Research, U.S.A. (P450 1A2) and by the Fundação para a Ciência e Tecnologia, Portugal (RNR) via project POCTI/ 35376/QUI/2000. (ii)We are further involved in the study of the catalytic and inhibition mechanisms of other enzymatic reactions, some of them also thought to be involved in cancer, such as superoxide dismutase, glutathione transferase Laboratório Associado para a Química Verde 7 1 and farnesyl transferase12-16. These studies have been financed by the Fundação para a Ciência e Tecnologia, Portugal (RNR) via project POCI/QUI/61563/2004. (iii)Continuation of the screening of 3,5 billion small molecules as potential inhibitors of 16 proteins, directly involved in cancer, which are current targets of the pharmaceutical industry. This project is part of the work carried out by the NFCR Centre for Computational Drug Design, based at Oxford, and directed by Prof. Graham Richard of which Maria João Ramos is an Associate Director, being financed by the National Foundation for Cancer Research, U.S.A 17. A.I.D.S. (iv) The study of the problem of new potential therapeutic agents for the treatment of acquired immunodeficiency syndrome (AIDS) by investigating the binding modes of different anti-AIDS chelators like ATA, HPH and other analogs to achieve a better design of anti-HIV metal chelators, continued. Several research works were published on this theme, as well as some reviews18-22. This project is being carried out in close collaboration with two experimental organic chemists, Dr. R. Sharma, in India, and Prof. M. Otsuka, in Japan. Hypertension (v) To help on the modelling of mimetic modifications of bioactive peptides by inclusion of novel synthetic amino acids, we have run extensive molecular dynamics on angiotensin II in both environments, water and DMSO23. This project is being worked on in close collaboration with an organic chemist (Prof. Hernâni Maia from the University of Minho, Portugal), whom has agreed to syntethise the novel peptides, and being financed by the Fundação para a Ciência e Tecnologia, Portugal, via projects POCTI/35380/QUI/2000 and POCI/QUI/ 55673/2004. II. Drug Design Complementary Studies (vii)Alanine scanning computational mutagenesis has been studied and a new methodology proposed. For this reason, extensive molecular dynamics simulations of protein complexes has been carried out and computational mutagenesis performed on the interface of these complexes, which has allowed the establishment of the respective hot spots 24-27. (viii) Continuation of the development of new formalisms to analyse the molecular interactions in association processes 28. This involves the partitioning of the physical observables of a molecular system into spatial and physical meaningful components. These decomposition methodologies can be used as powerful tools for molecular modelling and design of biological systems. (ix)Calix[4]arenes are molecules that possess 4 aromatic moieties which can form p-electron-rich cavities and behave as host compounds for ions and neutral species. Therefore, the adsorption of calix[4]arenes in electrode/electrolyte interfaces is an interesting field to be explored to get more insight about the role and properties of adsorbed molecules, as well as the nature of those interfaces and its applications to ionselective electrodes or potentiometric sensors. The work developed in this period has been concerned with the study of thialcalix[4]arenes and their complexation with transition metal ions. We are further involved in the design of new calixarene derivatives to help the experimental synthesis of potential host compounds of this type 29,30. (x)Efforts have also been made with imparting education on drug design 31 Laboratório Associado para a Química Verde 7 2 III. Computational Genomics We have started a line of research on Computational Genomics with the goal of expanding the team’s research experience on Bioinformatics, allowing the simultaneous integration of genomic 32,33 and proteomic data. The study of insertions of fragments of mtDNA into nuclear chromosomes is an important mechanism for the evolution of genomes, and when targeted to gene loci, such insertions can be mutagenic, causing disease. We have been studying this subject in more detail and additionally, we have been supervising research on the evolution and comparative genomics of genes with important biological functions, namely the gene coding the human enzyme ABAD that is linked to the mitochondrial toxicity in Alzheimer’s disease. III. Requimte’s collaboration projects Requimte itself has provided grants for collaborative projects between different groups in Porto and Lisbon and we have won two of those projects, with other different groups also belonging to Requimte, and have been investigating on: CBM11 Molecular determinants of ligand specificity in the family 11 Carbohydrate Binding Modules. Anthocyanin Blues A green approach towards food colorants and new pigments based on anthocyanins and synthetic flavylium salts. References: [1] R. Fonseca, M. Marini, A. Melo, M. C. Menziani; M. J. Ramos Medicinal Chemistry, 2005, 1, 355-360 [2] F. Iori; R. Fonseca; M. J. Ramos, Bioorganic Medicinal Chemistry, 2005, 13; 4366-4374 [3] N. M. F. S. A. Cerqueira; S. Pereira; P. A. Fernandes; M. J. Ramos, Current Medicinal Chemistry, 2005, 12, 1283-1294 [4] S. Pereira; P.A. Fernandes; M. J. Ramos, Journal of the Americal Chemical Society, 2005, 127; 51745179 [5] N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos, Biophysical Journal, 2005- In press [6] M. F. Lucas; M. J. Ramos, Journal of the Americal Chemical Society, 2005, 127; 6902-6909 [7] M. J. Ramos; P. J. Silva, Journal of Physical Chemistry B, 2005, Vol.109; 18195-18200 [8] A. P. Carvalho; P. Fernandes; M. J. Ramos, Progress in biophysics and molecular biology, 2005- In press [9] A. P. Carvalho; P. Fernandes; M. J. Ramos, Journal of Computational Chemistry, 2005- In press [10] A. P. Carvalho; P. Fernandes; M. J. Ramos, Journal of Physical Chemistry B, 2005- In press [11] A. P. Carvalho; P. Fernandes; M. J. Ramos, Mini-Reviews in Medicinal Chemistry, 2005- In press [12] R. J. F. Branco; P. A. Fernandes; M. J. Ramos, Journal of Molecular Structure: Theochem, 2005, 729, 141–146 [13] R. J. F. Branco; P. A. Fernandes; M. J. Ramos, Theoretical Chemistry Accounts, 2005- In press [14] S. F. Sousa; P. A. Fernandes; M. J. Ramos, J. Biol. Inorg. Chem., 2005, 10, 3-10 [15] S. F. Sousa; P. A. Fernandes; M. J. Ramos, Journal of Molecular Structure: Theochem, 2005,729, 125– 129 Laboratório Associado para a Química Verde 7 3 [16] S. F. Sousa; P. A. Fernandes; M. J. Ramos, Biophysical Journal, 2005, 88, 483-494 [17] http://www.chem.ox.ac.uk/cancer/ccdd.html [18] P. A. Fernandes et al, Theoretical Chemistry Accounts, 2005, 113; 197-204 [19] V. Pande; M. J. Ramos, Bioorganic & Medicinal Chemistry Letters, 2005, 15; 4057-4063 [20] V. Pande; M. J. Ramos, Current Medicinal Chemistry, 2005, 12; 357-374 [21] V. Pande; M. J. Ramos, Bioorganic & Medicinal Chemistry Letters, 2005, 15; 5129-5135 [22] R. K. Sharma; V. Pande; M. J. Ramos; H. K. Rajor; S. Chopra; K. Meguro; J. Inoue; M. Otsuka, Bioorganic Chemistry, 2005, 33; 67-81 [23] M. A. C. Preto; A. Melo; H. L. S. Maia; T. Mavromoustakos; M. J. Ramos, Journal of Physical Chemistry B, 2005, 109, 17743-17751 [24] I.S. Moreira; P. A. Fernandes; M. J. Ramos, Proteins, Structure, Function and Bioinformatics, 2005- In press [25] I.S. Moreira; P. A. Fernandes; M. J. Ramos, Int. J. Quantum Chem., 2005- In press [26] I.S. Moreira; P. A. Fernandes; M. J. Ramos, J. Phys. Chem. B, 2005- In press [27] I.S. Moreira; P. A. Fernandes; M. J. Ramos, Theor. Chem. Acc., 2005- In press [28] A. R. F. Carvalho; A. Melo, International Journal of Quantum Chemistry, 2005, 104, 240-248 [29] A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes, Journal of Molecular Structure: Theochem, 2005, 729, 83-90 [30] A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes, Chemical Physics, 2005, 310, 109-122 [31] M. J. Ramos; P. A. Fernandes, Journal of Chemical Education, 2005, 82; 1021-1025 [32] A. Antunes; M. J. Ramos, Genomics, 2005, 86; 708-717 [33] P. Gaubert; A. Antunes, Journal of Mammalogy, 2005, 86, 1068-1074. Laboratório Associado para a Química Verde 7 4 APPLIED BIOPHYSICS AND BIOCHEMISTRY Staff Members: Baltazar de Castro Full Professor José Costa Lima Full Professor Paula Gameiro Assistant Professor Maria Salette Reis Assistant Professor Eduarda Graça R. Fernandes Assistant Professor Mª José Feio Invited Assistant Professor Carla Manuela S. Matos Assistant Professor Catarina Mansilha Assistant Professor Ph.D. Students: Patrícia Neves, Sofia Costa Lima, Sónia garcia, Marlene Susana Dionísio Lucio, Helena Machado Ferreira, David Costa, Christophe Siquet, Ana Maria Gomes M.Sc. Students: Cláudia Daniela Oliveira de Lacerda Nunes, Célia Tavares de Sousa, Marisa de Freitas Project Grantee: Adriana Key Number of articles in scientific journals: 12 (218-229) Number of M.Sc. Thesis: 2 Quenching studies were performed in OmpF/DMPC proteoliposomes and a markedly difference in protein conformation (compared to that on mixed micelles) was observed. OmpF fluorescence, in the absence or presence of four different quinolones, was quenched by acrylamide or iodide and the differentiate accessibility of these quenchers to the OmpF tryptophans provide information on the possible entry of the quinolones into the cell showing that this entry is related to physico-chemical characteristics of the drugs. Proteoliposomes with other lipids are beeng performed and the interaction of fluoroquinolone analyzed by fluorescence spectroscopy. Ion current though single trimeric OmpF porins reconstituted into planar lipid bilayers were performed to study translocation of some quinolones of different size. Favorably interacting quinolones occlude the pore during their translocation and cause random transient blockages of the ion current through the channel. Analyzing these fluctuations, we observe relatively high affinity of moxifloxacin for OmpF. These studies will be performed for other fluroquinolones. The minimum inhibitory concentration (MIC) of copper(II)/norfloxacin, and copper(II)/ofloxacin and of the ternary complexes copper(II)/norfloxacin/phenanthroline copper(II)/ofloxacin/phenanthroline have been studied in several bacterial stains with series of porin mutants which lack one or several major outer membrane proteins and the results show that porins in general are very important for the uptake of these possible metalloantibiotics, and that their activity is much similar to that of the fluoroquinolones allow. Furthermore it seems that for ofloxacin and norfloxacin OmpC can also be important. Synthesis of other metal (Cu, Zn, Co, Ni) complexes with fluoroquinolonas and ternary metal/fluoroquinolone/phenanthroline complexes are beginning to be performed and also some studies to determine a possible activity of these new compounds in the bacteria stains. If these Laboratório Associado para a Química Verde 7 5 compounds show to be active some toxicological studies will be performed. Intrinsic Trp fluorescence of P-glycoprotein (Pgp) was used to perform fluorescence quenching studies with tuberculostatics in presence and absence of ATP and a resonance energy transfer was observed between protein and these drugs. Methodologies and formalisms of resonance energy transfer are being applied to the experimental results to extract information of the possible transport mechanism of Pgp. In vitro flow cytometry methods are being implemented to quantitatively assess inhibitors and/or modulators of P-glycoprotein. In cyanobacterial and some eukaryotic algal photosynthetic systems, electron transfer between the cytochrome b6-f complex and photosystem I is mediated by either plastocyanin or cytochrome c6 . The structure-function relationship that allows these two soluble proteins to carry out the same cellular role despite their structural differences has been the focus of considerable research efforts in recent years. The comparative study of the effect of temperature upon the stability of the plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and a mesophilic counterpart Synechoccystis sp. revealed considerable differences in the thermodynamic and kinetic properties of their unfolding process. Results previously obtained with cytochrome c6 from Nostoc sp. PCC 7119, a thermo tolerant cyanobacterium seem to suggest that a similar evolutionary pattern was followed. The work under way aims at studying the unfolding process of this protein functionally homologous to plastocyanin and check whether a similar stability pattern can be observed. We will therefore use cytochrome c6 from mesophilic and thermophilic cyanobacterial species overexpressed in E. coli and purified from periplasmic extracts to study their thermal unfolding process. We will use a combination of UV/vis, fluorescence and EPR spectroscopies to obtain thermodynamic and kinetic data. If the process proves to be reversible, we will also resort to DSC to obtain some of the process’ thermodynamic parameters. Biophysics and Biochemistry of organized media The main objective of this work is to study the role of the NSAIDs induced perturbation on the bilayer structure and dynamic properties, trying to clarify the mechanism of action of these drugs and to elucidate the role of these effects on their anti-inflammatory mechanisms of action. Liposomes were used as membrane mimetic models to study the effect of several non steroidal anti-inflammatory drugs (NSAIDs) on some biophysical parameters of biological membranes. It is well known that the principal mechanism of action for NSAIDs is the inhibition of the conversion of arachidonic acid through the cyclooxygenase or lipoxygenase pathways resulting in the formation of the prostaglandins or leukotrienes. Moreover, the development of inhibitors that selectively binds to COX-2 for which it is been expected anti-inflammatory action in vivo with minimal gastric side effects, shows that the sequence of events resulting from cyclooxygenase inhibition does not totally explain the overall gastric toxicity of NSAIDs. Such mechanisms are complex and the cascade of events leading to mucosal damage has yet to be characterized and can also be related to NSAIDs’ topical irritancy. We investigated the ability of NSAIDs to bind to surface-active phospholipids, as well as their effect on the lipid dynamic properties of membrane models, once the results may elucidate about the effects of these drugs compromising the integrity of gastric mucosal barrier. For this purpose, three different NSAIDs (indomethacin, acemetacin and nimesulide) were selected and their influence on the biophysical properties of 1, 2dipalmitoylphosphatidylcholine (DPPC) liposomes was studied using different and complementary techniques. Structural changes involved in the DPPC thermal transitions upon the interaction of the bilayers with NSAIDs were evaluated by small angle X-ray scattering (SAXS). The short-range organisation of the phases was characterised by using wide angle scattering (WAXS). These x-ray scattering techniques were also compared to the results yielded by differential scanning calorimetry studies (DSC) which have proven to be very useful for the study of the interactions between drugs and lipid membranes. Along with the studies in bilayers systems, models consisting of phospholipids spread at the air– water interface as a Langmuir monolayer were used. The interest in phospholipid monolayers is prompted by the fact Laboratório Associado para a Química Verde 7 6 that it allows varying such physical-chemical parameters of the interface as molecular density, lateral pressure, surface potential, or ionic conditions and thus, it provides a simple model for studying drugs penetration. Structural changes within the DPPC monolayer upon adsorption of indomethacin were investigated both by synchrotron X-ray experiments at grazing incidence (GIXD) and pressure–area isotherms. These experiments were carried out to obtain information on the structure of the penetrated monolayer and to gain additional knowledge to the interpretation of the penetration mechanism of indomethacin over the lipid phase, once this NSAID revealed a high perturbing effect both in the monolayer and bilayer systems. In these studies we have also evaluated the partition coefficient of NSAIDs, the location of that drugs on the membrane, their effect on membrane fluidity and also on the surface potential. The determinations of partition coefficient were performed by derivative spectrophotometry and by fluorescence. The effect of NSAIDs on surface potential was assessed by measure the zeta potential in the presence of different drug concentrations. This methodology was applied to the study of the NSAIDs concentration effects. Results revealed an intense membrane charging that was proportional to the amount of charged drug in media. Location and membrane fluidity were evaluated by fluorescence using fluorescent probe molecules. Some of these probes are capable of sensing “fluidity” gradient through the bilayer leaflet. The results obtained shows that all the NSAIDs studied increase the membrane fluidity. Only the diclofenac decrease the fluidity of the lipid membrane. It has also been suggested a relation between the ability of drugs to change membrane fluidity and their membrane antioxidant action namely their role on lipid peroxidation process. Lipid peroxidation results in an alteration of dynamic and structural properties of the membrane which could be the main reason for the impairment of cell and organism functioning. We have studied the effects of NAIDs on the lipid peroxidation using a hydrophilic initiator (AAPH) and fluorescence probes with different hidrophobicity properties. Fluorescein was used as a hydrophilic probe, DADF as a fluorescein derivative probe that have a lipophilic moisture and consequently the fluorescein part is located near the membrane surface, and DPH-PA as a hydrophobic probe that is located inside the lipid bilayer. The studies were performed in aqueous buffers and in liposomal systems trying to get information concerning to the NSAIDs scavenging activity to peroxyl radical and to the effect of their partition in lipid membrane. Results show that, to understand the effect of NSAIDs on lipid peroxidation it must be considered not only their scavenging activity but also the location of the drugs in the membrane and their interactions with the membrane surface. The 3-(4-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid (DPHPA) was also used as fluorescent probe to monitoring the antiperoxidation activity of the NSAIDs and the changes in membrane fluidity during the peroxidation. With the experimental assays developed it was possible to evaluate that NSAIDs can act by another synergic mechanism that involve changes on membrane fluidity and not only as free radical scavenging as the mechanism of protective action against peroxidative injury. We have also performed the study of the effect of NSAIDs on lipid peroxidation using a hydrophobic initiator (AMVN). The results obtained show that are a relation between the effect of the NSAIDs on the membrane fluidity and their protective effect on the lipid peroxidation. The studies of the effect of NSAIDs on their interaction with membrane enzymes that are usually involved in the inflammation processes were initiated. Phospholipase A2 (PLA2) plays an important role in the regulation of the phospholipid composition of biological membranes and in the release of physiologically and pathophysiologically important inflammation mediators as prostaglandines, leukotrienes and thromboxanes15. Although the structure of several PLA2s and their catalytic sites are known with atomic resolution, their mode of action on the larger scale of the membrane is not understood and one of the consequences is that their exact metabolic role also remains unresolved. Therefore, the investigation of the mechanism of the PLA2 enzymatic reaction and the search for opportunities to influence this process is of great interest for the therapy of inflammation and allergic diseases. In addition, drugs which are able of inhibiting PLA2 activity remain a potential target for the development of new drugs in the treatment of diseases like Alzheimer. Some NSAIDs like indomethacin, although regarded as cyclooxygenase inhibitors, also inhibit PLA2 but the precise mechanism by which indomethacin blocks the activity of PLA2 remains an unresolved issue. We have implemented to different methodologies (a spectrophotometric and a fluorimetric) to evaluate the Laboratório Associado para a Química Verde 7 7 PLA2 activity. We are also studding the effect of nature and composition of lipid matrix on the PLA2 activity using liposomes prepared with different kinds of phospholipids with or without cholesterol. Scavenging activity studies for reactive oxygen and nitrogen species by non-steroidal antiinflammatory drugs (NSAIDs), â-adrenergic antagonists and plant extracts Endogenously produced pro-oxidant reactive species are essential to life, being involved in several biological functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels of antioxidants become severely depleted, these reactive species become highly harmful, causing oxidative stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and cause cell death. The scientific research in the field of reactive oxygen species (ROS) and reactive nitrogen species (RNS) associated biological functions and/or deleterious effects is continuously requiring new sensitive and specific tools in order to enable a deeper insight on its action mechanisms. However, reactive species present some characteristics that make them difficult to detect, namely their very short lifetime and the variety of antioxidants existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies capable of overcoming this type of obstacles. Our group has been involved in the application of chemiluminiscent and fluorescent probes as sensors for ROS and RNS, with the objective of their use in scavenging assays. Once implemented, the methodologies have been subsequently applied in scavenging activity studies for ROS and RNS by non-steroidal anti-inflammatory drugs (NSAIDs), â-adrenergic antagonists and plant extracts, as briefly explained below. We have recently proposed that several NSAIDs may react with reactive oxygen species (ROS), namely peroxyl radical (ROO.), hydroxyl radical (HO.), superoxide radical (O2.-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl), and reactive nitrogen species (RNS), namely nitric oxide (.NO) and peroxynitrite (ONOO-) produced at sites of inflammation. This type of effect has potential therapeutic relevance since the antioxidant activity may strongly contribute for the final anti-inflammatory outcome to be attained by these compounds. The scavenging activity is being evaluated using non-cellular in vitro methodologies, as well as cellular systems, using Human neutrophils. The therapeutic effects of â-blockers are normally explained by their capacity to block the â-adrenoceptors, however, some of the beneficial cardiovascular effects shown by this group of compounds have already been associated with the antioxidant properties that some of them seem to possess. The â-blockers atenolol, labetalol, metoprolol, pindolol, propranolol, sotalol, timolol, and carvedilol were tested for their putative scavenging activity for ROS (O2.-, H2O2, HO., HOCl and ROO.) and RNS (.NO and ONOO-). Some of the studied compounds are effective ROS and/or RNS scavengers, these effects being possibly useful in preventing oxidative damage verified in hypertension as well as in other cardiovascular diseases that frequently emerge in association with oxidative stress. Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub with a reported wide range of healing properties. In Cuba, this species is traditionally used as a tincture in the treatment of stomatological infections, as stomatitis and gingivitis. A tincture from P. tithymaloides collected in Cuba has been evaluated for its in vitro scavenging effects on reactive oxygen species (ROS) (HOÿ, O2ÿ•, HOCl, ROOÿ and H2O2), reactive nitrogen species (RNS) (ONOO• and ÿNO). Laboratório Associado para a Química Verde 7 8 BIOINORGANIC AND MEDICINAL INORGANIC CHEMISTRY Head of Laboratory: Maria Rangel, Associate Professor Staff Members: Baltazar de Castro Full Professor Paula Gameiro Assistant Professor João Gomes Assistant Professor Ph.D. Students: Ana Claúdia Nunes Project Grantee: Andreia Daniela Leite, André Neto da Silva Number of articles in scientific journals: 1 (230) Design of orally active insulin-mimetic drugs. In order to compare the properties of complexes formed by multidentate and bidentate ligands we have initiated the synthesis of multidentate ligands derived from 3hydroxy-4-pyridinone type and their corresponding metal complexes with VO(IV) and Zn(II). The complexes formed are being characterized in the solid state and in solution. Evaluation of the insulin-like action of the synthesized complexes will be performed in collaboration with Prof. Hiromu Sakurai (Kyoto University). This work is being developed under contract POCI/QUI/56949/2004 (FCT, Portugal). Design of functionalized siderophores to target infection processes. The design of new chelators is crucial for treatment of diseases associated with iron overload and to deprieve bacteria and virus associated to infectious processes such as TUBERCULOSIS and AIDS, from iron which is one of its essential nutrients. The work that has been done in the current year and that will be pursued in the next one is focused on the design of novel molecules to target infection and act as intracellular fluorescent probes. The work can be summarized as the synthesis of functionalized units in order to produce macromolecules that can act as iron chelators and also as fluorescence probes. This work is being developed under contract POCI/QUI/56214/ 2004 (FCT, Portugal). Reactivity of B12 model compounds. The work in this project aims the establishment of structural factors that may be determinant on the reactivity of photolysis products of B12 model compounds. We have identified and characterized the photolysis products of cobaloximes and imino/oxime compounds with variable organic radicals and nitrogen and phosphorus bases. The results obtained evidence that the primary products are strongly dependent on the nature of the organic radical and are identical for the two different equatorial moieties. This work is being developed under contract POCTI/QUI/46231/2002 (FCT, Portugal). During the year of 2006 we will pursue the objectives of the above projects in which new compounds will be synthesized and characterized from the chemical and biological point of view. Laboratório Associado para a Química Verde 7 9 COMPUTATIONAL STUDY OF METAL SURFACES Head of Laboratory: José Ferreira Gomes, Full Professor Staff Members: Natália Cordeiro Associate Professor Post-Doct Fellows: Shuwen Yao Ph.D. Students: Hugo Santos, Akapong Suwattanamala M.Sc. Students: Ana Sofia Pinto Number of articles in scientific journals: 6 (231-236) We are particularly interested in understanding the physical and chemical behaviour of heterogeneous systems, looking in particular to the modelling of interfacial phenomena. The theoretical study of such systems and phenomena is interesting on its own and is of such difficulty that experimentalists find it very useful to complement their work with computational results; many of our studies are thus being carried out in close collaboration with experimentalists. We have been using quantum chemical methods, such as DFT, as well as molecular simulations techniques (MC and MD simulations). Representative of the studies performed in 2005 and to be pursued in 2006, follows: Modelling and reaction of species adsorbed on metal surfaces. We have been studying the interaction of methoxy radical with copper and ruthenium surfaces using a cluster model approach. The DFT results show that this approach gives a reasonable description of the surfaces allowing a good prediction of the structural and energetics features of the adsorbate. Furthermore, it shows that a reliable description of the infrared vibration spectra of adsorbed methoxy on the clean and low oxygen precovered Ru(0001) surfaces may be obtained. In addition, the preferential sites for the adsorption of OH (radical and anionic species) on the elemental gold surfaces, (111), (110) and (100), has been studied by the same approach. This work is now being extended to new reactions and different metals, namely to the study of the reactions pertaining to the methanol synthesis on single-crystal Cu and Ru surfaces as well as enantiomerically pure metal catalysts, which can transmit chirality information and thereby be applied in enantioselective synthesis/detection. In those studies, more realistic methods like periodic DFT methods will be applied. Modelling nanoporous materials. We have been applying molecular simulation methods (MC and MD), both at atomistic and at mesoscale levels, to study the structure and the synthesis of nanoporous materials. This research area is at its infancy in our laboratory. So far, we have applied Monte Carlo methods on a simple cubic lattice to model the spontaneous formation of silica nanoparticles during the early stages of zeolite synthesis. In spite of the simplicity of the model, it is able to describe the self-assembly of nanoparticles from clear solution, yielding results that are in close qualitative agreement with experimental observations. We have also begun to apply the above methods to study the synthesis of periodic mesoporous silica materials (e.g., MCM-41). The lattice model is being extended to cope with micelle formation from chain surfactants, Laboratório Associado para a Química Verde 8 0 which are precursors in the mesoporous silica synthesis. Molecular dynamics and Kinetic Monte Carlo will be employed to model micelle self-assembly in the presence of silica condensation. Recently, we have also started a collaboration with an experimental group, with the aim of developing a theoretical model for enantioselective catalytically active metal complexes grafted onto nanoporous supports. Information about the structure of the material, as well as the coverage and conformations of the complexes will be obtained from MC simulations. DFT methods will be used to model reactions catalysed by the immobilized complexes. Modelling and ion-transfer at liquid-liquid interfaces. Following our previous work, we have been using MD simulations to study liquid-liquid interfaces like the strongly associating liquid interfaces formed between water and a linear alcohol. Emphasis has been given to the effects of increasing the length of the alcohol chain on the structure and dynamics of such interfaces. Present work focuses on the study of other liquid interfaces currently being used in ion transfer experiments, such as those formed between water and 2-octanol, nitrobenzene (NB) or 2-nitrophenyloctyl ether (NPOE). Notice that 2-octanol is a chiral organic liquid which may specially favour the transfer of chiral ionic species. On the other hand, NPOE is quite interesting due to its low toxicity, which enables its use on large-scale industrial processes. We have recently completed a detailed comparative study of the pure and water-saturated NB and NPOE liquids, including thermodynamic and dynamic properties, as well as ion solvation properties. The simulation of the interfaces between water and these liquids is underway. Selected References: R Gulaboski, CM Pereira, MNDS. Cordeiro, I Bogeski, AF Silva, J. Solid State Electrochem., 2005, 9(6), 469474 R Gulaboski, CM Pereira, MNDS. Cordeiro, I Bogeski, E Ferreira, D Ribeiro, M Chirea, AF Silva, J. Phys. Chem. B, 2005, 109(25), 12549-12559 JE Rodrígez-Borges, X García-Mera, F Fernández, VHC Lopes, AL Magalhães, MNDS Cordeiro, Tetrahedron, 2005, 61(46), 10951-10957 M Jorge, SM Auerbach, PA Monson, J. Am. Chem. Soc., 2005, 127(41), 14388-14400 R Gulaboski, V Mirceski, CM Pereira, MNDS. Cordeiro, AF Silva, F Quentel, M L´Her, e M. Lovric, Langmuir, 2005, in press. Laboratório Associado para a Química Verde 8 1 NOVEL HETEROGENEOUS CATALYSTS Head of Laboratory: Ana Cristina Freire, Associate Professor / Baltazar de Castro, Full Professor Research Team: Eulália Pereira Assistant Professor Uwe Pischel Assistant Researcher Rita Ferreira Assistant Ana Rosa Silva Assistant Researcher Post-Doc Students: Pankaj Das, Iwona K-Biernacka, Iwona Kiersztyn, Peter Eaton, Sunita Arun Salunke, Elzbieta Skiba, Issam Oueslati Ph.D. Students: Magda Martins, Andrea Carneiro, João Tedim, Joana Fonseca, Adelaide Miranda, Marek Kluciar M.Sc. Students: Rosa Bessada, Miguel de Sousa, Bruno Jarrais, Jorge Teixeira, Tiago Borrego, Ana Isabel Cruz Project Grantee: Sónia Patrício, Frederico Calheiros Number of articles in scientific journals: 18 (237-254) Number of Ph.D thesis: 1 Number of Ms.C. thesis: 1 Novel catalysts by heterogenisation of metal complexes We pursued the preparation and characterisation of chiral manganese(III) salen complexes for immobilisation on new room temperature ionic liquids, activated carbons, carbon xerogels, new mesoporous aluminium pillared clays, clays and mesoporous silica-based materials. The catalytic activity of the new complexes in the asymmetric epoxidation of several alkenes (styrene, á-methylstyrene and 6-ciano-2,2-dimethylchromene) was evaluated in CH2Cl2 using NaOCl, m-chloroperoxybenzoic acid (m-CPBA) and PhIO (in CH3CN) as oxygen sources. The experiments were performed at low to room temperature and reaction time, the alkene conversion, chemical selectivity of products, respective yields and enantiomeric excesses (ee%) were determined by GC-FID. All the complexes showed catalytic activity in the experimental conditions used. The asymmetric epoxidation of 6-ciano-2,2-dimethylchromene by chiral manganese(III) salen complexes was performed in a series of new room temperature ionic liquids based on imidazolium and guanidinium cation using NaOCl, PhIO, H2O2 and H2O2.urea as oxygen sources. The new room temperature ionic liquids could be used as neoteric solvents for the 6-ciano-2,2-dimethylchromene asymmetric epoxidation, at room temperature; nevertheless, compared with the reaction performed on CH2Cl2 its shows a decrease in ee%, but could be reused up to 3 times, although with some decrease in ee% values. To understand and to model the membrane ûux and retention behaviour as a function of the solvent/catalyst/ membrane system and to optimise the integrated case-studies, preliminary studies on the nanofiltration of a manganese(III) salen complex were done. The SRNF membrane STARMEM 120 (Grace-Davison, USA) was chosen for this system. It has a MWCO of about 200 Da, and is stable for ethanol – water mixture. It was observed that catalyst rejection was higher than 95% for a 1 mM manganese complex solution, after processed by NF at different pressure values, which indicates that the catalyst is almost totally retained by the chosen membrane. Laboratório Associado para a Química Verde 8 2 Chiral Mn(III) salen complexes were anchored using several strategies developed in our lab onto several supports: activated carbons, carbon xerogels, mesoporous pillared clays (PILCs), clays (Laponite) and mesoporous silica materials (HMS, MCM-41 and FSM-16). All the materials were characterised by elemental analyses, XPS, XRD (inorganic materials), nitrogen adsorption at 77 K, temperature programmed desorption (carbon materials), FTIR (inorganic materials) and Uv-vis spectroscopies (inorganic materials). The catalytic properties in the asymmetric epoxidation of several alkenes of the new catalysts were determined using equivalent experimental conditions of those used in homogeneous phase. For these catalysts the leaching of the active phase and their reusabibility was evaluated. All the catalysts showed catalytic activity: the reaction times were larger than those observed in homogeneous phase, in some cases the alkene conversion decreased, as well as the ee%; in some cases ee% did not decrease with reuse. Copper(II) and vanadyl(IV) acetylacetones have also been heterogenised through Schiff condensation onto amine functionalised activated carbons, carbon xerogels, new mesoporous aluminium pillared clays, clays (Laponite and K10 montmorillonite) and hexagonal mesoporous silica (HMS). All the materials were characterised by elemental analyses, XPS, XRD, nitrogen adsorption at 77 K, termogravimetry, FTIR, Uv-vis spectroscopies and EPR. Their catalytic properties in the aziridination of styrene and epoxidation of allylic alcohols using tertbutylhydroperoxide as oxygen source, respectively, have been evaluated. All the new heterogeneous catalysts were active and could be reused in more catalytic cycles. Finally, 4-triethoxysilylaniline (TESA) was used as an organo-silane coupling reagent for surface functionalization of several inorganic materials with different textural properties: Laponite, K10 montmorillonite, silica gel, MCM-41 and FSM-16. All the materials were characterised by 13C and 29Si MAS NMR, FTIR and nitrogen elemental analysis. FUTURE WORK (1) Design and preparation of new chiral complexes based on the Schiff base ligands with other transition metals with homogeneous catalytic properties. (2) Determine the Reichardt’s e Kamlet-Taft polarity parameters of the new room temperature ionic liquids used as neoteric solvents for the asymmetric epoxidation of alkenes. Characterise the room temperature ionic liquids by XPS. (3) Explore the use of nanofiltration as a new method for the separation of homogeneous catalysts. (4) Anchoring/encapsulation of chiral Mn complexes onto several supports: mesoporous silica materials, mesoporous carbons, nanotubes, mesoporous PILCs and clays. (5) Homogeneous/heterogeneous catalytic reactions (epoxidation and aziridination of alkenes and epoxidation of allylic alcohols). For the heterogeneous catalysts, reutilisation of the catalysts will be also evaluated. (6) Use other organo-silane coupling reagents for surface functionalisation of several inorganic mesoporous matrixes. Chemosensors based on transition metal complexes We have pursed the preparation of nickel and copper complexes with Schiff base ligands functionalised with groups that can act as coordination sites for representative and lanthanide cations and for anions (halides, H2PO4-, HSO4-, NO3-) by methodologies developed in our laboratory. Several functionalities were introduced in the aldehyde moiety and in the diimine bridge: crown ether and pseudo crown ethers groups. These complexes were characterised by the usual techniques: elemental analyses, mass spectra, EPR, FTIR and UV-Vis spectroscopies, cyclic voltammetry. The sensing properties of the complexes were performed in several solvents, using cyclic voltammetry, UVvis; for the complexes that can polymerise and give stable films recognition studies were also done by cyclic voltammetry, in-situ FTIR and UV-Vis, electroacoustic impedance and electrochemical impedance. In solution, it was possible to determine equilibrium constants for the complexation of lanthanides and alkalineearth cations for the metal complexes functionalised with pseudo-crown groups by Uv-vis spectroscopy. Cyclic voltammetry allowed the semi-quantiative evalutation of the interaction of all the cations tested (+1, +2 and +3). For the complexes functionalised with crown ethers in the imine bridge, no interaction was detected Laboratório Associado para a Química Verde 8 3 by UV-Vis, since the complexation site does not influence the CT bands used to monitor the interaction between the complex and the cations. All the polymeric films adsorbed the metal cations tested (+1, +2), although they exhibited different behaviour: those with higher change induced higher changes in the electrochemical, UV-Vis and IR properties of the films. The coordination site was unambiguously identified as the crown ether or pseudo-crown ether by IR spectroscopy and XAS of Ni and Ba K-edge. The adsorption of cations by the polymeric films was also monitored by electrochemical quartz crystal microbalance: there was an increase in mass during the adsorption process and during reduction the cations were expelled into the solutions; this property was confirmed by the cyclic voltammetric studies of the films with adsorbed cations in free supporting electrolyte and confirm the films could be reuse in successive sensing experiments without loosing their electrochemical and chemical stability. FUTURE WORK (1) Design, preparation and characterisation of new complexes with sensing properties for cations and anions. (2) Preparation and characterisation of their corresponding films. (3) Evaluation of the association constants for the interaction of cations and anions with the complexes by new techniques: fluorescence, calorimetric titration and conductimetry (4) Study of the interactions of cations and anions with the polymeric films by new techniques: electrochemical quartz crystal microbalance, XAS and neutron reflectivity (5) Morphological characterisation of the films by SEM and AFM. (6) Cation and anion selectivity studies and for polymeric films reutilisation studies. Molecular materials with non-linear optical properties The synthesis of materials with non linear optical properties (NLO) is a current research area due to the potential application of these materials for the fabrication of high performance electro-optical switching elements for telecommunication and optical information processing. In this context, transition metal complexes, which show in some cases intrinsic NLO properties, are important building blocks for the preparation of material with this type of properties. During 2005 new nickel and copper complexes with salen type ligands functionalised with donor-acceptor groups, designated generically as [M(DA-salen)] were prepared. The new complexes were electropolymerised by methods developed in our group and the linear (UV-Vis) and 3d NLO properties of the compounds and films have been determined. We have started to prepare films with the complexes by using the self-assembly layerby-layer technique. The 3d NLO properties were evaluated using the z-scan technique, which is based on the self-focusing or defocusing of a converging beam of known direction (Nd:YAG laser, l=1064 nm). The technique permits the evaluation of non linear refractive index (n2) and the non linear absorption coefficient (b). The 3d NLO properties of the complexes, polymeric films and layer-by-layer films were measured. The complexes showed a great variation on their NLO properties; the differences were attributed to the donor/acceptor properties of the substituents within the ligand. The polymeric films show 3d NLO properties which have been strongly enhanced when compared to the monomers in solution, and are a result of the charge carriers generated during the oxidation of the polymeric films. The films prepared by layer-by-layer technique did not exhibit NLO properties due to their very thin thickness. FUTURE WORK (1) Design, preparation and characterisation of Pd and Pt complexes (2) Preparation and characterisation of their corresponding films. (3) Evaluation of linear (UV-Vis) and 3d NLO properties for Pd and Pt complexes and corresponding films. (4) Preparation and characterisation of new films by self-assembly layer-by-layer method. (5) Evaluation of 3d NLO properties for films prepared by self-assembly layer-by-layer method. Laboratório Associado para a Química Verde 8 4 Metallosurfactants. Deposition of monolayer and mutilayer films of the amphiphilic complexes [Fe(RR’bpy)2(CN)2] on ITO and mica was performed using the Langmuir-Blodgett technique, and the resulting modified surfaces were studied by UV/vis spectroscopy, cyclic voltammetry and AFM. In addition, the aggregation behavior of these compounds in methanol/water was studied by AFM in order to clarify the influence of the drying conditions on the morphology of the nanocapsules formed. Several novel ruthenium(II) and ruthenium(III) complexes with alkyl substituted bipyridine ligands were synthesized and characterized by the usual techniques (UV/vis, FTIR, NMR, FAB-MS, CV). Their aggregation behavior was studied in several different solvents, and solvent mixtures by SEM, AFM and DLS. The preliminary results obtained show that the morphology of the nanoparticles obtained is strongly dependent on solvent polarity. Ongoing and future work include further characterization of Langmuir-Blodgett films, including determination of Langmuir isotherms, and UV/vis, IR, CV and AFM studies. Metal Nanoassemblies. Application of a photocatalytic method to gold reduction enabled us to prepare gold sols with different nanoparticle morphology, namely triangular prisms, spheres and disks. TEM and AFM were used to study the growing process of the nanocrystals. The results obtained show that the morphology of the nanoparticles is strongly dependent on pH and amount of photocatalyst used. In addition, novel methods for the preparation of Pt and Pd nanoparticles with catalytic applications were developed using a similar approach. Homogeneous suspensions of 2-5 nm nanoparticles were prepared. The method is being modified in order to obtain larger nanoparticles. Immobilization of this nanoparticles in solid supports (graphite) was also used, but in this case the nanoparticles obtained showed a very broad size distribution. Ongoing studies aim the optimization of the method in order to minimize size dispersion and to understand the main factors controlling the morphological characteristics of nanoparticles. Application of nanoparticles to biological systems: A novel method for the detection of gene expression was developed in collaboration with Prof. Pedro Baptista and Prof. Ricardo Franco (FCT-UNL). The method is based on the different aggregation properties of gold nanoparticles derivatized with a specific oligonucleotide as the probe, in the presence or absence of the complementary sequence. We have used AFM to study the nanoparticle-probe-DNA interactions. Interestingly, hybridizing the DNA plasmids in the presence of gold nanoparticle-probe leads to extremely large concatemeric structures forming from individual DNA molecules. The synthesis of other nanoparticles with appropriate physical properties for detection and/or separation of DNA plasmids, namely FexOy/Au is in progress. The preliminary results obtained show that the method used is successful for the preparation of mixed FexOy/Au. Nevertheless the particles show a broad size distribution and irregular distribution of gold at the surface of the iron oxide core. The synthetic method is being optimized in order to obtain homogeneous size distribution and capping of the iron oxide core. Future developments include optimization of the synthetic procedures, microscopy characterization of the nanopartcicles (TEM and AFM), determination of the magnetic properties (Mossbauer, SQUID), and development of a new method for detection and removal of specific DNA plasmids from biological samples. Photoactive Compounds and Solid-State Materials The research activities in the year 2005 can be divided in four main projects: (a) development and photophysical characterization of novel lanthanide-calixarene complexes (published in 2006); (b) development of novel molecular logic gate functions by using phthalimide-terbium conjugates (published in 2006); (c) synthesis and characterization of bistable molecular fluorescent switches with aromatic dicarboximides (published in 2005); (d) investigation of energy transfer phenomena in organic-inorganic hybrid materials doped with europium (collaboration with Prof. Carlos, CICECO University of Aveiro). Furthermore, two more works related to the investigation of stereoselective phenomena in excited states were published in collaboration with Prof. Miranda in Valencia. Laboratório Associado para a Química Verde ANNEXES 2005 A - 2 ANNEX I RESEARCH TEAM Laboratório Associado para a Química Verde A - 3 A MEMBERS OF STAFF Laboratório Associado para a Química Verde A - 4 Alberto Sundaresan Prabhakar Ana Maria F. T. Lobo Baltazar Manuel Romão de Castro Fernando J. S. Pina Hugo Gil Ferreira Isabel Maria A. M. G. de Moura Jose Alberto Nunes Ferreira Gomes José J. G. de Moura Jose Luis Fontes Costa Lima Luis F. G. Sousa Lobo Manuel M. Nunes da Ponte Maria Lurdes Souteiro Bastos Maria Conceição B. S. M Montenegro Pedro Brito Correia Abel José de Sousa Costa Vieira Alberto Manuel Carneiro Sereno Alberto Nova Araujo Ana Cristina Moreira Freire Aquiles J. F.de Araújo Barros Duarte José da Costa Pereira João Paulo S. Goulão Crespo Jose F. M. Magalhâes Cardoso Karin Tonnies Gil Ferreira Maria Conceição S. S. Rangel Maria João Ribeiro Nunes Ramos Maria João L. R. M. C. Romão Maria Natália Dias Soeiro Cordeiro Maria Pilar Figueroa Gonçalves Maria Teresa Barros Silva Rosa Maria Moreira Seabra Pinto Rui Alexandre Santos Lapa Susana Filipe Barreiros Teresa Maria Fonseca de Moura Cristina Maria Delerue Alvim Matos Maria Leonor Oliveira Madureira Pinto Maria Carmo Veiga Fernandes Vaz Rui Alves Agostinho Almiro Almeida Alberta Paula Gameiro Santos Alexandre Lopes Magalhães Ana I. N. M. Aguiar Ricardo Ana M. F. C. Lourenço Ana Maria Martelo Ramos Andre Alberto Sousa Melo Ângela M. S. Relva António Gil de Oliveira Santos António Jorge Parola Carlos S. S. Pereira Lima Eduarda Graças Rodrigues Fernandes Elvira Maria M. Gaspar Eulália Fernanda Carvalho Pereira Eurico José da Silva Cabrita Felix Dias Carvalho Fernando Manuel Gomes Remião Helena Maria Neto Ferreira Helena Maria Vieira Monteiro Soares Henrique J. R. Guedes Isabel Borges Coutinho de Medeiro Isabel Maria Ligeiro da Fonseca Isabel Maria Viegas Oliveira Ferreira Isabel Maria Rola Coelhoso João Carlos S. B. Sotomayor João Carlos Lima João Luis Machado Santos João M. Aires de Sousa João Paulo C. Noronha Professor Catedrático Professora Catedrática Professor Catedrático Professor Catedrático Professor Catedrático Professora Catedrática Professor Catedrático Professor Catedrático Professor Catedrático Professor Catedrático Professor Catedrático Professora Catedrática Professora Associada Professor Catedrático Convidado Professor Associado Professor Associado Professor Associado Professora Associada Professor Associado Professor Associado Professor Associado Professor Associado Professora Associada Professora Associada Professora Associada Professora Associada Professora Associada Professora Associada Professora Associada Professora Associada Professor Associado Professora Associada Professora Associada Professora Coordenadora Professora Coordenadora Professora Coordenadora Professor Coordenador Professor Auxiliar Professora Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professora Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Laboratório Associado para a UNL UNL FCUP UNL UNL UNL FCUP UNL FFUP UNL UNL FFUP FFUP UNL UNL FEUP FFUP FCUP FCUP FCUP UNL ICBAS-UP UNL ICBAS-UP FCUP UNL FCUP FEUP UNL FFUP FFUP UNL UNL ISEP ISEP ISEP IPVC FFUP FCUP FCUP UNL UNL UNL FCUP UNL UNL UNL UNL FFUP UNL FCUP UNL FFUP FEUP FFUP FEUP UNL UNL UNL FFUP UNL UNL UNL FFUP UNL UNL Química Verde A - 5 Joaquim Silvério Marques Vital Jorge M. P. Lampreia Pereira José António Maia Rodrigues Jose Augusto Caldeira Pereira Jose Oliveira Fernandes José Paulo Barbosa Mota Luís Guilherme L. Ferreira Guido Luisa M. S. P. Ferreira Luísa Maria S. Vieira Peixe Marco Diogo R.Gomes da Silva Maria Alice S. Pereira Maria Ascenção Reis Maria Beatriz Prior Pinto Oliveira Maria Beatriz Guerra Junqueiro Maria Cristina O. Costa Maria D’Anjos L. Macedo Maria Gabriela Teles Cepeda Ribeiro Maria João Melo Maria José Feio Maria Lúcia Sousa Saraiva M. Madalena A.C.S.Dionísio de Andrade Maria Manuela M. A.Pereira Maria Margarida C.M. A. Cardoso Maria Salete Reis Dias Rodrigues Maria Teresa Avilés Perea Marco Diogo Silva Miguel Freire de A. Cabral Olivia Maria de Castro Pinho Patrícia Carla Ribeiro Valentão Paula Cristina Branquinho Andrade Paula Cristina S. Branco Paulina M. E. N. Mata Paulo Joaquim Ferreira Almeida Pedro António Brito Tavares Pedro Jorge Macedo Abreu Pedro Manuel Alexandrino Fernandes Pedro Miguel Calado Simões Ricardo Franco Rui Manuel Freitas Oliveira Susana Isabel Pereira Casal Ermelinda Manuela Jesus Garrido Jorge Manuel Pinto Jesus Garrido Alexandra Bernardo Adriana Martins Pimenta Carla Manuela Soares Matos Catarina Isabel Guerra Rodrigues Cristina Maria C. Morais Couto Ivone Valente Oliveira João Luís Tavares de Matos Gomes Francisco Jorge Fernandes Caldeira Lígia Maria da Silva Rebelo Gomes Luísa Lima Gonçalves Maria Alexandra Bernardo Maria Gabriela Almeida Maria da Graça Soveral Rodrigues Maria Helena Reis Prado de Castro Paula Cristina Gerónimo Rita Isabel Lemos Catarino Sara Isabel Xavier Candeias Stephane Besson Ana Maria Philips Maria Fernanda Cabral Adriano Fachini Agostinho Pereira Ana Bicho dos Santos Ana Luísa Carvalho Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Convidada Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Professor Auxiliar Convidado Professor Auxiliar Professor Auxiliar Professor Auxiliar Assistente Professora Adjunta Professor Adjunto Professora Associado Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Professor Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professora Auxiliar Professor Auxiliar Investigadora Principal Investigadora Principal Investigador Auxiliar Investigador Auxiliar Investigadora Auxiliar Investigadora Auxiliar Laboratório Associado para a UNL UNL FCUP ICBAS-UP FFUP UNL FCUP UNL FFUP UNL UNL UNL FFUP FFUP UNL UNL FCUP UNL FCUP FFUP UNL UNL UNL FFUP UNL UNL FFUP FCNAUP FFUP FFUP UNL UNL FCUP UNL UNL FCUP UNL UNL UNL FFUP ISEP ISEP ISCS UFP UFP ISCSN ISCSN ISCSN UFP ISCSS ISCSN ISCSS ISCSS ISCSS FFUL ISCSN UFP UFP U. Lusófona U. Lusófona UNL FCUP REQUIMTE REQUIMTE REQUIMTE REQUIMTE Química Verde A - 6 Ana Rosa Silva Carlos Lodeiro Espino Isabel Mafra João Pedro Lopes Marcela Alves Segundo Maria Manuel Marques Marta Andrade Peter Eaton Loïc Hilliou Owen Catchpole Serguey Bursakov Svetlozar Velizarov Uwe Pischel Manuel Rui Azevedo Alves Maria Isabel Branco Alves Martins Maria Teresa de Oliva Teles Moreira Ana Claro Helena Carmo Abel José Assunção Duarte Florinda F. Martins Hendrikus Petrus Antonius Nouws João Velho Prior Maria de Fátima de Sá Barroso Maria Goreti Ferreira Sales Maria Isabel Brito Limpo de Serra Maria João D. Ramalhosa Ferreira Maria Manuela Barbosa Correia Micaela de Sousa Olga Manuela Matos de Freitas Rita Isabel Simões Ferreira Simone Barreira Morais Salomé de Sousa Teixeira Sónia Adriana R. C. Figueiredo Valentina Maria FernandesDomingues Maria Elisa A. M. Fernandes Soares Eulalia Maria Bernardino Mendes Maria Isabel Afonso da Rocha Maria Isabel Almeida Cardoso José Tomás Soares de Albergaria Paula Paíga Carla Rodrigues Cecília Bonifácio Cristina Réu João Fraga João Rodrigo da Silva Santos Nelson Brito Pedro Assunção Rui Viegas Maria Aurora Soares da Silva Sérgio Cruz Monteiro de Morais Investigadora Auxiliar Investigador Auxiliar Investigadora Auxiliar Investigador Auxiliar Investigadora Auxiliar Investigadora Auxiliar Investigadora Auxiliar Investigador Auxiliar Investigador Auxiliar Investigador Auxiliar Investigador Auxiliar Investigador Auxiliar Investigador Auxiliar Professor Coordenador Professora Adjunta Professora Adjunta Assistente Convidada Assistente Assistente Assistente Assistente Assistente Assistente Assistente Assistente Assistente Assistente Assistente Convidada Assistente Assistente Assistente Assistente Assistente Assistente Assessora Principal Assessora Principal Assessora Assessora Técnico Técnica Téc. Sup. 2.ª Classe Téc. Sup. 2.ª Classe Téc. Sup. 2.ª Classe Téc. Informática Téc. Sup. 2.ª Classe Téc. Informática Téc. Informática Téc. Sup. 2.ª Classe Encarregada de trabalhos Encarregado de trabalhos REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE REQUIMTE IPVC ISEP ISEP UNL FFUP ISEP ISEP ISEP FFUP ISEP ISEP ISEP ISEP ISEP UNL ISEP IPVC ISEP ISEP ISEP ISEP FFUP FFUP FCUP FFUP ISEP ISEP REQUIMTE REQUIMTE FFUP REQUIMTE REQUIMTE REQUIMTE UNL REQUIMTE ISEP ISEP KEY UNL FCUP FFUP FCNAUP ISEP ISCSN UFP Universidade Nova de Lisboa Universidade do Porto - Faculdade de Ciências Universidade do Porto - Faculdade de Farmácia Universidade do Porto - Fac.Ciências da Nutrição Instituto Superior de Engenharia do Porto Instituto Superior de Ciências de Saúde (Norte) Universidade Fernando Pessoa FFUL FEUP ICBAS-UP UAl IPVC ISCSS U. Lusófona Universidade de Lisboa - Faculdade de Farmácia Universidade do Porto - Faculdade de Engenharia Universidade do Porto - Inst. Ciências Biomédicas Universidade do Algarve Inst. Politécnico de Viana do Castelo Instituto Superior de Ciências de Saúde (Sul) Universidade Lusófona Laboratório Associado para a Química Verde A - 7 B POST-DOCTORAL FELLOWS Laboratório Associado para a Química Verde A - 8 Adrian Oehmen Berta Covelo Cristina Timóteo Damián Fernández Elzbieta Skiba Ewa Bogel Lukasik Françoise Auchère Filomena Freitas Gilda Carvalho Isabel Esteves Issam Oueslati Iwona Biernacka Iwona Kiersztyn José Castanheiro José Trincão Krasimira Petrova Krisztina Istvan Luísa Serafim Ludwig Kripahal Mário Gomes Marta Eiroa Marta Isabel Silva Patrícia Sousa PauloLemos Pankas Das Pedro Rodrigues Sofia Pauleta Quingyou Zhang Raquel Fortunato Raquel Otero Ricardo Mendonça Roeland Boer Rui Duarte Rui Ruivo Shabir Najmudin Shuwen Yao Snezhana Bakalova Sonia Dopico Sunil Gupta Sunita Salunke Susan Matthews Svetlana Lyubchik Svetlozar Velizarov Teresa Casimiro Vesna Nasdanovic-Visak Vitor Alves Yonh Hong Liu Yuri Binev Laboratório Associado para a Química Verde A - 9 C Ph. D. STUDENTS Laboratório Associado para a Química Verde A - 10 Adelaide Miranda Akapong Suwattanamala Alexandra Carvalho Alexandra Marques Alexandre Carvalho Ana Freitas Ana Gomes Ana Nunes Ana Martins Ana Teixeira André Araújo André Pinheiro Andrea Carneiro Andreia Filipa Curto António Nunes Bárbara Ribeiro Bruno Tamames Juan Tamames Carina Machado Carla Brazinha Carla Portugal Carla Silva Carlos Martins Carlos Pinheiro Carlota Macedo Célia Amorim Christophe Siquet Claudia Galinha Cristina Alves Cristina Cordas Cristina Matos Cristina Vieira Daniel Dourado David Costa Diogo Latino Emília Santos Ema Alves Elizabete Machado Eunice Rodrigues Fábio Larotonda Fátima Lucas Filipe Duarte Filipe Folgosa Francisco SIlva Gabriela Rivas Gonçalo Carrera Graça Albuquerque Helena Ferreira Helena Pontes Hugo Oliveira Hugo Santos Inês Gomes Isabel Esteves Irina Moreira Joana Amaral Joana Ferreira Joana Fonseca Joana Raimundo João Lourenço João Dias João Fernandes João Capela João Tedim Jorge Dias José dos Santos José Marques Karine Marques Laila Ribeiro Leticia Giestas Liliana Guerreiro Luís Magalhães Luis Lopez Luís Pinto Magda Martins Márcia Guilherme Márcio Temtem Marco Preto Marek Kluciar Margarida Moncada Maria João Morgado Maria Luisa Soares Silva Maria Teresa Viciosa Mariana Costa Marieta Passos Mário Eusébio Marlene Lucio Marta Filipa Ribeiro Miguel Faria Miguel Lopes Mónica Matos Nuno Cerqueira Oriza Tavares Pablo Gonzalez Patrícia Antunes Patrícia Neves Patricia Oliveira Patrícia Raleiras Paula Rodrigues Paula Gomes Pinto Paula Silva Paulo Glória Pedro Manuel Santos Pedro Maia Pedro Vidinha Rafal Lukasik Ricardo Branco Ricardo Oliveira Rita Noronha Rute Fonseca Sara Cristina Cunha Sandra Quinteira Sérgio Alves Sérgio Sousa Sónia Garcia Sofia Barata Sofia Costa Lima Sofia Gomes Susana Pereira Susana Gaudêncio Teresa Santos Silva Valdemar Figueira Vasco Bonifácio Vera Costa Victor Rosa Vineet Pande Wancheng Sittikijyothin Zenaida Mourão Laboratório Associado para a Química Verde A - 11 D M. Sc. STUDENTS and other research students Laboratório Associado para a Química Verde A - 12 M. Sc. Students Aida Reis Alexandra Soares Ana Castro Ana Filipa Gomes Ana Isabel Cruz Ana Maria Gomes Ana Sofia Pinto Aúrea Roxo Branca Teixeira Bruno Jarrais Carla Barbosa Célia Sousa Claúdia Nunes Cristina Silvestre Cristina Soares Daniel Osório Daniel Ribeiro Eva Cunha Filipa Grosso Jorge Páscoa Jorge Teixeira Lúcia Maia Luís Correia Maria Resende Marisa de Freitas Marta Sofia Pires Miguel Sousa Renata Silva Ricardo Mendes Rosa Bessada Sergio Teixeira Sílvia Barros Sónia Mendes Tiago Borrego Other Research Students Adriana Key Aldino Viegas Américo Duarte Ana Brás Ana Fins Ana Lopes André Neto da Silva Andrea Mestre Andreia Barateiro Andreia Leite Ângela Machado Artur Abreu Bruno Pedras Carlota Macedo Catarina Coelho Catarina Soares Célia Silveira Celina Santos Cristiano Mota Duarte Alves Filipe Freire Frederico Calheiros João Dias Lídia Barata Maria Baleizão Marisa Silva Marta Vilela Miriam Sousa Natércia Brás Patrick Nunes Sara Berguet Sónia Patrício Ricardo Alves Ricardo Couto Ricardo Pais Rui Tostões Simonne dell’Acqua Vanessa Cabral Laboratório Associado para a Química Verde A - 13 E Profiles of the new researchers contracted under the Associate Laboratory Program Laboratório Associado para a Química Verde A - 14 Dr. Peter Eaton studied chemistry at the University of York, UK and obtained his BSc in 1994. He then obtained an MSc in Polymer Technology from Loughborough University, before beginning his PhD at the Materials Research Institute, Sheffield Hallam University. His doctorate title was “ATR-FTIR Spectroscopy and Raman Microscopy Studies of Organosilane Diffusion and Hydrolysis on PVC Films”. Since 1998 Peter has been using Atomic Force Microscopy(AFM). Firstly, at the University of Portsmouth, UK, he developed novel surface analysis techniques with the AFM; including adhesion force and nanoindentation mapping.These novel techniques were applied to a variety of samples, including ultralow surface energy polymers. Then he moved to the IIQ, Seville, Spain, where he used AFM to study glyconanoparticle aggregation, and began to study the interactions between nanoparticles and DNA. This work has been continued since 2004 in the REQUIMTE at the department of chemistry, Porto, Portugal, where he is working as an associate researcher and also using the AFM to study a wide variety of chemical and biological systems including electroactive polymer films, organometallic vesicle structure, protein binding by biosensors and model proteoliposome surfaces. Dr. Ana Rosa Silva started her research work in her late undergraduate Chemistry studies (1995) and Master (1996/1998) at LAQUIPAI (FCUP) synthesising transition metal complexes with biologically important ligands and characterising their spectroscopic and magnetic properties. Then, in her Ph.D. (1998/2002) at CEQUP (FCUP) besides of synthesising and characterising transition metal complexes with Schiff base ligands, she anchored these transition metal complexes onto activated carbons, using several methodologies, and applied them as successful heterogeneous catalysts in the epoxidation of alkenes. The Post-Doctoral research work (2003/2005) at CEQUP (FCUP) was focused on the heterogenisation of chiral homogeneous catalysts using activated carbon, mesoporous hexagonal silica and room temperature ionic liquids for the heterogeneous asymmetric epoxidation of alkenes. During this period she visited for 9 months the Green Chemistry Group/Clean Technology Centre of the University of York (2004/2005) where she also became interested in the use of renewable resources as supports and the use of microwave technology in Chemistry. In September of 2005 joined REQUIMTE and her current research interests are the synthesis of new chiral homogeneous catalysts, mainly transition metal complexes with Schiff base ligands, and the development of sustainable chemical catalytic processes by heterogenisation of homogeneous catalysts in several solid supports (activated carbons, mesoporous carbons, carbon nanotubes, mesoporous silicas, clays, pillared clays), by using non-conventional solvents and by using nanofiltration membranes for separation and recycling of homogeneous catalysts. Dr. Marta Morais Saraiva de Andrade obtained her degree in Organic Chemistry in 1999 at the Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa. In 1997 carried out a research period in the Institut für Organische Chemie der Universität Wien (Viena, Austria). Since 2003/2004 she is an Invited Assistant in the Structural Organic Chemistry Group. In 2005 she obtained a doctoral degree in Chemistry, specialization in Organic Chemistry, from the Universidade Nova de Lisboa. Since December 2005 she is an Assistant Researcher at REQUIMTE - Laboratório Associado - Centro de Química Fina e Biotecnologia. Her main research interests are selective organic synthesis, with special attention to the development of new strategies in green organic chemistry. She has published, as main author/co-author, 2 papers in international scientific periodicals with referees and presented 2 oral communications by invitation and 9 posters in scientific meetings. She has co-supervised undergraduate students. At the moment, she is involved, as a team research member, in two different research projects, namely the development of carbohydrates-based chiral auxiliaries as green raw materials for new methodologies in Organic Synthesis and in the synthesis, complexation and biodegradation studies of chiral chelators for industrial and domestic application. These studies are directed toward the development of cleaner (greener) organic chemistry. Laboratório Associado para a Química Verde A - 15 Dr. Rui O. Duarte, born in Lisbon, 1968, graduated in 1991 on Applied Chemistry (Biotechnology) from the Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (FCT/UNL). He completed his PhD thesis in 2002 in Inorganic Chemistry, titled “Proteínas contendo Molibdénio e Tungsténio”. From 1998 to 2002, he was Invited as Assistant in the Departamento de Química e Bioquímica of Universidade do Algarve. From 2002 to 2005 he had a post-doctoral position in the BioVanadium research group, in collaboration with the Bioquímica Física de Proteínas group headed by Prof. Isabel Moura (FCT/UNL). He is currently Invited Assistant Professor in Departamento de Química (FCT/UNL) and has recently (2005) joined the Associated Lab Requimte as an Assistant Researcher. Within the present research interests are the isolation and characterization of metalloproteins, the use of molecular biology techniques for cloning and overexpression and characterization of novel Iron-Sulfur centers by Electron Paramagnetic Resonance (EPR) and Mössbauer spectroscopies. He also has a special interest in spectroscopic studies applied to medical and biological problems. He is the responsible person for the Bruker X-band EPR spectrometer located at FCT/UNL. Dr. Ulrich Scheven obtained a bachelors degree in Physics at the University of California at Berkeley in 1989, a masters degree in Physics from Princeton University in 1991, and a Ph.D. degree in Physics from Princeton University in 1995. His experimental Ph.D. research focussed on the electronic properties of low dimensional organic metals at cryogenic temperatures, in high magnetic fields. After a two year post-doctoral appointment at Princeton, during which he continued work with organic metals, he joined Schlumberger Doll Research in Ridgefield, Connecticut, to work in Schlumberger's NMR group doing research relevant to the oilfield services industry. This led to in the development of novel measurement techniques. Later on he pursued NMR studies of fluid flow and dispersion in porous media, first in in collaboration with Professor Cory's group at MIT in Boston and, after moving to Schlumberger's laboratory in Cambridge (UK), in collaboration with members of the Magnetic Resonance Research Centre at Cambridge University. At MIT and Cambridge he has cosupervised Ph.D. and postdoctoral researchers. Dr. Scheven has (co-)authored 14 publications, presented at numerous conferences, and is co-inventor on two patents. In December 2005 he joined REQUIMTE, where he intends to use NMR techniques to study non-linear spatiotemporal phenomena associated with flow (non-Newtonian / multiphase) and reactions in porous media, such as rocks or packed bed rectors employed in heterogeneous catalysis. Dr. João Almeida Lopes joined the REQUIMTE (Laboratório Associado) in December 2005 as assistant researcher in chemometrics and process spectroscopy. He graduated in Chemical Engineering (biotechnology branch) in 1997 (Instituto Superior Técnico). He completed his PhD in Biotechnology in the area of chemometrics in the pharmaceutical industry in 2001 working in chemometrics modelling of industrial pharmaceutical production processes (Instituto Superior Técnico). Since then, he consolidated the development and application of chemometrics and spectroscopy for systems identification in the chemical, biochemical and pharmaceutical fields working as post-doc researcher at Instituto Superior Técnico. In 2004, he and three co-workers at the time in the Center for Biological and Chemical Engineering (CEBQ) of IST were awarded with the first prize in the entrepreneurship contest Bioempreendedor (http://www.bioempreendedor.com) where a FTIR spectroscopy/ chemometrics based methodology was proposed for rapid and cost effective identification of human bacteria pathogens. Vibrational spectroscopy (Near infrared, Mid infrared and Raman spectroscopy) in biotechnology has been there since one of his major research interests. Current research activity is focused on the development and application of chemometrics and vibrational spectroscopy systems in the context of green-chemistry (non-destructive, non-invasive, reagent-free): 1) pharmaceutical processes and pharmaceutical quality control, 2) analytical methods development and optimization, 3) biochemical, biological and environmental systems monitoring and 4) improved microrganisms identification for infection control. Laboratório Associado para a Química Verde A - 16 ANNEX II PUBLICATIONS Laboratório Associado para a Química Verde A - 17 A Scientific Articles —— Book Chapters —— Articles in Procedings Laboratório Associado para a Química Verde A - 18 Articles in Scientific Journals 1. “Ultra fast trypsin digestion of proteins by high intensity focused ultrasound” D. Lopez-Ferrer; JL Capelo; J. Vazquez Journal of Proteome Research, 2005, Vol. 4, 1569-1574 2. “Micro-focused ultrasonic solid-liquid extraction (mu FUSLE) combined with HPLC and fluorescence detection for PAHs determination in sediments: optimization and linking with the analytical minimalism concept” J.L. Capelo, M.M. Galesio, G.M. Felisberto, C. Vaz and J. Costa Pessoa Talanta, 2005, Vol.66, 1272-1280 3. “Focused ultrasound versus microwave digestion for the determination of lead in must by electrothermalatomic absorption spectrometry” J.L.Capelo; S. Catarino; A.S. Curvelo-Garcia and M. Vaiao Journal of AOAC International, 2005, Vol. 88, 585-591 4. “Discussion of parameters associated with the ultrasonic solid-liquid extraction for elemental analysis (total content) by electrothermal atomic absorption spectrometry. An overview” J.L. Capelo; C. Maduro; C. Vilhena Ultrasonics Sonochemistry, 2005, Vol. 12, 225-232 5. “Effect of zeolites on lipase catalyzed esterification in nonaqueous media” C. Peres; N. Harper; M. D. R. Gomes da Silva; S. Barreiros Enzyme and Microbial Technology, 2005, Vol. 37 145-149 6. “Cutinase activity in supercritical and organic media: water activity, solvatation and acid-base effects” S. Garcia; P. Vidinha; H. Arvana; M. D. R. Gomes da Silva; M.O. Ferreira; J.M.S. Cabral; E.A. Macedo; N. Harper; S. Barreiros The Journal of Supercritical Fluids, 2005, Vol. 35, 62-69 7. “Ultrasonication for Analytical Chemistry” J.L. Capelo and A. Mota Current Analytical Chemistry, 2005, Vol. 1, 193-20 8. “A Zinc(II)-based receptor for ATP binding and hydrolysis” C. Bazzicalupi; A. Bencini; A. Bianchi; A. Danesi; C. Giorni; C. Lodeiro; F. Pina; S. Santarelli; B. Valtancoli Chem. Commun. 2005, 2630-2632 9. “A New Zn(II) Chemosensor Based on a Tweezer Pyridine-Naphthalene System. An Off-On-Off System Working in a Biological pH window” R. Aucejo; J. Alarcón; E. García-España; J. M. Llinares; K. L. Marchin; C. Soriano; C. Lodeiro; M. A. Bernardo; F. Pina; J. Pina; J. S. Melo Eur. J. Inorg. Chem. 2005, 4301-4308 10. “Spectroscopy and Coordination Chemistry of a New Tetranucleating Tetra(fluorophoric) Ligand Displaying Sensing Ability for Metal Cations” J. Pina; J. S. de Melo; F. Pina; C. Lodeiro; J. C. Lima; A. J. Parola; M. P. Clares; Mª T. Albelda; C. Soriano; R. Aucejo; E. García-España Inorg. Chem., 2005, 44, 7449-7458 Laboratório Associado para a Química Verde A - 19 11. “New Fluorescence PET System Based on N2S2 Pyridine-Anthracene-Containing Macrocyclic Ligands. Spectrophotometric, Spectrofluorimetric, and Metal Ion Binding Studies” A. Tamayo; C. Lodeiro; L. Escriche; J. Casabó; B. Covelo; P. González Inorg. Chem., 2005, 44, 8105-8115 12. “Write-Read-Erase Molecular Switching System Trapped in a Polymer Hydrogel Matrix” F. Galindo; J. C. Lima; S. V. Luis; A. J. Parola; F. Pina Adv. Funct. Mat., 2005, 15, 541-545 13. “Water/humidity, and ammonia sensor, based on a polymer hydrogel matrix containing a fluorescent flavylium compound” F. Galindo; J. C. Lima; S. V. Luis; M. J. Melo; A. J. Parola; F. Pina J. Mater. Chem., 2005, 15, 2840 – 2847 14. “Bio-Inspired Multistate Networks Responsive to Light, pH and Thermal Inputs. An Example of a Multistate System Operating Through Different Algorithms” L. Giestas; F. Folgosa; J. C. Lima; A. J. Parola; F. Pina Eur. J. Org. Chem., 2005, 4187-4200 15. “One-dimensional fluorescent stacking structure based on zinc mixed-complex salt encapsulated within an ‘ice-like’ three-dimensional hydrogen-bonded water network” R. Carballo; B. Covelo; C. Lodeiro; E. M. Vazquez-López Cryst Eng. Commun., 2005, 7(48) 294-296 16. “Self-Assembly of a Hydrophobically Modified Naphthalene-Labeled Poly(acrylic acid) Polyelectrolyte in Water:Organic Solvent Mixtures Followed by Steady-State and Time-Resolved Fluorescence” T. Costa; M. G. Miguel; B. Lindman; K. Schillen; J. C. Lima; J. Seixas de Melo J. Phys. Chem. B., 2005, 109, 3243-3251 17. “Charge-Transfer Complexation as a General Phenomenon in the Copigmentation of Anthocyanins”P. F. da Silva; J. C. Lima; A. A. Freitas; K. Shimizu; A. L. Maçanita; F. H. Quina J. Phys. Chem. A, 2005, 109, 7329-7338 18. “Identification of a Novel Small-Molecule Inhibitor of the Hypoxia-Inducible Factor 1 Pathway” C. Tan; R. G. de Noronha; A. J. Roecker; B. Pyrzynska; F. Khwaja; Z. Zhang; H. Zhang; Q. Teng; A. C. Nicholson; P. Giannakakou; W. Zhou; J. J. Olson; M. M. Pereira; K.C. Nicolaou; E. G. V. Meir Cancer Research, 2005, Vol. 65, 605-612 19. “Total Synthesis of Halipeptins: Isolation of Halipeptin D and Synthesis of Oxazoline Halipeptin Analogues” K. C. Nicolaou; D. Schlawe; D. W. Kim; D. A. Longbottom; R. G. de Noronha; D. E. Lizos; R. Rao Manam; D. John Faulkner Chemistry European Journal, 2005, Vol. 11, 6197–6211 20. “Total Synthesis of Halipeptins A and D and Analogues” K. C. Nicolaou; David W. Kim; D. Schlawe; D. E. Lizos; R. G. de Noronha; D: A. Longbottom Angewandte Chem. Int. Ed., 2005, Vol. 44, 2–7 21. “New Enantioselective Method for Hydration of Alkenes Using Cyclodextrins as Phase Transfer Catalyst” A. R. Abreu; I. Costa; C. Rosa; L. M. Ferreira; A. Lourenço; P. P. Santos Tetrahedron, 2005, Vol. 61, 11986-11990 Laboratório Associado para a Química Verde A - 20 22. “Estimation of melting points of pyridinium bromides ionic liquids with decision trees and neural networks” G. Carrera; J. Aires-de-Sousa Green Chemistry, 2005, Vol. 7, 20-27 23. “Prediction of Enantiomeric Excess in a Combinatorial Library of Catalytic Enantioselective Reactions” J. Aires-de-Sousa; J. Gasteiger J. Comb. Chem., 2005, Vol. 7, 298-301 24. “Automatic assignment of absolute configuration from 1D NMR data” Q.-Y. Zhang; G. Carrera; M. J. S. Gomes; J. Aires-de-Sousa J. Org. Chem., 2005, Vol. 70, 2120-2130 25. “Prediction of enantioselectivity using chirality codes and Classification and Regression Trees” S. Caetano; J. Aires-de-Sousa; M. Daszykowski; Y. Vander Heyden Anal. Chim. Acta, 2005, Vol. 544, 315-326 26. “Structure-based classification of chemical reactions without assignment of reaction centers” Q.-Y. Zhang; J. Aires-de-Sousa J. Chem. Inf. Model., 2005, Vol. 45, 1775-1783 27. “Antioxidant Phenolic Glycosides from Moricandia arvensis” H. Braham, Z. Mighri, H. Ben Jannet, S. Matthew, P. M. Abreu J. Nat. Prod., 2005, Vol 68, 517-522 28. “New Ceramides from Rantherium suaveolens” M. H. Oueslati, H. Ben Jannet, Z. Mighri, P. M. Abreu Lipids, 2005, Vol 40, 1075-1079 29. “High-Performance Liquid Chromatographic Determination of Glycoalkaloids in Potatoes from Conventional, Integrated, and Organic Crop Systems” P. Abreu, A. Relva, S. Matthew, Z. Gomes, Z. Morais Food Control, 2005, (in press, doi:10.1016/j.foodcont.2005.08.005) 30. “The Cahn, Ingold and Prelog System: eliminating ambiguity in the comparison of diastereomorphic and enantiomorphic ligands” P. Mata; A. M. Lobo Tetrahedron: Asymmetry, 2005, 16, 2215-2223 31. “Phosphorus Pentoxid”’ S. M. P. Gaudêncio Synlett, 2005, 16, 2545-2546 32. “Antimony Pentachloride” Paulo M. C. Glória Synlett, 2005, 9, 1486-1487 33. “Stereoselective Synthesis of Optically Active Mono and Diaminoalcohols” M. T. Barros; M. Adilia Charmier, Christopher D. Maycock and Thierry Michaud, Tetrahedron, 2005, 61, 7960-7966 Laboratório Associado para a Química Verde A - 21 34. “Novel chiral bis(oxazolines): synthesis and application as ligands in the copper-catalyzed enantioselective conjugate addition of diethylzinc to enones” M. T. Barros, C. D. Maycock, and A. M. Faísca Phillips Tetrahedron: Asymmetry, 2005, vol. 16, 2946-2953 35. “Contrasting behavior in azide pyrolyses: an investigation of the thermal decomposition of methyl azidoformate, ethyl azidoformate and 2-azido-N, N-dimethylacetamide by ultraviolet photoelectron spectroscopy and matrix isolation infrared spectroscopy” J.M. Dyke, G. Levita, A. Morris, J.S. Ogden, A.A. Dias, M. Algarra, J.P. Santos, M.L. Costa, P. Rodrigues, M. Andrade, M.T. Barros Chem. Eur. J., 2005, vol. 11, 1665-1676 36. “Intermolecular Interactions as Investigated by NOE and Diffusion Studies” T. Brand; E. J. Cabrita; S. Berger Progress in Nuclear Magnetic Resonance, 2005, v46, 159-196 37. “A Novel Pentacyclic Triterpene from Leontodon filii” Z. Tostão, J. P. Noronha; E. J. Cabrita; J. Medeiros; J. Justino; J. Bermejo; A. P. Fitoterapia, 2005, 76, 173-180 38. “New Chiral Auxiliaries for Dynamic Kinetic Resolutions. From Theory to Practice” A. G. Santos; J. Pereira; C.A.M. Afonso; G. Frenking Chem. Eur. J., 2005, 11, 330-343 39. “ESR and ENDOR Investigations on Various Wurster’s Radical Cations in Solution. Experimental Results, Theoretical ab initio, and DFT Calculations” Grampp, G.; Kelterer, A-M.; Landgraf, S.; Sacher, M.; Niethammer, D.; Telo, J.P., Dias, R.M.B.; Vieira A.J.S.C., Monathshefte für Chemie, 2005, 136, 519-536 40. “A new dihydroxysterol from the marine phytoplankton Diacronema sp. (Haptophyta)” Tostão, Z.; Noronha, J.P.; Cabrita, E.J.; Medeiros, J.; Justino, J; Bermejo, J., Rauter, A.P. Fitoterapia, 2005, 76, 433-438 41. “Solubility of the nematic liquid crystal E7 in supercritical carbon dioxide” ARE Bras; T Casimiro; J Caldeira; A Aguiar-Ricardo Journal of Chemical and Engineering Data, 2005, 50, 1857-1860 42. “Heterogeneous Polymerisation of Diethylene Glycol Dimethacrylate in Supercritical Carbon Dioxide” T Casimiro; AM Banet-Osuna; AMRamos; M Nunes da Ponte; A Aguiar-Ricardo European Polymer Journal, 2005, 41, 1947-1953 43. “Phase behavior studies of a perfluoropolyether in high-pressure carbon dioxide” T Casimiro; A Shariati; CJ Peters; M Nunes da Ponte; A Aguiar-Ricardo Fluid Phase Equilibria, 2005, 228, 367-371 44. “Hydration of alpha-pinene over molybdophosphoric acid immobilized in hydrophobically modified PVA membranes” JE Castanheiro; IM Fonseca; AM Ramos; R Oliveira; J Vital Catalysis Today, 2005, 104, 296-304 Laboratório Associado para a Química Verde A - 22 45. “Thermophysical and thermodynamic properties of ionic liquids over an extended pressure range: [bmim][NTf2] and [hmim][NTf2]” RG Azevedo; JMSS Esperanca; J Szydlowski; JP Visak; PF Pires; HJR Guedes; LPN Rebelo Journal of Chemical Thermodynamics, 2005, 37, 888-899 46. “Thermophysical and thermodynamic properties of 1-butyl-3-methylimidazolium tetrafluoroborate and 1-butyl-3-methylimidazolium hexafluorophosphate over an extended pressure range” RG Azevedo; JMSS Esperanca; V Najdanovic-Visak; ZP Visak; HJR Guedes; M Nunes da Ponte; LPR Rebelo Journal of Chemical and Engineering Data, 2005, 50, 997-1008 47. “Mathematical modelling of a mixed culture cultivation process for the production of polyhydroxybutyrate” JML Dias; LS Serafim; PC Lemos; MAM Reis; R. Oliveira Biotechnology and Bioengineering, 2005, 92, 209-222 48. “Glass transition dynamics of poly(L-lactic acid) during isothermal crystallisation monitored by realtime dielectric relaxation spectroscopy measurements” M Dionisio; MT Viciosa;YM Wang; et al Macromolecular Rapid Communicationd, 2005, 26, 1423-1427 49. “Sonocatalysis and Alkaline Doped Carbons. An Efficient Method for the Synthesis of Chalcones in Heterogeneous Media” CJ Durán-Valle; IM Fonseca; V Calvino-Casilda; M Picallo; AJ López-Peinado; RM Martín-Aranda Catalysis Today, 2005, 107-108, 500-5006 50. “Automatic filtering and reodorization of adsorbed natural gas storage systems” IAAC Esteves; MS Lopes; MP Nunes, PM; et al Adsorption-Journal of the International Adsorption Society, 2005, 11: 905-910 Suppl 51. “Biodegradation of thiomersal containing effluents by a mercury resistant Pseudomonas putida strain” R Fortunato; JG Crespo; MAMReis Water Research, 2005, 39, 3511-3522 52. “Stability of supported ionic liquid membranes as studied by X-ray photoelectron spectroscopy” R Fortunato;CAM Afonso;J Benavente; JG Crespo Journal of Membrane Science,2005, 256, 216-223 53. “Preparation and catalytic testing of sulfonic acid functionalized activated carbons” l. Guerreiro; I Fonseca; RM Martin-Aranda; AM Ramos;AM Botelho do Rego; JVital Phosphorus Sulfur and Silicon and the Related Elements, 2005, 180 1485-1486 54. “Enantioselective separation of propranolol by chiral activated membranes” T Gumi; C Palet; Q Ferreira; JG Crespo; IM Coelhoso; C Palet Separation Science and Technology, 2005, 40 773-789 55. “Enhanced esterification conversion in a room temperature ionic liquid by integrated water removal with pervaporation” P Izak; NMM Mateus; CAM Afonso; JG Crespo Separation and Purification Technology, 2005, 41, 141-145 Laboratório Associado para a Química Verde A - 23 56. “Ternary-phase equilibria for CO2+3-methyl-1-butanol+2-phenylethanol” JA Lopes; MN da Ponte Journal of Supercritical Fluids, 2005, 34, 189-194 57. “Deviations from ideality in mixtures of two ionic liquids containing a common ion” JNC Lopes; TC Cordeiro; JMSS Esperanca; HJR Guedes; S Huk;LPN Rebelo; K Seddon Journal of Physical Chemistry B, 2005, 109 : 3519-3525 58. “Biphasic hydrogenation of alpha-pinene in high-pressure carbon dioxide” A Milewska; AMB Osuna; IM Fonseca; M. Nunes da Ponte Green Chemistry, 2005, 7, 726-732 59. “Single-column simulated-moving-bed process with recycle lag” JPB Mota; JMM Araujo Aiche Journal, 2005, 51, 1641-1653 60. “Molecular simulation of gas separation by equilibrium-based adsorption processes” JPB Mota; IAAC Esteves; RCR Rodrigues; et al. Adsorption-Journal of the International Adsorption Society, 2005, 11 319-324 . 61. “Oxidation of limonene over carbon anchored transition metal Schiff base complexes: Effect of the linking agent” P Oliveira; AM Ramos;I Fonseca; A Botelho do Rego; J Vital Catalysis Today, 2005, 102: 67-77 62. “Direct integration of pervaporation as a sample preparation method for a dedicated “electronic nose” C Pinheiro; T Schafer; JG Crespo Analytical Chemistry, 2005, 77, 4927-4935 63. “Sorption of aroma compounds in poly (octylmethylsiloxane) (POMS)” T Schafer; A Heintz; JG Crespo Journal of Membrane Science, 2005, 254, 259-265 64. “Elucidating interactions of ionic liquids with polymer films using confocal Raman spectroscopy” T Schafer; RE Di Paolo; R Franco; JG Crespo Chemical Communications, 2005, 2594-2596 65. “Dynamic model of a supercritical carbon dioxide heat exchanger” PC Simoes;J Fernandes; JP Mota Journal of Supercritical Fluids, 2005, 35, 167-173 66. “Modelling the enantioselective hydrolysis of a meso-diester using pig liver esterase in a two-phase hollow fibre reactor” HA Sousa; CAM Afonso;JPB Mota; JG Crespo Chemical Engineering Research & Design, 2005, 83, 285-294 67. “Removal of inorganic charged micropollutants in an ion-exchange membrane bioreactor” S Velizarov;C Matos; MAM Reis; JG Crespo Desalination, 2005, 178, 203-210 Laboratório Associado para a Química Verde A - 24 68. “Changes in molecular dynamics of the tri-ethylene glycol dimethacrylate monomer upon polymerization” MT Viciosa; CM Rodrigues; M Dionisio Journal of Non-Crystalline Solids, 2005, 351, 14-22 69. “Non-mechanistic modelling of complex biofilm reactors and the role of process operation history” G Wolf;JS Almeida; MAM Reis; JG Crespo Journal of Biotechnology, 2005, 117, 367-383 70. “Structural Characterization of Single-Walled Carbon Nanotube Bundles by Experiment and Molecular Simulation” S Agnihotri; JPB Mota; M Rostam-Abadi; MJ Rood Langmuir 2005, 21, 896-904 71. “Accessing Gelling Ability of Vegetable Proteins using Rheological and Fluorescence Techniques” AP Baptista; CAM Portugal; I Sousa;JG Crespo; A Raymundo Int. Journal of Biological Macromolecules, 2005, 36, 134-143 72. “Characterization of a Nematic Mixture and its Components by Reversed-Phase HPLC and UV Spectroscopy Analysis: an Application to Phase Behavior Studies in Liquid Crystal-CO2 Systems” Brás, A.R. E.; Henriques, S; Casimiro, T; Aguiar-Ricardo A.; Sotomayor J., Caldeira J., Santos C., Dionísio M Electronic Liquid Crystals Communications, Online 2005 73. “N2O Conversion Using Manganese Binary Mixtures Supported on Activated Carbon” SA Carabineiro; f Brás Fernandes; JS Vital;AM Ramos;. IM Fonseca Applied Catalysis B, 2005, 59, 185-190 74. “Microbial Population Response to Changes of the Operating Conditions in a Dynamic Nutrient Removal Sequencing Batch Reactor” F Freitas; M Temudo; MAM Reis Bioprocess And Biosystems Engineering 2005, 28, 3-8 75. “Evidence For Lower Critical Solution Behavior In Ionic Liquid Solutions” J Lachwa; J Szydlowski; J Najdanovic-Visak; LPN Rebelo; KR Seddon; M Nunes da Ponte; JMSS Esperança; HJR Guedes Journal American Chemical Society, 2005, 127, 6542-6543 76. “Optimization of the Conditions of Cr (III) Adsorption on the Activated Carbon” SB LyubchiK; I Perepichka.; OL Galushko; AI Lyubchik; ES Lygina; IM Fonseca Adsorption, 2005, 11, 581-593 77. “Removal of bromate, perchlorate and nitrate from drinking water in an ion exchange membrane bioreactor” CT Matos; S Velizarov; JG Crespo; MAM Reis Water Science And Technology :Water Supply, 2005, 5, 9-14 78. “Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation” R Oliveira; JJ Clemente;AE Cunha; MJT Carrondo J. Biotechnol., 2005, 116, 35-50 Laboratório Associado para a Química Verde A - 25 79. “Deuterium Isotope Differences In 2-propanone, (CH3)2CO/(CD3)2CO: A High-Pressure Sound-Speed, Density, and Heat Capacities Study” J Szydlowski; R Gomes de Azevedo; LPN Rebelo;JMSS Esperança; HJR Guedes J. Chem. Thermodyn, 2005, 37, 7, 671-683 80. “Modelling and optimisation of a recombinant BHK-21 cultivation process using hybrid grey-box systems” A Teixeira; H Cruz; A Cunha; J Clemente; P Alves; MJT Carrondo; R Oliveira J. Biotechnol., 2005, 118, 290-303 81. “Modelling of the Extractive Membrane Bioreactor Process Based on Natural Fluorescence Fingerprints and Process Operation History” G Wolf;JS Almeida; MAM Reis; JG Crespo Water Science And Technology, 2005, 51, 51-58 82. “Rotational Mobility in a Crystal studied by Dielectric Relaxation Spectrocopy. An Experiment for the Physical Chemistry Laboratory” MSC Dionísio; HP Diogo; JPS Farinha; J Moura-Ramos, J. Chem. Edu, 2005, 32, 1335-1339 83. “Clean osmium-catalyzed asymmetric dihydroxylation of olefins in ionic liquids and supercritical CO2 product recovery” Luís C. Branco; Ana Serbanovic; Manuel Nunes da Ponte; Carlos A. M. Afonso Chem. Comm., 2005, 107-109 84. “Liquid membranes using ionic liquids: the influence of water on solute transport” Raquel Fortunato; Maria Jesus González-Muñoz; Monika Kubasiewicz; Susana Luque; J. R. Alvarez; Carlos A. M. Afonso; Isabel M. Coelhoso; João G. Crespo Journal of Membrane Science, 2005, 249, 153–162 85. “Osmium catalyzed asymmetric dihydroxylation of methyl trans-cinnamate in ionic liquids, followed by supercritical CO2 product recovery” Ana Serbanovic; Luis C. Branco; Manuel Nunes da Ponte and Carlos A.M. Afonso, J. Organometallic Chemistry, 2005, 690, 3600-3608 86. “Purification and characterisation of tetraheme cytochrome c3 and adenylylsulfate reductase from the peptidolytic sulfate_reducing bacterium desulfovibrio aminophilus DSM 12254” A.L. Cortés, S. Bursakov, A. Figueiredo, A.E. Thapper, S. Todorovic, J.J.G. Moura, B. Ollivier, I. Moura and G. Fauque Bioinorg Chem Applic, 2005, 3, 81-91 87. “Synechocystis ferredoxin/ferredoxin – nadp+ reductase/nadp+ complex: structural model obtained by nmr-restrained docking” P.N. Palma, B. Lagoutte, L. Krippahl, J.J.G. Moura, F. Guerlesquin FEBS, 2005,579, 4585-4590 88. “Study of the spin – spin interactions between the metal centers of desulfovibrio gigas aldehyde oxidoreductase: identification of the reducible sites of the [2fe-2s]1+,2,+ clusters” C. More, M. Asso, G. Roger, B. Guigliarelli, J. Caldeira, J.J.G. Moura and P Bertrand Biochemistry, 2005, 44, 11628-35 Laboratório Associado para a Química Verde A - 26 89. “Superoxide reductase from the syphilis spirochete treponema pallidum: crystallization and structure determination using soft x-rays” T. S. Santos, J. Trincão, A. Carvalho, C. Bonifácio, F. Auchere, I. Moura, J.J.G. Moura and M.J. 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Bastos Toxicology Letters, 2005, 158S: S76 183. “Are reactive oxygen species (ROS) involved in transcriptional repression induced by sodium arsenate in bladder cancer (ECV 304) cell line?” R. Pires-Das-Neves; M. L. Pereira; F. Carvalho; M. L. Bastos; F. J. Iborra Toxicology Letter, 2005,158S: S173 184. “Detection of GSH adducts of catecholamines in biological samples” R. Silva; V. Costa; S. Boldt; H. Carmo; F. Carvalho; M. Carvalho; R. Carvalho; M. L. Bastos; F. Remião Toxicology Letters, 2005, 158S: S189 185. “Influence of sodium depletion on the kinetics of paraquat in the isolated rat lung” R. J. D. Oliveira; M. J. Valle; M. L. Bastos; F. Carvalho; A. S. Navarro Toxicology Letters, 2005, 158S: S213 186. “Acute paraquat poisoning: report of a survival case following intake of a potential lethal dose” R. J. D. Oliveira; A. M. Sarmento; P. Reis; A. Amaro; F. Remião; M. L. Bastos; F. Carvalho Toxicology Letters, 2005, 158S: S241 187. “E-learningand risk communication. Report of an experience at the Faculty of Pharmacy of the University of Porto” F. Remião; H. Carmo; F. Carvalho; M. L. Bastos Toxicology Letters, 2005, 158S: S76 188. “Effect of the NADPH oxidase inhibitior, apocynin, on systolic blood pressure and on vascular antioxidant defenses in hypertensive rats with activation of the renin-angiotensin system” T. Sousa; M. Morato; D. Pinho; E. Fernandes; F. Carvalho; A. Albino-Teixeira Journal of Hypertension, 2005, 23, S361: P3.305 189. “Voluntary fatal intoxication with inorganic mercury. Evaluation of the distribution of the metal in several autopsy samples” P. Triunfante; M.E. Soares; M.L. Bastos Toxicology Letters, 2005, 158S: S169 Laboratório Associado para a Química Verde A - 35 190. “The Drinking of a Salvia officinalis Infusion Improves Liver Antioxidant Status in Mice and Rats” C. F. Lima; P. Andrade; R. Seabra; M. Fernandes-Ferreira; C. Pereira-Wilson J of Ethnopharmacology, 2005, vol 97, 383-389 191. “Acute Toxicity of Widely Used Pharmaceuticals in Aquatic Species: Gambusia holbrooki, Artemia parthenogenetica and Tetraselmis chuii” B. Nunes; F. Carvalho; L. Guilhermino Ecotoxicology and Environmental Safety , 2005, vol 61(3), 413-419 192. “Scavenging of Nitric Oxide by an Antagonist of Adenosine receptors” T. Sousa; E. Fernandes; C. Nunes; J. Laranjinha; F. Carvalho; D. Pinho; M. Morato, A. Albino-Teixeira Journal of Pharmacy and Pharmacology, 2005, vol 57, 399–404 193. “Characterization and Use of the Total Head Soluble Cholinesterases from Mosquitofish (Gambusia holbrooki) for the Assessment of SDS Anticholinesterasic Activity“ B. Nunes; F. Carvalho; L. Guilhermino Journal of Enzyme Inhibition and Medicinal Chemistry ,2005, vol 20(4), 369–376 194. “Do Metals Inhibit Acetylcholinesterase (AChE)? Implementation of Assay Conditions for the Use of AChE Activity as a Biomarker of Metal Toxicity” M. F. Frasco; D. Fournier; F. Carvalho; L. Guilhermino Biomarkers, 2005, vol 10(5), 360-375 195. “A Collection of Papers Presented at the 30th International Congress of Theoretical Chemists of Latin Expression, Porto, Portugal, 8-12 September 2004- Foreword” M. J. Ramos; A. L. Magalhães; A. S, Melo; P. A. Fernandes Journal of Molcular Structure – Teochem, 2005, Vol.729, 1-2 196. “Computational Insight into Anti-mutagenic Properties of CYP1A Flavonoid Ligands” R. Fonseca, M. Marini, A. Melo, M. C. Menziani e M. J. Ramos Medicinal Chemistry, 2005, Vol.1, 355-360 197. “Molecular Dynamics Simulations of Angiotensin II in Aqueous and Dimethyl Sulfoxide Environments” M. A. C. Preto; A. Melo; H. L. S. Maia; T. Mavromoustakos; M. J. Ramos Journal of Physical Chemistry B, 2005, Vol. 109, 17743-17751 198. “Computer Modeling and Research in the Classroom” M. J. Ramos; P. A. Fernandes Journal of Chemical Education, 2005, Vol.82 (7); 1021-1025 199. “Density-Functional Calculations of the Cu, Zn Superoxide Dismutase Redox Potential: The Influence of Active Site Distortion” R. J. F. Branco; P. A. Fernandes; M. J. Ramos Journal of Molecular Structure: Theochem, 2005, Vol. 729, 141–146 200. “Cu, Zn Superoxide Dismutase: Distorted Active Site Binds Substrate without Significant Energetic Cost” R. J. F. Branco; P. A. Fernandes; M. J. Ramos Theoretical Chemistry Accounts, 2005, DOI: 10.1007/s00214-005-0672-x Laboratório Associado para a Química Verde A - 36 201. “Overview of Ribonucleotide Reductase Inhibitors: An Appealing Target in Anti-1Tumor Therapy” N. M. F. S. A. Cerqueira; S. Pereira; P. A. Fernandes; M. J. Ramos Current Medicinal Chemistry, 2005, Vol.12, 1283-1294 202. “Accuracy of the Numerical Solution of the Poisson–Boltzmann Equation” I. S. Moreira; P. A. Fernandes; M. J. Ramos Journal of Molecular Structure: Theochem, 2005, Vol. 729, 11–18 203. “Theoretical Study on the Inhibition of Ribonucleotide Reductase by 2 -Mercapto-2 -Deoxyribonucleoside5 -Diphosphates” S. Pereira; P.A. Fernandes; M. J. Ramos Journal of the Americal Chemical Society, 2005, Vol.127; 5174-5179 204. “Unraveling the Mechanism of the Farnesyltransferase Enzyme” S. F. Sousa; P. A. Fernandes; M. J. Ramos J. Biol. Inorg. Chem., 2005, Vol. 10, 3-10 205. “Farnesyltransferase: Theoretical Studies on Peptide Substrate Entrance Thiol or Thiolate Coordination?” S. F. Sousa; P. A. Fernandes; M. J. Ramos Journal of Molecular Structure: Theochem, 2005, Vol. 729, 125–129 206. “Farnesyltransferase – New Insights into the Zin-Coordination Sphere Paradigm: Evidence for a Carboxylate-Shift Mechanism” S. F. Sousa; P. A. Fernandes; M. J. Ramos Biophysical Journal, 2005, Vol. 88, 483-494 207. “New Designs for Inhibitors of the NF-kappa B: DNA Binding” P. A. Fernandes; A. R. R. Maia; A. A. S. Almeida; B.F.B.Silva; C.M.S.Ribeiro; C.F.B.Ribeiro; D.S.M. Ribeiro; D.A.P.Fonseca; E.S.Henriques; E.M.S.Cunha; F.R.N.C.Maia; J.A.A.Pereira; J.P.G.Pacheco; J.A.A.D.Ferreira; L.R.C.Matos; M.A.B.P.Pinto; M.C.S.Borges; P.J.C.R.Magalhães; P.F.R.D.Teixeira; P.N.B.C.Veloso; R.J.F.Ferreira; S.S.Gomes; T.F.Barros; T.S.J.T.Selão; V.Pande; V.M.M.C.Fernandes; M. J. Ramos Theoretical Chemistry Accounts, 2005, Vol.113; 197-204 208. “Discovery of a Large Number of Previously Unrecognized Mitochondrial Pseudogenes in Fish Genomes” A. Antunes; M. J. Ramos Genomics, 2005, Vol.86; 708-717 209. “Theoretical Quantitative Structure-Activity Relationships of Flavone Ligands Interacting with Cytochrome P450 1A1 and 1A2 Isozymes” F. Iori; R. Fonseca; M. J. Ramos Bioorganic Medicinal Chemistry, 2005, Vol.13; 4366-4374 210. “Theoretical Study of the Suicide Inhibition Mechanism of the Enzyme Pyruvate Formate Lyase by Methacrylate” M. F. Lucas; M. J. Ramos Journal of the Americal Chemical Society, 2005, Vol.127; 6902-6909 211. “Molecular Recognition of 15-deoxy-Delta(12,14)-Prostaglandin J(2) by Nuclear Factor-kappa B and other Cellular Proteins” V. Pande; M. J. Ramos Bioorganic & Medicinal Chemistry Letters, 2005, Vol. 15; 4057-4063 Laboratório Associado para a Química Verde A - 37 212. “NF-kB in Human Disease: Current Inibitors and Prospects for De Novo Structured Based Design of Inhibitors” V. Pande; M. J. Ramos Current Medicinal Chemistry, 2005, Vol.12; 357-374 213. “Structural Basis for the GSK-3b Binding Affinity and Selectivity Against CDK-2 of 1-(4Aminofurazan3yl)-5-Dialkylaminomethyl- 1-H-[1,2,3] Triazole-4-Carboxylic Acid Derivatives” V. Pande; M. J. Ramos Bioorganic & Medicinal Chemistry Letters, 2005, Vol. 15; 5129-5135 214. “Inhibitory Activity of Polyhydroxycarboxylate Chelators Against Reconbinant NF-kappa B p50 ProteinDNA Binding” R. K. Sharma; V. Pande; M. J. Ramos; H. K. Rajor; S. Chopra; K. Meguro; J. Inoue; M. Otsuka Bioorganic Chemistry, 2005, Vol.33; 67-81 215. “Density-Functional Study of Mechanisms for the Cofactor-Free Decarboxylation Performed by Uroporphyrinogen III Decarboxylase” M. J. Ramos; P. J. Silva Journal of Physical Chemistry B, 2005, Vol.109; 18195-18200 216. “Energy Partitioning in Association Processes” A. R. F. Carvalho; A. Melo International Journal of Quantum Chemistry, 2005, Vol.104, 240-248 217. “Assessing the Taxonomic Status of the Palawan Pangolin Manis Culionensis (Pholidota) Using Discrete Morphological Characters” P. Gaubert; A. Antunes Journal of Mammalogy, 2005, Vol.86, 1068-1074 218. “Interaction between quinolones antibiotics and bacterial outer membrane porin OmpF” P. Neves, E. Berkane, P. Gameiro, M. Winterhalter, B. de Castro Biophys. Chem., 2005, Vol. 113, 123-128 219. “Surfactant removal for reconstitution of ATPase activity prtoteoliposomes of mouse MDR3 P-glycoprotein” Sofia A.C. Lima, Anabela Cordeiro-da-Silva, Paula Gameiro, Baltazar de Castro Anal. Biochem., 2005, Vol. 338, 350-353 220. “Antioxidant properties of prepared blueberry (Vaccicium myrtillus) extracts” Faria A. Oliveira J., Neves P., P. Gameiro, Santos-Buelga C., Freitas V. Mateus N J. Agricul. Food Chem., 2005, Vol. 53, 6896-6902 221. “Fluorescence probes used for detection of reactive oxygen species” A. Gomes; E. Fernandes; J. L.F.C. Lima J. Biochem. Biophys. Meth., 2005, Vol. 65, 45-80 222. “Sensitive sequential injection determination of naproxen based on interaction with b-cyclodextrin” E. P. Zisiou; P. C.A.G. Pinto; M. L. M. F. S. Saraiva; C. Siquet; J. L.F.C. Lima Talanta, 2005, Vol. 68, 226-230 Laboratório Associado para a Química Verde A - 38 223. “Evalution of the total antioxidant capacity by using a multipumping flow system with chemiluminescent detection” S. R.P. Meneses; K. L. Marques; C. K. Pires; J. L.M. Santos; E. Fernandes; J. L.F.C. Lima; E. A.G. Zagatto Anal. Biochem., 2005, Vol. 345, 90-95 224. “Structure-property studies on the antioxidant activity of flavonoids present in diet” S. Teixeira; C.e Siquet; C. Alves; I. Boal; M. P. Marques; F. Borges; J. L.F.C. Lima; S. Reis Free Rad. Biol. Med., 2005, Vol. 39, 1099-1108 225. “Pindolol is a potent scavenger of reactive nitrogen species” E. Fernandes; A. Gomes; D. Costa; J. L.F.C. Lima Life Sci., 2005, Vol. 77, 1983-1992 226. “Effects of diclofenac on EPC liposome membrane properties” H. Ferreira; M. Lúcio; J. L.F.C. Lima; C. Matos; S. Reis Anal. Bioanal. Chem., 2005, Vol. 382, 1256-1264 227. “Interaction of clonixin with EPC liposomes used as membrane models” H. Ferreira; M. Lúcio; J.L.F.C. Lima; C. Matos; S. Reis J. Pharm. Sci., 2005, Vol. 94, 1277-1287 228. “Hydrogen peroxide scavenging activity by non-steroidal anti-inflammatory drugs” D. Costa; A. Gomes; S. Reis; J.L.F.C. Lima; E. Fernandes Life Sciences, 2005, Vol. 76, 2841-2848 229. “Effect of anti-inflammatory drugs on splenocyte membrane fluidity” H. Ferreira; M. Lúcio; J. L.F.C. Lima; A. Cordeiro-da-Silva; J. Tavares; S. Reis Anal. Biochem., 2005, Vol. 339, 144-149 230. “Photolysis secondary products of cobaloximes and imino/oxime compounds controlled by steric hindrance imposed by the Lewis base” M. Rangel; A. Leite; J. Gomes; B. Castro Organometallics, 2005, 24, 3500-3507 231. “A New Strategy to Model the Si(100) Surface” H. R. R. Santos; M. J. Ramos; J. A.N. F. Gomes Comptes Rendus Chimie, 2005, Vol. 8, 1461–1468 232. “Adsorption of Protected and Unprotected Amino-Cyclopentene at the Si(100) Surface Modeled with a Hybrid Quantum Mechanical Cluster Technique” H. R. R. Santos; M. J. Ramos; J. A. N. F. Gomes Physical Review B, 2005, Vol. 72; 075445 233. “Comparative Study of Novel Sulfonylcalix [4] Arene Derivative Compounds” A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes Journal of Molecular Structure: Theochem, 2005, Vol.729, 83-90 234. “Computational Study of calix [4] Arene Derivatives and Complexation with Zn2+” A. Suwattanamala; A. L. Magalhães; J. A. N. F. Gomes Chemical Physics, 2005, Vol.310, 109-122. Laboratório Associado para a Química Verde A - 39 235. “Enzymatic Formation of Ions and their Detection at a Three-Phase Electrode” R. Gulaboski; C. M. Pereira; M. N. D. S. Cordeiro; et al. Journal of Solid State Electrochemistry, 2005, Vol.9; 469-474 236. “Electrochemical Study of Ion Transfer of Acetylcholine Across the Interface of Water and a LipidModified 1,2-Dichloroethane” R. Gulaboski; C. M. Pereira; M. N. D. S. Cordeiro; et al. Journal of Physical Chemistry B, 2005, Vol. 109; 12549-12559 237. “Catalytic Properties of a Mn(III) Salen Complex Immobilised in a Pillared Clay by Simultaneous Pillaring/ Encapsulation Procedures” B. Cardoso; J. Pires; A. P. Carvalho; M. B. Carvalho; I. Kuzniarska-Biernacka; A. R. Silva; C. Freire; B. de Castro Eur. J. Inorg. Chem., 2005, 837-844 238. “Synthesis of Ferrocenyldiimine Metal Carbonyl Complexes and an Investigation of the Mo Adduct Encapsulated in Cyclodextrin” Z. Petrovski; S. S. Braga; S. S. Rodrigues; C. C. L. Pereira; I. S. Gonçalves; M. Pillinger; C. Freire; C. C. Romão New J. Chem., 2005, Vol. 29, 347-354 239. “Copper(II) Acetylacetonate Anchored onto an Activated Carbon as a Heterogeneous Catalyst for the Aziridination of Styrene” A. R. Silva; J. L. Figueiredo; B. de Castro; C. Freire Cat. Today, 2005, Vol. 102-103C, 154-159 240. “Styrene Epoxidation Catalysed by Manganese(III) Salen Complex Supported onto Activated Carbons” M. Cardoso; A. R. Silva; B. de Castro; C. Freire Apply Cat. A, General, 2005, Vol. 285, 110-118 241. “Synthesis and Characterisation of Salen-Type Ligands Functionalised with Pyrrole Derivative Pendant Arms” M. Andrade; C. Sousa; J. E. Borges; C. Freire J. Phys. Org. Chem., 2005, Vol.18, 935-940 242. “Chiral Manganese(III) Schiff Base Complexes Anchored onto Activated Carbon as Enantioselective Heterogeneous Catalysts for Alkene Epoxidation” A. R. Silva; V.y Budarin; J. H. Clark; B. de Castro; C. Freire Carbon, 2005, Vol. 43, 2096-2105 243. “Spectroelectrochemical Characterisation of Copper(II) Salen-Based Modified Electrodes” M. Martins; M. Vilas Boas; C. Freire; A. R. Hillman Electrochimica Acta, 2005, Vol. 51, 304-314 244. “Asymmetric Epoxidation of Alkenes by a Chiral Manganese(III) Salen Complex Anchored onto a Functionalised Hexagonal Mesoporous Silica” A. R. Silva; K. Wilson; J.H. Clark; C. Freire Stud. Surf. Sci. Catal., 2005, Vol. 158, 1525-1532 245. “Anchoring of Vanadyl Acetylacetonate onto Amine Functionalised Activated Carbons and Catalytic Activity in the Epoxidation of an Allylic Alcohol” B. Jarrais; A. R. Silva; C. Freire Eur. J. Inorg. Chem., 2005, 4582-4589 Laboratório Associado para a Química Verde A - 40 246. “Mn(III) Salen Complex Immobilised into Pillared Clays by in Situ and Simultaneous Pillaring/ Encapsulation Procedures. Application in the Heterogeneous Epoxidation of Styrene” B. Cardoso; J. Pires; A. P. Carvalho; I. Kuzniarska-Biernacka; A. R. Silva; B. de Castro; C. Freire Microp. Mesoporous Mat., 2005, Vol.86, 295-302 247. “(Salen)Nickel-Catalysed Epoxidations in the Homogeneous and Heterogeneous Phase: The Implication of Oxygen on the Efficiency and Product Selectivity” R. Ferreira; H. Garcia; B. de Castro; C. Freire Eur. J. Inorg. Chem., 2005, 4272-4270 248. “Organo-Laponites as Novel Mesoporous Supports for Manganese (III) Salen Catalysts” I. Kuzniarska-Biernacka; A. R. Silva; A. P. Carvalho; J. Pires; C. Freire Langmuir, 2005, Vol. 21, 10825-10834 249. “Thermodynamic Evidence for Ca2+-Mediated Self-Aggregation of Lewis X Gold Glyconanoparticles. A Model for Cell Adhesion via Carbohydrate- Carbohydrate Interaction” J. M. de la Fuente, P. Eaton, A. G. Barrientos, M. Menéndez, S. Penadés J. Am. Chem. Soc., 2005, Vol 127, 6192-6197 250. “Tribological and Structural Studies of Nanocrystalline Ti-C-N Composites Coatings Prepared by Reactive Sputtering” D. Martínez-Martínez, J.C. Sánchez-López, T.C. Rojas, A. Fernández, P. Eaton, M. Belin Thin Solid Films, 2005, Vol 472 (1-2), 64-70 251. “Structural Characterization of Self-Assembled Monolayers of Neoglycoconjugates using Atomic Force Microscopy” C. Tromas, P. Eaton, J. Mimault, J.Rojo, S. Penadés Langmuir, 2005, Vol 21, 6142 - 6144 252. “Intramolecular electron transfer in diastereomeric naphthalene-amine dyads: a fluorescence and laser flash photolysis study” S. Abad; U. Pischel; M. A. Miranda Photochem. Photobiol. Sci., 2005, Vol. 4, 69-74 253. “Wavelength-dependent stereodifferentiation in the fluorescence quenching of asymmetric naphthalenebased dyads by amines” S. Abad; U. Pischel; M. A. Miranda J. Phys. Chem. A, 2005, Vol. 109, 2711-2717 254. “Proton-induced fluorescence switching in novel naphthalimide-dansylamide dyads” S. Abad; M. Kluciar; M. A. Miranda; U. Pischel J. Org. Chem., 2005, Vol. 70, 10565-10568 Laboratório Associado para a Química Verde A - 41 Books and book chapters 1. P.M. Abreu; P. Branco; S. Matthew “Natural product-like combinatorial libraries towards the discovery of lead compounds” in Combinatorial Synthesis of Natural Product Based Libraries, Ed. A.M Boldi in Combinatorial Chemistry Series, CRC Press, 257-352, 2005 2. Afonso, Carlos A. M.; Crespo, João P. S. G. “Green Separation Processes: Fundamentals and Applications” 1nd ed. Wiley-VCH, Weinheim, 2005 3. T Schafer; LC Branco; R Fortunato; P Izak; CM Rodrigues; CAM Afonso; JG Crespo “Opportunities for membrane separation processes using ionic liquids” in ACS Symposium Series, 902: 97-110 (2005) 4. V Najdanovic-Visak; A Serbanovic; JMSS Esperanca; HJR Guedes; LPN Rebelo; M Nunes da Ponte “Multiphase equilibrium in mixtures of [C(4)mim][PF6] with supercritical carbon dioxide, water, and ethanol: Applications in catalysis” in ACS Symposium Series, 901: 301-310 (2005) 5. A Banet Osuna; A Serbanovic; M Nunes da Ponte “Supercritical Fluids” in Green Separation Processes, eds. C.A.M. Afonso and J. G. Crespo Eds., Wiley-VCH, Weinheim (2005) 6. T Schäfer, JG Crespo “Vapour Permeation and Pervaporation” in: Green Separation Processes, Carlos M. Afonso and João G. Crespo (Eds), Wiley-VCH Publishers (2005) 7. LPN Rebelo; V Najdanovic-Visak; R Gomes de Azevedo; JMSS Esperança; M Nunes da Ponte; HJR Guedes; ZP Visak;. HC de Sousa; J Szydlowski; JN Canongia Lopes; TC Cordeiro “Phase behavior and thermodynamic properties of ionic liquids, ionic liquid mixtures, and ionic liquid solutions” ACS Symposium Series, 901: 270-291 (2005) 8. CA Cerdeiriña; J Troncoso; C Paz Ramos; L Romani; V Najdanovic-Visak; JMSS Esperança; M Nunes da Ponte; HJR Guedes; ZP Visak; LPN Rebelo “Criticality Of The [C4mim][Bf4] + Water System” ACS Symposium Series, 901: 175-186, (2005) 9. M. Sousa, M.J. Melo, A. Aguiar-Ricardo, P. Cruz, “A Green Approach to Antique Textile Cleaning” in The 14th Triennal Meeting The Hague Preprints, Vol II, Paterakis, A.B. (Ed.), ICOM Committee for Conservation, 944-953 (2005) 10. JPB Mota; IAAC Esteves “Fundamentos dos Processos de Adsorção” in Descontaminación Ambiental Mediante Adsorbentes, ed. F. Rodriguez-Reinoso, Cap. 1, 1-23, Ediciones CYTED, Madrid, Spain (2005) Laboratório Associado para a Química Verde A - 42 11. P. Valentão; P. Andrade; R. Seabra “Análise Macroscópica e Microscópica em Farmacognosia” in Farmacognosia e Fitoquímica, ed. A. Proença da Cunha, Fundação Calouste Gulbenkian, 17-35 (2005) 12. M.P.L.V.O. Ferreira “RP-HPLC methods for separation of milk proteins: application on quality control and detection of species adulteration in dairy products” in Encyclopedia of Chromatography, eds. J. Cazes, Taylor & Francis, 1457 (2005). 13. I.S. Moreira; P. A. Fernandes; M. J. Ramos “Computational Determination of the Relative Free Energy of Binding- Application to Alanine Scanning Mutagenesis” in Molecular Materials with Specific Interactions: Modeling and Design, ed W.Andrzej Sokalski, Springer, (2005) Laboratório Associado para a Química Verde A - 43 Articles in Proceedings “Synthesis of Polyurethanes in Supercritical CO2” C. Veiga de Macedo, T. Casimiro, A. C. Fernandes, C. Romão, A. Aguiar-Ricardo in Proceedings of the 10th Meeting on Supercritical Fluids, ed. M. Perrut, ISASF: Strasbourg / Colmar, France (2005). “Membrane Formation with CO2-Assisted Phase Inversion Method. Solvent Power and Depressurization Effect” M. Temtem, T. Casimiro, J. F. Mano, A. Aguiar-Ricardo in Proceedings of the 10th Meeting on Supercritical Fluids, ed. M. Perrut, ISASF: Strasbourg / Colmar, France (2005). “Nonisothermal Dynamic Model of a SFE Structured Packing Column” J. B. Fernandes, R. Ruivo, J. P. Mota, P. C. Simões in Proceedings of the 10th Meeting on Supercritical Fluids, ed. M. Perrut, ISASF: Strasbourg / Colmar, France (2005). “Solubility of Liquid Crystal, E7, in Supercritical Carbon Dioxide” A.R.E. Brás, T. Casimiro, J. Caldeira, M. Dionísio, A. Aguiar-Ricardo in Proceedings of the 9th International Chemical Engineering Conference-Chempor, Coimbra, (2005). ”Evaluation of CO2-philicity of Perfluoropolyethers using High-pressure NMR Spectroscopy” M. Temtem, T. Casimiro, E. J. Cabrita, A. L. Macedo, A. Aguiar-Ricardo in Proceedings of the 9th International Chemical Engineering Conference - Chempor, Coimbra (2005). “Study Of Cold-Set Whey Protein Gels Prepared With Mg2+” M. Vázquez da Silva, L. Hilliou, M.P. Gonçalves in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento de Engenharia Química da Universidade de Coimbra, paper QL056-6 pages (2005) (ISBN 972-8055-13-7). “Extraction of Biopolymers From Natural Resources And Industrial Wastes For Edible Films Applications” A.M. Ramos, H. Coelho, L. Guerreiro, A.M. Relva, L. Hilliou, F.D.S. Larotonda and A.M. Sereno in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento de Engenharia Química da Universidade de Coimbra, paper QL078-6 pages (2005) (ISBN 972-8055-13-7). “Characterization Of Model Edible Films Using Hydrocolloids And Pectins” V.D. Alves, L. Hilliou, F.D.S. Larotonda, I.M. Coelhoso, M.P. Gonçalves, A.M. Sereno in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento de Engenharia Química da Universidade de Coimbra, paper QL096-6 pages (2005) (ISBN 972-8055-13-7). “Effect Of Seasonal Variation On The Chemical And Physical Properties Of Carrageenans Extracted From Mastocarpus Stellatus And Gigartina Pistillata” L. Hilliou, F.D.S. Larotonda, P. Abreu, A.M. Ramos, M.P. Gonçalves, A.M. Sereno, in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento de Engenharia Química da Universidade de Coimbra, paper QL086-6 pages (2005) (ISBN 972-8055-13-7). “Trypsin immobilisation in zeolites” C. Rocha, M.P. Gonçalves, J.A.Teixeira in Proceedings of the 9th International Chemical Engineering Conference CHEMPOR 2005, Departamento de Engenharia Química da Universidade de Coimbra, paper QL0102- 6 pages (2005) (ISBN 972-8055-13-7). Laboratório Associado para a Química Verde A - 44 ”Simultaneous Determination of Tocopherols and Tocotrienols in Virgin Olive Oils by Normal Phase HPLC Coupled with Diode-Array / Fluorescence detector” S.C. Cunha; J.S. Amaral; JO. Fernandes; MBPP. Oliveira in Proceedings of INTRADFOOD 2005 Innovations in Traditional Foods, Valência, 2005, 353-356. ”Tocopherols and Tocotrienols Composition of Hazelnuts” SC. Cunha; JS. Amaral; JO. Fernandes, MBPP. Oliveira in Proceedings of INTRADFOOD 2005 Innovations in Traditional Foods, Valência, 2005, 353-356. Laboratório Associado para a Química Verde A - 45 B Dissertations Laboratório Associado para a Química Verde A - 46 Ph. D. Disertations “Caracterização Química do Aroma de Azeites Virgens com Atributos Positivos e Negativos” Laila H. Ribeiro, Organic Chemistry PhD; Dec 2005 Supervisors: M. D. R. Gomes da Silva. “Monitoring of the structural alterations of proteins induced during ultrafiltration processes using fluorescence techniques” Carla Portugal, UNL, Dec 2005 Supervisors: J.G.Crespo, J.C. Lima “Synthesis of Terpene Esters In Compressed Gases” Célia Maria Simões Peres, UNL, 2005 Supervisor: S. F. Barreiros “Medidas Acústicas Aplicadas à Determinação de Pontos Críticos de Misturas” Nuno Rui Alves Ribeiro, UNL, Jun 2005 Supervisor: Ana Aguiar Ricardo “Gas separation processes by integrated adsorption and Permeation technologies” Isabel Esteves, UNL, Jul 2005 Supervisor: José Paulo Mota “Mistura de Fluidos e Transferência de Calor em escoamentos Laminares Caóticos” António Rodrigo, UNL, Jul 2005 Supervisors: José Paulo Mota, Esteban Saatdjian “Superoxide Reductases: Structural anf Functional Aspects” Patricia Raleiras, UNL, 2005 Supervisor: J. J. G. Moura “Sensores Ópticos de Matriz Sol-Gel com Interesse na Análise Farmacêutica” Paula Cristina de Almeida Jerónimo, Dez de 2005 Supervisors: Alberto Araújo and Conceição Montenegro “Sistemas Automáticos em Fluxo Continuo para o Controlo de Formulações Farmacêuticas” João Alexandre Velho Prior, Dez de 2005 Sistemas Automáticos em Fluxo Continuo para o Controlo de Formulações Farmaceutricas Supervisors: João Luís Santos and Costa Lima “Marmelo (Cydonia oblonga Miller) e Marmelada: Perfil em Compostos Fenólicos, Ácidos Orgânicos e Aminoácidos Livres e Avaliação do Potencial Antioxidante” Branca Maria Cardoso Monteiro da Silva, UP, Jun de 2005 Orientadores: M A Ferreira, R Seabra e P Andrade “Desenvolvimento de métodos de biologia molecular para a identificação de castas e clones de videira – aplicação ao estudo da autenticidade de mostos e vinhos” Miguel Ângelo Rodrigues Pinto de Faria, FFUP, Dez. de 2005 Supervisor: Margarida A. Ferreira Laboratório Associado para a Química Verde A - 47 “Epidemiologia da resistência a antibióticos em Enterococcus spp portugueses de origem humana, animal e ambiental” Carla Novais, UP, Dez de 2005 Supervisors: L. V. Peixe and T. C. Coque. “Ulilização de um produto natural (cortiça) como adsorvente de pesticidas piretróides em água” Valentina Maria Fernandes Domingues Faculdade de Engenharia da Universidade do Porto, 22 Dezembro de 2005 Supervisor: Cristina Maria Fernandes Delerue Alvim Matos “Filmes Poliméricos do Tipo [M(salen)] Funcionarizados para o Reconhecimento de Catiões Representativos” Andrea da Conceição Ferreira Carneiro, UP, Dezembro, 2005 Supervisor: C. Freire Laboratório Associado para a Química Verde A - 48 M. Sc. Dissertations “Classificação geográfica automática de petróleos brutos” Ana Margarida Pereira da Fonseca, UNL 2005, (Mai 2005) Supervisors: J. Aires-de-Sousa, Ana M. Lobo , J. L. Biscaya “Avaliação do Efeito de Poluentes Orgânicos na Membrana Celular” Joana Moutinho de Veiga Marcelino, UP, Jan de 2005 Orientador (es): Carla Matos e Salette Reis “Avaliação da Actividade Captadora de Espécies Reactivas de Oxigénio e de Azoto por Antagonistas BetaAdernégicos” Ana Maria de Carvalhais Mendes Gomes, UP, Jan de 2005 Orientador (es): Eduarda Fernandes e Costa Lima “Determinação de Parâmetros Físico-Químicos em Xantonas” Ana Bela Ferreira Gomes Orientador: Carlos Afonso e Salette Reis, UP, Abr de 2005 “Determinação de ião brometo em águas através de um sistema automático baseado em multi-seringa” Liliana Sara Melo Oliveira, UP, Dez de 2005 Orientador: Marcela Segundo “Hidrólise Enzimática de Concentrados de Proteínas do Soro: Separação e Caracterização de Péptidos – Contribuição para a Valorização da Fracção Proteica do Soro” Maria Virgínia Custódio Moreira Teixeira da Mota, FFUP, Jul. de 2005 Supervisor: IMPLVO Ferreira “Estudo Comparativo da Fracção Protéica de Maltes, Mostos e Cervejas Provenientes das Variedades de Cevada Scarlett e Prestige” José Filipe Campos da Silva, FFUP, Set. de 2005 Supervisor: IMPLVO Ferreira “Validação de um método analítico para a quantificação de Ocratoxina A em cerveja” Sofia Catarina Cunha Silva Nogueira, FFUP, Nov. de 2005 Supervisors: MBPP Oliveira and Arminda Alves “Contributo para a Caracterização do Queijo Terrincho: Estudo da Proteólise e Avaliação da Autenticidade por HPLC/UV” Carla Beatriz Rodrigues Vieiros, FFUP, Nov. de 2005 Supervisor: IMPLVO Ferreira “Estudo do efeito da exposição de geotêxteis a soluções de catiões metálicos, por voltametria de redissolução anódica” Marta Sofia Roma Pires, FCUP, 2005. Supervisor: Paulo Joaquim Ferreira de Almeida. “Gelificação Térmica de Hidrolisados Enzimáticos de Proteínas do Soro do Leite Bovino. Comportamento de Sistemas Aquosos Mistos Péptidos-Polissacarídeos” Duarte Paulo Martins Torres, UMinho, Mai de 2005. Supervisors: Maria do Pilar F. Gonçalves and José A. C. Teixeira Laboratório Associado para a Química Verde A - 49 “Fermi Resonance Coupling in the C–H Stretching Region of Methoxide Adsorbed on Clean Ru(0 0 1)” Ana Sofia Pinto, UP, Mar de 2005 Supervisor: Natália Cordeiro “Estudo Computacional da Decomposição de Energia em Processos de Associação Molecular” Alexandre Rodrigues Faria de Carvalho, UP, Mar de 2005 Supervisor: André Melo “Líquidos Iónicos como Solventes Neotéricos para a Epoxidação Asimétrica de Alcenos Catalisada por Complexos de [Mn(III)salenX]” Fernando Jorge de Sousa Teixeira, UP, Outubro, 2005 Orientadora: C. Freire Laboratório Associado para a Química Verde A - 50 C Patents Laboratório Associado para a Química Verde A - 51 Patents “Ultra-fast protein enzymatic digestion by high intensity focused ultrasound” J. L. Capelo Martinez; I. Moura; J. Vasquez and D. Lopez; Patent Nº: 23848, code: 0198, 2005/07/07-16:12:38 (103303 C) “Método para a obtenção de um concentrado natural rico em hidroxitirosol a partir de resíduos da produção de azeite utilizando tecnologias limpas”, submission of Portuguese patent, process nº 103326 K. Inventors: Manuel Nunes da Ponte, Catarina Duarte, João Goulão Crespo, Svetlozar Velizarov, Ana Matias, Ana Nunes, José Santos. “New Catalysts for Producing N-phosphonomethylglycine” P.B. Correia Patent PCT 2005 3773 “Novel Higher Eficiency Formulation of Glyphosate with Lower Attending Time Before Rain” P.B. Correia National Patent, filed, 2005 “Process for Producing N-phosphonomethylglycine” P.B. Correia Patent PCT IB 2005 003201 “Glyphosate Containing Mixtures with Cell Membrane Disrupture Compounds and Ammonium Sulphate for Herbicide Application in Normal Crops and in Genetic Modified Crops” P.B. Correia Patent WIPO, filed, 2005 Laboratório Associado para a Química Verde A - 52 ANNEX III RESEARCH PROJECTS Laboratório Associado para a Química Verde A - 53 International funding “New approach to waste recovery into selective adsorbents of heavy metals” Project NATO Science for Peace Program nº 977984, 2002-2006 SUPERGREENCHEM Green Chemistry in Supercritical Fluids: “Phase Behaviour, Kinetics and Scale-up” Marie Curie Research Training Network (MCRTN-2004-504005) “Membrane bioreactor technology (MBR) with an EU perspective for advanced municipal wastewater treatment strategies for the 21st century” EUROMBRA,European Commission,2005-2008 European Research Network TMR: (HPRN-CT-1999-00084) - “The Mechanism, Specificity and Inhibition of Enzymes belonging to the Xantine Oxidase family: Medical and Industrial Applications” EA-BIOFILMS: “Electrochemical control of biofilm-forming micro-organisms : screening, identification, and design of new knowledge-based technologies” NEST – 508866 (STREP) (2005 – 2008) COST Action D31 - “Organizing Non-Covalent Chemical Systems with Selected Functions” Internal Project N. D31/0016/05 “Non-Covalent Interactions between Functional Abiotic Receptors and Ion Pairs” COST Action D31 - “Organizing Non-Covalent Chemical Systems with Selected Functions” Internal Project N. D31/0011/04 “Supra-Biomimetics: Towards Bio-Inspired Photoadressable Supramolecular Systems. Synthesis, LightEmission, Dynamics, Biomedical Applications” Funded by FCT “Natural Vegetal Antioxidants” Ref. POCTI/QUI/47343/2002 Coordinator: Abel José de Sousa Costa Vieira “Hopanoid composition of sediments from Lower Tagus Basin” POCTI/QUI/45720/2002 Coordinator: Angela M. S. Relva “New carbohydrate-based compounds from ethnopharmacological plants: bioactivity against pathogenic yeasts” POCTI/1999/FCB/35863 Coordinator: Elvira Maria Mendes Sardão Monteiro Gaspar “Volatile phenol yeast producers: a great concern in wine industry to be understood” POCTI/AGR/56771/2004 Ciências Agrárias e Florestais. Coordinator: Manuel Malfeito Ferreira, Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia. REQUIMTE participation: M.D.R. Gomes da Silva. “Chemical and Sensorial Characterization of Malolactic Fermentation impact in Wines”, POCTI/AGR/55432/2004 - Ciências Agrárias e Florestais Coordinator: Ana Costa Freitas, Dep. Fitotecnia, U. Evora. REQUIMTE participation: M.D.R. Gomes da Silva and Angela M.S. Relva. Laboratório Associado para a Química Verde A - 54 “EnvironMetalControl: Fast monitoring in environmental matrices of total and speciation of elements and its control by a new method based on enzyme probe sonication” POCTI/AMB/58045/2004 Coordinator: Luis Fonseca; IST, Universitade Técnica de Lisboa. Participation: J. L. Capelo Martinez. “Towards New Supramolecular Devices: from new synthetic methods to multifunctional applications”, POCTI/QUI/55519/ Coordinator: Carlos Lodeiro Espinho, FCT-UNL, REQUIMTE. Participation: J. L. Capelo Martinez. “Sensores químicos baseados em receptores poliamínicos ligados a unidades fluorescentes” Projecto POCTI/47357/QUI/2002 Coordinator: Fernando Jorge da Silva Pina “Photochromism of Flavylium Compounds in Water/Ionic Liquid Biphasic Systems” POCTI/QUI/57735/2004 Coordinator: Fernando Jorge da Silva Pina POCI/QUI/55519/2004 “Towards New Supramolecular Devices: from new synthetic methods to multifunctional applications” . Coordinator: Carlos Lodeiro Espiño “Alcalóides Naturais como sintões quirais” POCTI/QUI/44501/2002 Coordinator: Prof. A. M. Lourenço “Volatile phenol yeast producers: a great concern in wine industry to be understood.” POCTI/AGR/56771/2004 Coordinator: Prof. Manuel Malfeito Ferreira (Instituto Superior de Agronomia) Coordinator: Prof. João Aires de Sousa “Algicidas naturais contra microalgas nocivas” POCI/AMB/60351/2004 Coordinator: Dr. P. Pereira (Instituto Nacional de Saúde) Coordinator: Prof. A. M. Lobo POCTI/QUI/37423/2001 “New Synthetic methodology towards chiral gamma-amino acids useful in the design of new drugs” Coordinator: Prof. M. Pereira “Towards New Supramolecular Devises: from new synthetic methods to multifuntional applications” POCTI/QUI/55519/2004. “Tailored Synthesis of Biopolymers by mixed microbial cultures from molallse”, Fundação para a Ciência e Tecnologia, POCI/BIO/55789/2004 “Saccharose as a Water Soluble Chiral Auxiliary and a Linker for Solid Phase Chemistry” Fundação para a Ciência e a Tecnologia, POCTI/QUI/47973/2002. “Rationalising Stereoselectivity in Organic Chemistry: From Theory to Practice” POCTI/QUI/42983/2001 Laboratório Associado para a Química Verde A - 55 “High-Pressure NMR Spectroscopy of Polymers and biopolymers in CO2 Emulsions” POCTI/QUI/42313/2001 “Catalytic Depolymerisation Of Current Plastics Residues” POCTI/EQU/37946/2001 “Mechanisms Of Drug Controlled Release From Microsphere” POCTI/EQU/46715/2002 “Technology For Obtention of Edible Films and Coatings for Foods Obtained From Low Value National Resources” POCTI/EQU/45595/2002 “Transesterification Of Vegetable Oils. Application To The Production of Biodiesel” POCTI/EQU/48879/2002 “Development Of Catalytic Polymeric Membranes For The Oxidation Of Terpenic Olefins” POCTI/CTM/45449/2002 “Technology to produce edible films and coatings for foods obtained from low value national resources” POCTI/EQU/45595/2002, 2003-2006. “Removal of Trace Heavy Metals from Drinking Water Supplies in an Ion Exchange Membrane Bioreactor” POCI/AMB/57356/2004, 2005-2008 “Bioremediation of soil and water contaminated with pesticides” POCI/AMB/59836/2004, 2005-2008. “Enzymatic reduction of perchlorate” POCTI/QUI/55435/2004, 2005-2008 “New biodegradable and environmental friendly chelating agents for industrial applications” POCTI/QUI/57891/2004, 2005-2008. “Metabolism and characterization of mixed cultures in wastewater processes for simultaneous removal of nitrogen and phosphorus” POCTI/AMB/56075/2004, 2005-2008 “Síntese de biopolímeros “á medida” por culturas microbianas mistas a partir de melaços” POCTI/BIO/55789/2004, 2005-2008 “Advanced Bioprocess Automation Based on Hybrid Grey-Box Systems” POCTI/BIO/56571/2004, 2005-2008 “Advanced Control of BHK cultivation processes based on intracellular metabolic fluxes” POCTI/BIO/57927/2004, 2005-2008 “Production and characterisation of a PDLC. New strategies for polymerisation and incorporation in supercritical CO2” POCTI/CTM/47363/2002 Laboratório Associado para a Química Verde A - 56 “Static mixers as heat exchangers in supercritical fluid extraction processes - computational fluid dynamics and process simulation studies” POCTI/EME/61713/2004 “Gas separation by cyclic process combining pressure swing adsorption and membrane permeation” POCTI/EQU/45102/2002 “Viscous fluid mixing and chaotic heat transfer in spatially and time-periodic flows” POCTI/EME/40057/2001 “Chromatographic separation of anthocyanins by simulated moving bed” POCTI/EQU/39391/2001 “Process integration of supercritical fluid extraction and membrane separation to recover “vegetal” squalene from olive oil residues” POCTI/EQU/61550/2004. “Estudo por espectroscopia dieléctrica das alterações na dinâmica molecular durante a formação de um cristal líquido disperso numa matriz polimérica” POCTI/CTM/37435/2001 “RenH2 – Stand-Alone Energy System Supported by Totally Renewable Hydrogen Production” POCTI/ENR/59623/2004 “Bacterial cytochrome c peroxidases- Activation, Enzymatic Mechanism And Structure” POCTI/QUI/42309/2001 Coordinator: Isabel Moura “Enzimas chave aa reduçâo do Nitrato. Redutase do Óxido Nítrico e Redutase do Óxido Nitroso - As duas enzimas terminais” POCTI/BME/42265/2001 Coordinator: Isabel Moura “High Pressure NMR spectroscopy of polymers and biopolymers in CO2 emulsions” POCTI/QUI/42313/2001 Coordinator: Maria dos Anjos Macedo “Estudos Electroquímicos Dinâmicos em Proteínas de Transferência Electrónica” POCTI / QUI / 42277 / 2001 Coordinator: José J. G. Moura “NMR structural studies of the active center of 5-aminolevulinate synthase, the first enzyme of the mammalian heme biosynthetic pathway” POCTI/BME/39184/2001 Coordinator: Maria dos Anjos Macedo “Orientation of Proteins By Liquid Crystals” POCTI/QUI/42279/2001 Coordinator: Francisco Jorge Caldeira Laboratório Associado para a Química Verde A - 57 “Structural and mechanistic studies of fatty acid desaturases. Looking for reactivity of diiron clusters” POCTI/QUI/37413/2001 Coordinator: Pedro Tavares “Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins” POCTI/QUI/47273/2002 Coordinator: Alice S. Pereira “Protease de Plasmodium chabaudi como alvo na terapia da malária” POCTI/43637/BME/2000 Coordinator: Jorge Lampreia “As proteases de parasitas da malária como alvo na quimioterapia” POCTI/ESP/42223/2001, Coordinator:Jorge Lampreia “Mechanisms and Energetics of Electron Transport in Metalloproteins by Dynamic Voltammetry” POCT/QUI/55743/2004 Coordinator:José J. G. Moura (FCT-UNL) and Margarida Santos (IST-UTL) “Transient Protein Complexes – Soft Docking and NMR” POCT/QUI/57741/2004 Coordinator:José J. G. Moura “Development of enzyme biosensors for multi-parametric control and monitoring of environmental samples” POCTI/QUI/58026/2004 Coordinator:M. Gabriela Almeida. “New molybdo and copper cofactors in proteins isolated from sulphate reducers” POCTI/QUI/55350/2004 Coordinator:Isabel Moura “Cobalt in metabolism of sulfate reducers. Noncorrin Co/Zn ATPS, AK, and a new Co-corrinoid protein” POCTI/QUI/59119/2004 Coordinator:Sergey Bursakov “(Per)chlorate enzymatic reduction” POCTI/QUI/55435/2004 Coordinator:Cristina Costa “Spectroscopy of Oriented Proteins on Conductive Polymers” POCTI/QUI/58973/2004 Coordinator:Francisco Caldeira “Studying kinetics and mechanism of superoxide reduction” (POCTI/QUI/57475/2004) Coordinator:Alice Pereira “Análise estrutural e funcional de um novo citocromo multihémico: um modelo para redutases do fumarato” POCTI/QUI/42902/2001 Coordinator:C. Salgueiro Laboratório Associado para a Química Verde A - 58 “Caracterização Bioquímica de Proteínas com Actividade Bioremediativa” POCI/QUI/60060/2004 Coordinator:C. Salgueiro “Caracterização de CymA: uma proteína chave na respiração anaeróbia de Shewanella” POCI/BIA-PROQUI/58722/2004 Coordinator:C. Salgueiro “Desenvolvimento de biossensores enzimáticos para controlo e monitorização multiparamétrica de amostras ambientais” (POCI/QUI/58026/2004). Coordinator: M. Gabriela Almeida. “Structural and Functional Characterization of Nitrate Reductases and Formate Dehydrogenases” POCTI/QUI/57641/2004 “Molecular determinants of ligand specificity in carbohydrate-binding modules and cohesin-dockerin complexes” POCTI/BIA-PRO/59118/2004 (with Prof Carlos Fontes and Prof. José Prates, FMV-UTL) “Bacterial Cytochrome c Peroxidases- Activation, Enzymatic Mechanism and Structure”. POCTI/QUI74230972001 “Development and Application of in Vitro Methodologies for Evaluation of Anti-Inflammatory and Antioxidant Mechanisms of Non-Esteroidal Anti-Inflamatory Drugs” FCT (POCTI/FCB/47186/2002) Coordinator: Salette Reis “Assessing the dietary exposure of Portuguese consumers to acrylamide. Improvemrnt, development and validation of rapide screening procedures and confirmatory analytical methodologies” FCT (POCTI/AGR/61543/2004) Coordinator: José Fernandes e João L. M. Santos “Síntese de cromonas e avaliação in vitro das propriedades antioxidantes e anti-inflamatórias” FCT (POCTI/QUI/59284/2004) Coordinator: José L.F.C. Lima “Desenvolvimento de equipamentos automáticos e metodologias expeditas para controlo ambiental na perpectiva de um desenvolvimento sustentado” FCT/GRICES/CNPq (Projecto Bilateral Portugal/Brasil) Coordinator: José L.F.C. Lima “Desenvolvimento de biossensores enzimáticos para a determinação de nitratos e nitritos em amostras complexas” FCT (POCTI/QUI/58026/2004) Coordinator: M. Conceição Montenegro “Caracterização fitoquímica e actividade antioxidante de culturas in vivo e in vitro de Brassica oleracea var costata” POCTI/AGR/57399/2004 Coordinator: Paula Andrade Laboratório Associado para a Química Verde A - 59 “Tecnologias para a valorização de bio-produtos da Salvia sp. (SalvaBiotech)” POCTI/AGR/62040/2004 Coordinator: Manuel Ferreira (Universidade do Minho) “Stress Oxidativo na Doença Cardíaca: Acção das Catecolaminas” POCI/SAU-OBS/55849/2004 Coordinator: Fernando Manuel Gomes Remião “Estudos farmacogenéticos para avaliação da influência do metabolismo na expressão da toxicidade de derivados anfetamínicos usados correntemente como drogas de abuso” POCI/SAU-FCF/57187/2004 Coordinator: Maria de Lourdes P.A.S. Bastos “Indicators of swine industry pollution: antibiotics and antibiotic-resistant bacteria” POCI/AMB/61814/2004 Coordinator: Luísa Peixe “Study of factors implicated in the stability and dissemination of multi-resistant Enterococcus spp in distincts metagenomes” POCTI/SAU-ESP/61385/2004 Coordinator: Luísa Peixe “Biological Markers of contamination by hospital wastewater” POCTI/ESP/39885/2001 Coordinator: Helena Maria Neto Ferreira “Metodologias para controlo de solos contaminados com pesticidas” POCTI/AGR/44491/2002 Coordinator: Simone Barreira Morais “Bioremediation of organic pollution in soil by augmentation with robust methylotrophic bacteria” POCI/AMB/57353/2004 Coordinator: Paolo De Marco (IBMC/UP) “Experimental Protocols for Estimating the Design Parameters of Permeable Reactive Barriers” POCI/ECM/59779/2004 Coordinator: António Fiúza “Respirometry of Rock Acid Drainage and the Usage of Oxygen Consuming Coatings as a Mean of Reducing Environmental Emissions from Tailings Disposals” POCI/ECM/60438/2004 Coordinator: António Fiúza (FEUP) “Previsão do tempo de remediação de solos contaminados utilizando a extracção de vapor” POCI/AMB/61315/2004 Coordinator: Maria da Conceição M. Alvim Ferraz “Study of geosynthetics durability in order to obtain a better definition of their safety factors” POCTI/ECM/42822/2001 Coordinator: Maria de Lurdes Lopes (FEUP) Laboratório Associado para a Química Verde A - 60 “Tecnologia para a obtenção de filmes e revestimentos comestíveis para alimentos a partir de recursos nacionais de baixo valor” POCTI/EQU/45595/2002 Coordinator: Alberto M. Sereno COMFOOD: “Controling the microstructure of Food products by rheo-optical methods” POCTI/EQU/58064/2004 Coordinator: Loic Hilliou “Tailored synthesis of biopolymers by mixed microbial cultures from molasses” POCTI/BIO/55789/2004 Coordinator: Loic Hilliou “Interaction between metal ions and buffers for the environmental and biological pH ranges” POCTI/QUI/39950/2001 Coordinator: Helena Soares “Development of a clean technology for heavy metals removal and recovery from industrial effluents” POCTI/CTA/47875/2002 Coordinator: Helena Soares “New potentially biodegradable chelating agents for industrial and domestic applications environment-friendly” POCI/QUI/57891/2004 Coordinator: Helena Soares POCTI/ESP/39885/2001 Coordinator: Helena Maria Neto Ferreira Luísa Peixe, Diana Tuna Silva, Carla Novais “Experimental Protocols for Estimating the Design Parameters of Permeable Reactive Barriers” POCI/ECM/59779/2004 Coordinator: António Fiúza “Respirometry of Rock Acid Drainage and the Usage of Oxygen Consuming Coatings as a Mean of reducing Environmental Emissions from Tailings Disposals” POCI/ECM/60438/2004 Coordinator: António Fiúza “Previsão do tempo de remediação de solos contaminados utilizando a extracção de vapor” POCI/AMB/61315/2004 Coordinator: Maria da Conceição M. Alvim Ferraz “Metodologias para controlo de solos contaminados com pesticidas” POCTI/AGR/44491/2002 Coordinator: Simone Barreira Morais “Tecnologia para a obtenção de filmes e revestimentos comestíveis para alimentos a partir de recursos nacionais de baixo valor” POCTI/EQU/45595/2002 Coordinator: Alberto M. Sereno Laboratório Associado para a Química Verde A - 61 COMFOOD: “Controling the microstructure of Food products by rheo-optical methods” POCTI/EQU/58064/2004 Coordinator: Loic Hilliou “Tailored synthesis of biopolymers by mixed microbial cultures from molasses” POCTI/BIO/55789/2004 Coordinator: Loic Hilliou “Interaction between metal ions and buffers for the environmental and biological pH ranges” POCTI/QUI/39950/2001 Coordinator: Helena Soares “Development of a clean technology for heavy metals removal and recovery from industrial effluents” POCTI/CTA/47875/2002 Coordinator: Helena Soares “New potentially biodegradable chelating agents for industrial and domestic applications environment-friendly” POCI/QUI/57891/2004 Coordinator: Helena Soares “Ácidos Gordos Trans em Bolachas: Contribuição para a Ingestão Total de Gorduras Trans da População Portuguesa e Consequente Risco Cardiovascular” Segurança Alimentar Susana Isabel Pereira Casal Vicente (FFUP) “Study of geosynthetics durability in order to obtain a better definition of their safety factors” POCTI/ECM/42822/2001 Maria de Lurdes Lopes (FEUP) Bilateral cooperations Acção Integrada Luso-Britânica-2006 Nº B-16/06. “Towards novel flexible fluorescence chemosensors for metal ion detection” Portuguese Coordinator: Carlos Lodeiro Espiño British Coordinatior: Dr. Emília Bértolo Pardo Department of Geographical and Life Sciences Canterbury Christ Church Univeristy 2005-2007 Acção Integrada Luso-Espanhola Nº E-68/05 “Multistate/Multifunctional Systems Trapped in Polymer Hydrogel Matrices” Portuguese Coordinator: António Jorge Dias Parola Spanish Coordinator: Prof. Santiago V. Luis Departamento de Quimica Inorganica y Organica Universidade Jaume I de Castellón, Spain 2005-2006 Acção Integrada Luso-Alemã A-11/04 “Computer Prediction of Biological Activity: Chirality in QSAR Applications” Coordinator of Portuguese team: Prof. João Aires de Sousa (Universidade Nova de Lisboa, Portugal) Coordinator of German team: Prof. Johann Gasteiger (University of Erlangen-Nürnberg, Germany) Laboratório Associado para a Química Verde A - 62 Acção Integrada Luso-Espanhola E-59/06 “Compostos Ogânicos azotados: desenho de moléculas com actividade biológica baseado em sugestões da natureza (actividade e biotransformações) Coordinator of Portuguese team: Prof. A. M. Lobo (Universidade Nova de Lisboa, Portugal) Coordinator of the Spanish team: Prof. M. Amat (Univ. Barcelona, Espanha) “SciencEduc” project http://www.cienciaviva.pt/projectos/scienceduc/ SciencEduc is a European project aimed at improving science education in primary education schools by creating a network which will stimulate and support the development of good practices in Science teaching at this level of education and will foster interaction between the project partners at a national and crossborder level. The project is coordinated by the Ecole Normale Supérieure (Paris) in partnership with the following institutions: University of Tartu (Estonia), La main à la pâte – Académie des sciences (France), Apor Vilmos Catholic College (Hungary), Ciência Viva (Portugal), NTA – and the Royal Swedish Academy of Sciences (Sweden). Advisory of the Portuguese Partner: Prof. P. Mata “Biochemical and Spectroscopy Studies on the Enzyme Nitrate Reductase (NAP) from the sulfate-reducing organism Desulfovibrio desulfuricans” Projecto De Cooperação Científica E Tecnológica Portugal / Argentina, Programa 2003-2004 (J.J.G.Moura e Carlos Brondino) “Thermodynamic Structural Studies of The Temperature Induced Unfolding of Plant Thermophylic and Bacterial Mesophylc Enzymes” Acçoes Itegradas Luso-Espanholas 2006 Action N0 E-62/06 Sergy Bursakov CRUP “Study of a multihemic nitrite reductase by direct electrochemistry and electronic paramagnetic ressonance spectroscopy (EPR)” M. Gabriela Almeida and Bruno Guigliarelli. CQFB, Dept. Química, FCT/UNL, Monte da Caparica and CNRS/Université de Provence, Aix-Marseille I (France) “Nanoenssambled based electroactive biointerfaces for environmental and food analysis of nitrates and nitrites” M. Gabriela Almeida and Serge Cosnier. CQFB, Dept. Química, FCT/UNL and ICM, Grenoble, UMR CNRS 5630 GRICES “Desenvolvimento de equipamentos automáticos e metodologias expeditas para controlo ambiental na perpectiva de um desenvolvimento sustentado” FCT/GRICES/CNPq (Projecto Bilateral Portugal/Brasil) Responsável: José L.F.C. Lima “Influence of NSAIDs on the activity of enzymes at interfaces” CRUP (Acção Integrada Luso-Alemã A-6/06) Responsável: Salette Reis “Novos Compostos para um Futuro Ambientalmente Limpo” Acção Integrada Luso-Britânica Laboratório Associado para a Química Verde A - 63 Acções Luso-Alemãs Ref. A-5/04 “Metabolism and toxicity of the designer drugs 4-MTA and 2C-B: identification of hypersensitive individuals by pharmacogenetic studies” Investigador Responsável pela equipa portuguesa: Maria de Lourdes P.A.S. Bastos “Ecology and evolution of local genetic elements implicated in the antibiotic resistance of enterococci: study of factors determining the stability and spread of multiresistant bacteria in distinct metagenomes” Acções integradas Luso-Espanholas (CRUP). Acção nº 52/05. Coordinator: Luísa Peixe Collaborators: Carla Novais, Teresa Coque Biological Markers of contamination by hospital wastewater “Rheological behaviour and morphology of protein-polysaccharide mixed systems” GRICES/ CAPES (Portugal/ Brazil) Maria do Pilar Gonçalves “Frutas tropicais de alta humidade: processamento, embalagem sob atmosfera modificada e avaliação da qualidade” GRICES/CAPES (Portugal/ Brazil) Alberto M. Sereno “Tecnologia de películas biodegradaveis para alimentos en ibero-américa” “Technology for food grade biodegradable films in Ibero-America” CYTED project XI.20 Alberto M. Sereno International Co-ordinator: Dr. Paulo José do Amaral Sobral (Brazil) “Characterization of biopolymer mixtures for food applications” GRICES/DAAD (Portugal/Germany) 2005/2006 Loic Hilliou Projects financed by “Agência de Inovação” BEERVOLT (3 years project submitted to Agência de Inovação, which was signed in November 2004 and initiated in April 2004). Proposing Institution – Unicer, Bebidas de Portugal, SGPS S. A. Other institutions involved: Faculty of Science of the University of Porto, Carlsberg S/A and CAI, Controle e Automação Industrial, L.da. Project Director – Aquiles Barros. Projects financed by “Fundação Calouste Gulbenkien” Projecto Referência FCG 10/04 “Efeitos comportamentais e bioquímicos resultantes da exposição à MDMA (ecstasy) durante a adolescência”, Entidade Financiadora: Fundação Calouste Gulbenkian Data de início do Projecto: 4/Janeiro/2005 Investigador Responsável: Professor Doutora Maria Amélia Ferreira e Doutora Teresa Summavielle Laboratório Associado para a Química Verde A - 64 Food safety and public health: definition of the risks associated to E. coli (VTEC), Listeria monocytogenes e Salmonella spp. Fundação Calouste Gulbenkian. 2002. Coordinator: Xavier Malcata Collaborators: Luísa Peixe, Patrícia Antunes, Paula Teixeira, Nazaré Pestana Laboratório Associado para a Química Verde
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