report 2006

Transcrição

report 2006

Laboratório Associado
para a Química Verde
Tecnologias e Processos Limpos
REPORT
2006
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 - 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 2006 was the fifth 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 more to
carry out activities under the contract of Laboratório Associado.
The activities of REQUIMTE were presented on a joined meeting between CQFB and CEQUP in
Fátima, Mar 31/Apr 1, 2006. The book of abstracts of the meeting is delivered together with this
report.
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 400
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.
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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 429 researchers (235 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) organic synthesis and chemistry
of natural products, (ii) food quality and safety, (iii) clean technologies and clean processes, (iv)
environmental control and (bio) 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.
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REQUIMTE in 2006
2006 was the fifth 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.
2006 was the fourth year when the funding contract with FCT-MCTES was operational and
REQUIMTE hired new researchers and technicians in order to carry out activities included in the
contract with the Ministry.
The scientific production was also considered a key aspect of the activity of REQUIMTE, and the
total number of articles in international scientific journals has grown to 307, much to the dedication
of its graduate students, 32 of which have completed their doctoral thesis and 17 their master
thesis in 2006.
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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
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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.
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Events
The Day of Chemistry is organized every year bringing more than 600 students to the Campus
of Caparica. In 2006 it occurred on the XXXXX. 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).
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
Meeting of REQUIMTE was organized in JXXXXXXXX. This event brought together all researchers
in REQUIMTE and sessions included: i) a main session in which the achievements of the
Laboratório Associado were reported, highlights on the green chemistry field were presented and
discussed and in which the Investigadores Auxiliares employed by REQUIMTE presented their
work and projects; ii) a poster session which made possible the presentation of the work developed
by all the reaserchers.
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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 2006.
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RESEARCH REPORT
2006
Part A
2
AREA 1
ORGANIC SYNTHESIS AND CHEMISTRY OF NATURAL PRODUCTS
- A new chiral aziridination reaction, with potential application in the synthesis of avian flu antivirals,
developed and the absolute configuration of the 3-membered ring defined through X-Ray analysis.
- 5-(Glutathion-S-yl)-adrenaline, an adrenaline adduct of glutathione, for the first time synthesized
and used as standard for the identification and quantification in toxicity studies involving isolated
calcium tolerant cardiomyocytes.
- Identification of anti-inflammatory and antioxidant flavonoids, cytotoxic phenolics, and cytotoxic
diterpenes with reversal effect on multidrug resistance, isolated from plants collected in Cuba,
Guinea-Bissau, and Portugal.
- A metabolite of the anti-HIV drug Nevirapine found to give covalent adducts with cysteine and
glutathione.
- Portuguese wines and musts characterised by high intensity focused ultrasound combined
with inductively coupled plasma mass spectrometry.
- The developed MOLMAP molecular descriptors of products and reagents successfully applied
to genome-scale classification of metabolic reactions.
- Research in alginate technology applied to Portuguese food distinguished with an international
price in Paris.
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AREA 2
FOOD QUALITY AND SAFETY
- Chemical characterization of food matrices by classical and emergent techniques.
- Correlation of compositional analysis, beneficial food effects, and potential therapeutic properties
of vegetables.
- Molecular biology techniques (real-time PCR) for food safety, authenticity and biodiversity
evaluation.
- Genetically modified organisms in foodstuffs – Studies for their traceability.
- “Acrylamide” in different food matrices (SPE and MSPD/GC-MS).
- Implementation of collaborative projects with agro-industry and consumer’s associations, in
order to promote traditional food products and the development of new ones.
- In vitro screening assays for the evaluation of safety/toxicity/protection of food elements and
related drugs
- Elaboration of toxicity/safety reports of food additives requested by Industry in order to obtain
Market Authorization.
- Patented drugs and/or mechanisms for preventing or treating intoxications.
- Construction of equipment for determination of “diacetyl” on line in collaboration with several
beer industries.
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AREA 3
CLEAN TECHNOLOGIES AND CLEAN PROCESSES
- Membrane Processes, including pressure-driven (nanofiltration) and electromembrane (Donnan
dialysis) methods, for separation.
- Application of “Green” metrics in comparisons.
- CFD used for simulation and optimization of flow patterns.
- New selective Nafion membranes prepared by incorporation of Room Temperature Ionic Liquids.
- Biopolymer production by mixed cultures, and production of biopolymers from glycerol, a
byproduct of biodiesel, contracted with industrial sponsors.
- Development of thermoresponsive acrylamide based hydrogels and new “green” strategies to
coat scaffold surfaces with thermo and pH sensitive polymers in supercritical CO2.
- Preparation of membranes of chitosan, polysulphone and mixed blends with polycaprolactone
with controlled morphology and porosity by CO2-assisted phase inversion method.
- Application of dynamic modelling and CFD techniques to the study of fractionation with
supercritical CO2.
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AREA 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 recycling of materials.
AREAS OF IMPACT (ACHIEVEMENTS)
- Development of transducers
- Automation and instrumentation
- Chemosensors and photochemical transducers
- Development of enzyme biosensors
- (Bio) remediation
- Wastewater treatment - Organic mercury
- (Bio) Hydrogen - Bioenergy - Biocorrosion - Biofilms
DEVELOPMENT OF TRANSDUCERS
Different transducing schemes such us optical and electrochemical were investigated. 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 walljet electrodes dedicated to the implementation of flow systems.
AUTOMATION AND INSTRUMENTATION
Continuous flow systems and dedicated equipment were developed and applied in automatic
laboratorial determinations or in on-line control of industrial processes.
CHEMOSENSORS - PHOTOCHEMICAL TRANSDUCERS
New chemosensors based on polyamine receptors bearing a luminescent unit have been
developed. Polyamine receptors are very versatile due to their capacity of binding not only metals,
but also anions.
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DEVELOPMENT OF ENZYME BIOSENSORS
Several electrode configurations have been tested, using a variety of immobilization materials,
such as non-conducting polymers, electropolymerized films functionalized with redox groups,
sol-gel glasses and modified clays. As expected, the analytical performance (detection limit,
linear range, sensitivity and operational stability) of each system is strongly dependent on the
electrode design. Application to Nitrate and Nitrite reductases – determination of Nitrate and
Nitrite.
(BIO)REMEDIATION
Denitrification and the Dinitrogen Biocycle
Structural studies on the enzymes involved in denitrification and applications to bioremediation
are two different and complementary aspects that were pursue.
Wastewater Treatment - Organic mercury
Study of the removal of emerging water pollutants (perchlotate, ionic mercury, arsenic containing
species by pure and mixed microbial cultures. Microbial competition on enhanced biological
phosphorus removal: DPAO and GAO. Production of new biodegradable environmental-friendly
chelating agents for industrial and domestic applications: biodegradability studies. New
biodegradable polymers from industrial wastes.
(BIO)HYDROGEN - BIOENERGY - BIOCORROSION - BIOFILMS
Hydrogen is a clean fuel and considered to be the fuel of the future. Bacterial systems contain
elaborate enzymatic systems for dihydrogen production and consumption.
Another important aspect is the involvement of micro-organisms in the corrosion of metals. The
understanding of the mechanisms of bacterial corrosion is essential in order to envisage procedures
to avoid the process. Biofilm studies are relevant and being pursued.
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AREA 5
CATALYSTS, SOLVENTS AND NON-TOXIC COMPOUNDS
- A library of chiral amines and diamines was developed, and one of them, bis-Nmethylmethanamine, was successfully used to catalyze the diastereo- and enantioselective
addition of cyclohexanone to (E)-b-nitrostyrene.
- An efficient strategy to attach vinylic groups to the sugar nucleus by two different procedures
was developed (esterification and Wittig olefination), both under microwave irradiation.
- Multifunctional oxazoline based polymers with therapeutic applications were developed in
supercritical CO2. Matrices of imprinted acrylated co-polymers were synthesised in scCO2 for
controlled drug delivery.
- Novel fluorescent probes for intra-cellular iron and insulin-enhancing complexes were
synthesised.
- The screening of enzymes for the synthesis of important building block for drugs with
physiological activity in an ionic liquid has been carried out, in a process allowing the recycling
of both biocatalyst and medium.
- The production of biodiesel by catalytic transesterification of soybean oil with methanol is being
studied.
- Gold and silver nanoparticles with controlled size/shape and different plasmon band maxima
were used as probes in gene detection methods and in studies of the interaction of nanoparticles
with proteins.
- Heterogeneous catalysts based on zeolites, activated carbons and mesoporous silicas were
developed for the use in hydration and oxidation of olefins and esterification/transesterification
reactions.
- Molecular simulation of the synthesis of porous materials was performed; and quantum modelling
of adsorption and reaction on metal substrates, (chiral gold surfaces) was carried out.
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RESEARCH REPORT
2006
Part B
2
ANALYTICAL AND BIOORGANIC CHEMISTRY
Head of Laboratory: Elvira Maria M. Gaspar, Assistant Professor
Staff Members:
Marco Diogo R.G. da Silva
Assistant Professor
José Luiz Capelo Martinez
Assistant Researcher
Luz Catarina Neves Fernandes
Technician
Post-Doct Fellows:
Raquel Rial Otero
Ph.D. Students:
Marco Miranda Galésio, Pedro Manuel Antunes, Francisco Miguel Cordeiro, Ricardo Jorge Carreira
Number of articles in scienticical journals: 12 (1-12)
Number of patents: 1 (1)
Scope and description of on-going research
The field of research is the development of accurate, less time consuming, more efficient and reliable analytical
methods, based on GC, GC/qMS, GC/TOFMS, GCxGC/TOFMS, HPLC and HPLC/MS, for the detection,
identification and quantification of organic compounds in complex matrices. Several sample preparation
methodologies have been used such as LLE, SPE and SPME. Special works have been developed:
1. Characterization of the volatile fraction (VF) composition of wines, cheese and olive oils, establishing
correlations with sensorial evaluations and electronic nose.
2. Chemical characterization of the volatiles emitted by trees, namely pines, eucalyptus and cork oak (Quercus
suber), including the study of temporal and spatial variations, as well as the enantiomeric composition of the
terpenes emitted; volatiles with potential biological activity, regarding insect behaviour in connection with host
selection will be identified, separated and purified.
3. Analysis of pollutants, such as dioxins, PCBs, PAHs, pesticides, herbicides and their metabolites, in
different environmental compartments (air, water, soils, sediments).
Also new natural oligossacharides with potential pharmacological properties were isolated from an European
Convolvulaceae plant and new chiral HPLC and enantioselective GC separation methods have been developed
as new separation techniques too. New clean analytical methodologies for organometallic compounds
speciation in food and environmental samples were used.
Future Activities
During 2007 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. 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 GCxGCTOF MS in a collaborative program. New analytical methodologies to control the quality of food samples,
specially antibiotics and pesticides will be focused. Furthermore methods for rapid protein identification based
on MALDI-TOF-MS will be assayed.
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PHOTOCHEMISTRY AND SUPRAMOLECULAR CHEMISTRY
Head of Laboratory: Fernando Pina, Full Professor
Staff Members:
Alexandra Bernardo
Associate Professor
A. Jorge Parola
Assistant Professor
J. Carlos Lima
Assistant Professor
M. João Melo
Assistant Professor
Carlos Lodeiro
César Laia
Micaela de Sousa
Assistant
Ana Claro
Assistant
Post-Doct Fellows:
Vesselin Petrov, Laura Rodríguez, Solange Muralha
Ph.D. Students:
Margarida Moncada, Sérgio Alves, Letícia Giestas, Carlos Pinheiro, André Vidal Pinheiro, Raquel Gomes,
Bruno Pedras, Joana Ferreira
M.Sc. Students:
Elisabete Oliveira, Maria Heitor, Rita Marques
Project Grantee:
Catarina Miguel
Number of articles in scientific journals: 19 (2, 11, 13-29)
Number of Ph. D. Thesis: 2 (1,2)
Flavylium network of chemical reactions
This subject has been a recurrent area of research in our group. We have tried to go a step further on this
chemistry, and thus a large investment on new ideas and approaches was made; synthesis of new compounds,
and the study of these systems in micelles and ionic liquids was carried out. During the present year, a paper
was published in this area, paper 21, concerning the behaviour of the flavylium network in organic solvents and
the consequences for the colouring phenomenon in plants.
Concerning perspectives for the future, 5 papers on this subject have been already published or are in press
in 2007, reflecting the results of our investment in this area. Our main objective is to produce photochromic
systems based on the interaction of the flavylium network with micelles, polymers and ionic liquids, pointing
out to industrial applications.
Chemosensors
In the framework of the Projects POCTI/47357/QUI/2002, “Chemosensors based on polyamine receptors
containing fluorophoric units” and POCI/55519/QUI/2004, “Towards New Supramolecular Devices: from new
synthetic methods to multifunctional applications” we have privileged our collaboration with the group of Prof.
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Enrique Garcia-España from the University of Valencia, Prof. António Bianchi and Andrea Bencini from the
University of Florence, Prof. Jaume Casabó e Lluis Escriche, from the University Autónoma de Barcelona,
Prof. Rufina Bastida, from the University of Santiago de Compostela and Prof. Emília Bértolo from the Cantebury
Christ Church University.
In the case of the two first collaborations (Valencia and Florence) the chemosensor is constituted by a
polyamine receptor linked to a fluorophoric unit. Paper 13 shows an interesting macrocyclic ligand with two
pendant naphthalenes, which recognizes citrate ion selectively out of all carboxilic acids of the Krebs Cycle
in water.
Manuscript 14 presents a bismacrocycle molecule to recognize metal ions in water solution, such as, Cu(II),
Zn(II), Pb(II), Cd(II) and Hg(II) with important role in environmental problems. A study of a phosphate ester bond
present in BNPP cleavage was reported using the Zn(II) complexes.
With the group of Professor Casabó and Lluis Escriche, the receptor used is always a macrocycle having
nitrogen and sulphur as donor atoms in order to modify the coordinative properties and extend these
chemosensors to other target atoms or anions different from those achieved through the polyamine receptors.
In paper 15 the synthesis of a new ligand containing a tetraamine chain linked in the 6,6" positions of a
terpyridine unit is reported. Cu(II), Zn(II), Cd(II) and Pb(II) complexation was studied in aqueous solution by
means of potentiometric, spectrophotometric and spectrofluorimetric measurements.
In the case of papers 16, 17 and 18, some molecular devices provided with pyridine or anthracene units have
been studied. In detail, paper 16 has been one of the most downloaded full papers in Inorganic Chemistry
2006. The system reported changes in colour upon complexation with an external ligand, opening their use as
a potential colorimetric device. Paper 17 concerns Pd(II) complexation and its recognition in solution by
fluorescence quenching. New magnetic materials have been reported in paper 18. Here some halide anions
modulate the magnetic properties of Ni(II) complexes, changing from antiferromagnetic, to paramagnetic and
ferromagnetic compounds.
In the framework of the Project POCI/55519/QUI/2004, “Towards New Supramolecular Devices: from new
synthetic methods to multifunctional applications”, we worked in the development of new chemosensors
provided with 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) and the trivalents Cr(III)
and Al(III). This subject was developed mainly with the collaboration with the group of Prof. Rufina Bastida,
from the University of Santiago de Compostela. Paper 19 deals with this subject as well as the studied of an
example of Energy Transfer form a macrocyclic device to an Europium(IIII) and Terbium(III) metal center. With
the group of Prof. Manuela Raposo and Susana Costa, from the University of Braga, Portugal we have continued
a study of new bithiophene-phenanthroline systems and their Ru(II) complexes; part of this work was presented
in several congresses reported here.
Towards “multifunctional applications” we have explored the interaction of new pyrene derivatives with barbituric
acids, parabanic and cianuric acids (Paper 11). Several macrocyclic compounds based on pyridine were
characterized in the solid state in collaboration of Dr. Emília Bértolo from the Canterbuy Christ Church University
(Paper 20).
Into the Task 3 of the Project POCI/55519/QUI/2004, concerning the Analytical applications and Bioanalytical
studies, paper 2 has been published, with the collaboration of Dr. José Luis Capelo (Fellow Researcher of
REQUIMTE) and with Prof. Isabel Moura (BIOPRO group of REQUIMTE). A complete review of sample treatment
for protein identification by MS techniques was developed.
A quiet sensitive sensor for the detection of Water in Aprotic Solvents at Concentrations Below 50 ppm was
exploited Using Hydrogen Bonding Specific Interactions of a Ru(II) complex containing one 2,2’-bipyridyl and
four cyanide ligands (Paper 25)
In 2007 we will develop new molecules in the frame of the project in course POCI/QUI/55519/2004 incorporating
the O, N and S as donor atoms as heavy metal ion chemosensors. New green synthetic methods will be
tested using clays and zeolites, and using ultrasound as reaction power. New Ru(II), Os(III) and Ir(III) complexes
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will be developed for energy transfer studies. A new research area covering bio-applications of fluorescence
chemosensor will be started in collaboration of Prof. David Parker from the University of Durhum in UK as well
as will be keep working all other national and international collaborations mentioned above.
The study of ternary systems consisting in coordinatively unsaturated metal-ligand complexes and an anion
will be explored towards anion sensing using ligands from the Group of Prof. Enrique García-España (Valencia,
Spain) and Prof. Antonella dalla Cort (Rome, Italy) in the framework of an undergoing COST programme.
Chromogenic materials
In 2006 we have applied for a FCT-MCTES project entitled “Chromogenics”. It aims at the development of
compounds (or devices) whose colour changes upon an external stimulus of light, electric current, or temperature,
defining respectively photochromic, electrochromic and thermochromic materials. This project arose in the
frameworkof an undergoing collaboration with the company YDreams and anchors on our work in flavyliumbased photochromic systems as models fopr optical memories. While work on photochromism evolves in our
lab in the flavylium research, we started with the basics in electro- and thermochromism.
Several transparent conducting supports, such as ITO coated glass and PET, were used to develop techniques
leading to deposition of classic electrochromic systems (Prussian Blue, methylviologen) and new
lelectrochromic films synthesized by the Group of Prof. Cristina Freire (Porto) of REQUIMTE. These coated
electrodes were tested in solution and in the solid state using in this case hydrin as solid electrolyte and were
characterized electrocoulorimetrically (results presented in international meetings and submitted to publication).
These preliminary results prompted the submission of three applied projects to ADI (Innovation agency,
www.adi.pt) based on electrochromism applied to writing boards, cellulose-based paper and clothing involving
Associate Labs REQUIMTE and I3N (Institute of Nanostructures, Nanomodelling and Nanofabrication), YDreams
company and industrial partners specialized in each support.
Thermochromism occurs when a color change is observed on a given material when the temperature changes.
Several materials can undergo thermochromic transitions, like leuco dyes (where the transition is due to a
chemical reaction) and liquid crystals (where the transitions occur due to the change of the physical organization
at a molecular level). Leuco dyes such as crystal violet lactone are able to undergo reversible thermochromic
reactions in the presence of a so-called developer (normally a weak acid) and of a co-solvent with a melting
point near room temperature (e.g., dodecanol). These materials were studied in collaboration with YDreams®,
in order to achieve new thermochromic formulations that could be used on technological applications. The
research was focused mainly on encapsulating techniques in order to produce immobilized pigments and
also on the nature of the interaction between the leuco dye and the developer. New developers were found and
will be subject of future work, with applications also in other phenomena, like electrochromism, rendering
systems with different chromic properties. In 2007 the development of supramolecular structure with
thermochromic reactions, like liquid crystals or ionic liquids, will also be pursuit, taking into account previous
results obtained with the leuco dyes.
Cultural Heritage
An important part of our Cultural Heritage is based on material objects. The spiritual legacy is only possible
to pass on to the new generations if we know how to preserve the material object, and the complex evolution
it has endured with time. Since 2000, we have been involved in the photochemical and photophysical
characterization of historical dyes, by studying their molecular mechanism of degradation and proposing
strategies to improve their lifetimes (4 papers submitted, from which 2 were accepted for publication 2006 and
1 in 2007). Testing of the potentiality of confocal microfluorescence spectroscopy as an in situ analytical
method for lake pigments used in medieval manuscript illuminations and Impressionist painters was also
carried out (1 paper submitted). Concerning modern art conservation, we have also continued the study of the
photodegradation mechanisms in vinyl paints (1 paper accepted for publication in 2007).
For 2007, we plan to continue the research in these subjects.
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Spectral codification of molecular information
This is a starting research subject that deals with the use of probes with differentiated absorption properties
associated to specific molecules/functions which allow the use of light to initiate and control the course of
molecular events selectively, using photochemical (e.g. photocleavage) or photophysical (e.g. energy transfer)
processes.
Presently the research is focused in two main subjects:
1 - Single pot detection of single nucleotide polymorphisms (SNP’s) using probes labeled with chromophores
that are able to transfer excitation energy to chosen fluorophores. In such a way the final step of a single base
extension (SBE) reaction is signaled by the presence of an energy transfer process that was absent before
addition of the base. The excitation energy transfer from the probe donor label to the incoming nucleotide
acceptor label changes the fluorescent signature of the sample and simultaneously identifies inequivocally
the incoming nucleotide. This research work is part of a Ph.D research program (Letícia Giestas) and is done
in collaboration with the University of Algarve (Prof. Guilherme Ferreira) in the framework of the research
project POCTI/BIO/38922/2001 “Randomly ordered DNA sensors based on direct questioning of immobilised
probes with wavelength shifting pairs” financed by FCT-MCTES.
2 - Synthesis of nucleosides bonded to protect groups that can be cleaved with light at positions 5´ and/or 3´.
Presently phosphate esters of coumarin derivatives are beeing used as protecting groups with the aim of
achieving photocleavable protecting groups whose absorption bands cover the visible region. This will allow
to associate different chromophores to different nucleosides (natural or synthetic) for selective release under
monochromatic irradiation. Developments of near-infrared absorbing photocleavable groups for in vivo applications
are a goal for the near future, as well as the development of solvatochromic photocleavable groups, that will
allow introducing selectivity to the environment in the release of the nucleosides.
This research work is part of a Ph.D research program (André Vidal Pinheiro) and is done in colaboration with
the Seccção Autónoma de Biotecnologia of University Nova de Lisboa (Prof. Pedro Baptista) in the framework
of a research project submited to FCT-MCTES for finantial support in 2006 and wayting for evaluation.
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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 / Invited Assistant Professor
Mário Gomes
Post-Doct Fellows:
Yuri Binev, Sunil Gupta, Qingyou Zhang, Ricardo Mendonça, Susan Matthew
Ph.D. Students:
Luís Pinto, Vasco Bonifácio, Valdemar Figueira, Goncalo Carrera, Diogo Latino
M.Sc. Students:
Diana Almeida
Undergraduate Students:
Mónica Estevão, Ana Marta, Ana Luísa
Number of articles in scientific journals: 22 (30-51)
Number of Ph. D. Thesis: 4 (3-6)
Scope and description of on-going research
Novel rearrangements of the indole framework were studied. N-alkylation of N-acetyl L-tryptophan methyl
ester with 3,3-dimethylallyl bromide gave (S)-methyl 2-acetamido-3-[1-(3-methylbut-2-enyl)-1H-indol-3yl]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 towards a considerable ionic character of this rearrangement.
Several syntheses of Tamiflu, the antiviral for avian flu treatment, rest upon the ring opening of a chiral aziridine.
A new chiral aziridination reaction was developed and the absolute configuration of the 3-membered ring
defined through X-Ray analysis.
The chiral induction in new g-amino acids prepared by a radical pathway from the a-amino acids was study.
The use of different Lewis acids as catalysts was tested.
In the last few years, the phytochemical research efforts focused on the characterization of bioactive natural
products from medicinal plants of diverse origin (http://www.dq.fct.unl.pt/qoa/abreu). Following the screening
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of medicinal plant extracts from Guinea-Bissau, and using a methodology of bioassay-guided isolation, several
bioactive metabolites were isolated and identified by chemical and spectroscopic methods, as antimalarial
alkaloids from Sarcocephlalus latifolius, cytotoxic trichotecenes from Holarrhena floribunda, leishmanicidal
proanthocyanidins from Khaya senegalensis, and a 41-member library of alkyl and alkenylphenols from Ozoroa
insignis. From the species Moricandia arvensis and Pedilanthus tithymaloides, collected in Tunisia and Cuba,
respectively, the identification of antioxidant phenolic glycosides was disclosed. Macrocyclic diterpenes that
inhibit multidrug resistance (MDR) in cancer cells, were isolated from Portuguese Euphorbia species.
Some species of tthe Solanum genus show added value due to their nutritional and medicinal properties. The
interest on Solanum cernuum Vell. species (Brazil) largely contributes its traditional use due to antiinflamatory
effect and to the action in hepatic and gastric injuries. The dichlorometane extract produces dose dependent
gastric protection induced by ethanol.
Along with the chemical screening of the dichloromethane extract (mainly composed of alkanes, steroids and
a xanthophyll) different steroidal fractions were assayed for their antineoplastic activity against tumour cellular
lines. By structure analysis of the tested fractions the 29-norcycloartane components seems to be responsible
for the observed action against lung cellular lines that justifies further studies.
The acidic extraction method of quinolizidine alkaloids from Ulex Jussiaei was optimized. Several acids were
used, in different concentrations and extraction times (ambient temperature). It was verified that the most
efficient method is the use of acetic acid 1M (2,5h) meaning an increment of 33% in the extracted material
when compared with previously published results.
As alternative method for the alkaloids extraction supercritical fluid was assayed (SFE). It was carried through
sets of experiences using as fluid of extraction CO2 and CO2 modified with methanol or water. The
chromatographic comparison of for the classic method and SFE discloses the inability of the last one for the
desired extration.
Catecholamines comprise a distinct class of biomolecules, of which, the tyrosine derivatives dopamine,
noradrenaline, adrenaline and DOPA are the most familiar. Cardiovascular diseases are related to high values
of mortality and morbidity in the world, every year. High concentrations of catecholamines are found in several
cardiovascular diseases. Adrenaline is a reactive molecule that can oxidise to form ROS and reactive products,
as semi-quinones, quinones and aminochromes. 5-(Glutathion-S-yl)-adrenaline, an adrenaline adduct of
glutathione, was for the first time synthesized and used as standard for the identification and quantification in
toxicity studies involving isolated calcium tolerant cardiomyocytes.
The mechanisms by which MDMA (Ecstasy) catecholamine metabolites produce selective serotonergic
neurotoxicity are unclear, specially, the identity of a specific chemical neurotoxic entity (if there is one) have
yet to be determined. 3-Methoxy-α-methyldopamine (HMA) and 3-metoxi-N-methyl-α-methyldopamine (HMMA)
are two of the four principals MDMA metabolites. The synthesis of the HMA and HMMA were approach by two
different methodologies: the first, involving the chemical oxidation of 3-metoxi-α-metildopamina by AgO and
PbO2, lead to the production of a dimmer of the starting compound as a result of oxidative coupling; the
second involving the enzymatic methylation of 5-(GSH)-α-MeDA by COMT e SAM is still under evaluation.
The MOLMAP molecular descriptors of products and reactants was applied to genome-scale classification of
metabolic reactions. QSAR of antioxidant properties of phenolic compounds was derived from MOLMAP
descriptors and neural networks. Automatic classification of reactions was also studied on the basis of
calculated 1H NMR spectra by the program SPINUS.
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) through Prof. Paulina Mata, who belongs now to the Advisory Subcommittee
of the Chemical Nomenclature and Structure Representation Division (VIII) http://www.iupac.org/organ/members/
m/mata.html. In 2006 the collaboration was intensive, particularly in the revision of IUPAC recommendations
on specifications of molecular configuration to be included in the next IUPAC Book of Nomencalture of Organic
Compounds. The final draft was concluded.
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Molecular Gastronomy was also developed, through international connections with researchers in the area,
particularly Dr. Hervé This ( Institut National de Recherche Agronomique), national collaborations (Prof. M. C.
Loureiro Dias – Instituto Superior de Agronomia). Prof. P. Mata received in Paris, with her team, an international
price in Molecular Gastronomy for adapting the alginate technology to specific Portuguese cuisine.
Work continued in the science teaching and divulgation front which hopes 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: a) collaboration with Ciência Viva (participation in national and international
projects) and b) the publication of science divulgation articles in the press.
Material Safety Data Sheet of several hazardous chemical compounds were elaborated in Portuguese following
the REACH directives.
Plans for 2007
Applications of the chiral aziridination will be sought.
Use of the 3,3-sigmatropic rearrangement for the obtention of labile allenes and polysubstituted pyridines will
be amplified.
Substituted algicides derived from bacillamide will be synthesised and tested in the most common freshwater
and marine algae.
Phytochemical study of medicinal plants of diverse origin, oriented towards the characterization of antioxidant,
anti-inflammatory, and cholinesterase inhibitors.
The synthesis of the monomethoxylated cysteine and glutathione adducts of 4-hydroxy-3methoxymethamphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) will be continued for
neurotoxic evaluation.
A prorogation of the project POCTI/QUI/44501/2002 was allowed and is intented to conclude the proposed
synthesis of fenanthroquinolizidines.
Application of the MOLMAP representation of chemical reactions and compounds to the automatic learning
of reactivity in databases of chemical reactions. Improvement of genome-scale classification of metabolic
reactions. Application of the SPINUS system for the generation of databases of simulated 1H NMR spectra.
Development of materials for science education will continue towards the population at large, and the blinds
in particular. In collaboration with the Check Republic, Slovakia and Norway a new project was launched to
developed ICT materials to teach chemistry to blind students.
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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
Ana Maria Madeira Faísca Phillips
Principal Researcher
Post-Doct Fellows:
Krasimira Petrova, Miglena Georgieva, Christo Roussev, Galya Ivanova Ivanova
Ph.D. Students:
Vitor João Salgueiro Rosa, Paula Correia da Silva, Maria João Morgado, Filipe José dos Santos Duarte,
Bruno Pedras
M.Sc. Students:
Catarina Soares, Ricardo Barata
Aldino Viegas, Daniel Jana, Rui Pinto
2006 Activities
Chiral amines are important organic compounds, present in many pharmaceuticals, and often used as chiral
auxiliaries and as ligands for metal-catalyzed reactions. Since about 2000, they have become the subject of
much interest in another area: organocatalysis. The use of small organic molecules to catalyze organic
reactions is now well established as a new area of research. The organocatalysts used are metal-free, often
robust and usually non-toxic and friendly to the environment. The majority of organocatalysts developed up to
now are chiral amines. Our research during 2006 was centred in this area. The development of methods to
synthesize novel chiral amines and diamines, as well as some sulfur-containing analogs, from naturally
occuring (2R, 3R)-(+)-tartaric acid, already under way in 2005, was concluded and the work was published. A
library of chiral amines and diamines was developed, and one of them, bis-N-methylmethanamine, was
successfully used to catalyze the diastereo- and enantioselective addition of cyclohexanone to (E)-βnitrostyrene.
During this period, chiral piperazines derived from natural amino acids were also applied in organocatalysis.
It was found that C2-symmetric piperazines, used in substoichiometric amounts, could promote the addition
of unmodified aldehydes to aromatic nitroalkenes with high yields and in a highly diastereoselective and
enantioselective manner. This work has also been published.
The study of stereoselective methods in organic synthesis starting from renewable feedstocks is a major
target in organic synthesis. We have continued to explore the chemical transformation of low molecular
weight carbohydrates, as renewable natural chiral starting materials, into value added materials. By exploring
new organic reactions in solventless conditions and by using microwave technology as alternative source
of energy, we are contributing to the development of cleaner and more efficient methodologies.
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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 to the sugar nucleus (D-glucose,
D-xylose and sucrose) by two different procedures (esterification and Wittig olefination), both under microwave
irradiation. Water soluble dienes were also prepared by exposure of the hydroxyl groups of the sugar auxiliary.
1,3-Dipolar cycloaddition reactions have the ability to generate rings containing several contiguous
stereocenters, in one single synthetic operation, with a maximum of atom economy. The vinylic systems
attached previously to carbohydrates are excellent targets as dipolarophiles in these reactions, whether by
addition of azides for the preparation of 1,2,3-triazolines or nitrones for the synthesis of chiral isoxazolidines.
The need of biodegradable plastics has increased during the past decades, not only due to increasing
environmental concerns, but also for their biomedical applications. Vinylsugar starting materials, obtained by
regioselective synthesis via protection-deprotection sequence starting from sucrose, glucose, and xylose,
have been copolymerized with olefin monomers using focused microwave reactor, thus exploring new approaches
for performing the reactions as solvent-free or water-soluble procedures in demand for free of wastes and toxic
organic solvents.
By attaching the sugar to a polymer it is possible to form amphiphilic materials, which find widespread use in
the chemical, pharmaceutical, and food industry, because of their unique ability to self-assemble. We are
working on the synthesis of amphiphilic polymer with hydrophobic olefin backbone and hydrophilic sugar
moieties as side chains.
As another approach to green polymer materials, the use of α- and β-pinenes as comonomers for the synthesis
of polyolefins containing side chain sugar moieties have been investigated. By introduction of azide group
other applications of the carbohydrate based polymers could be achieved – performing 1,3-cycloadditions
with vinyl substrates in stereoselective manner, and using the method for “click-chemistry” identification or
separation procedures.
Also, the synthesis of new environment-friendly and readily biodegradable chelating agents was accomplished
by chemical transformation of stereospecific amino acids, which were used in the asymmetric synthesis of
natural aminopolycarboxilates (APCAs) and derivates. We have focused our attention in controlling the
stereochemistry during syntheses, for providing stereoisomerically pure compounds.
Part of the work concerning the synthesis of cobalt (II) complexes of general formula CoX2(á-diimine) was
completed and published. (This work was the result of collaboration between the following Universities: Centro
de Química Estrutural, Instituto Superior Técnico (Prof. Pedro Teixeira Gomes, polymerization studies))
Laboratoire DECOMET, ILB Université Louis Pasteur Strasbourg, France (Prof. Richard Welter, X-Ray)
Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas and INTEC, Universidad Nacional del
Litoral, Santa Fe, Argentina. (Prof Carlos Brondino, EPR)
We have synthesized new α -diimine ligands of the type Ar-BIAN (Ar = naphthyl) by reacting naphthylamine with the corresponding dicetone, with the aim of obtaining multifunctional ligands, in order to test on
one hand the catalytic properties of the complexes and also to explore their capacity as chemosensors. We
have accomplish the preparation and characterization of the new
ligands and its reactions with PdCl2 and the naked nickel compound Ni(BF4)2. This two reactions afforded compounds of different stoichiometry, with one or two ligands per metal cation respectively. In the case of the palladium complex we were able
to determine the solid state structure by single crystal X-ray diffraction, shown in the figure. The structure shows that, of the two
possible isomers anti- and syn- , the anti- isomer is formed. All
complexes have been characterized by elemental analyses, IR
and FAB spectroscopies and by NMR spectroscopy. ( this work is
been done in collaboration with the following Universities
Universidade de Vigo, Spain.(Dr. Berta Covelo, X-Ray) Departament de Química Inorgànica and Centre de
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Recerca en Química Teòrica, Barcelona, Spain. ( Prof Gabriel Aullon, theoretical calculations) and Prof Carlos
Lodeiro, REQUIMTE (photophysical and photochemical studies)
New Schiff base ligands have been synthesised based on a new group of bithiophene systems and crown
ethers. Also tripodal (3+1) imine and amine derivatives have been prepared, via the condensation of the
appropriate carbonyl and polyamine precursors. These new ligands have been complexated to 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).
Their photophysical and photochemical properties were studied. (In collaboration with Prof Carlos Lodeiro,
REQUIMTE)
The reactions of the P,O ligands (diphenylphosphino)acetophenone Ph 2 PCH 2 C(O)Ph and
(diphenylphosphino)acetamide Ph2PNHC(O)CH3 with the cobalt cyclopentadienyl precursor [(η5-C5H5)CoI2(CO)]
have been investigated. These results will be published briefly.
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.
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.
Stereoselective free radical reactions. Theoretical work has been developed aiming the rationalisation of
chiral auxiliary based free radical mechanisms, with or without Lewis acid catalysis. Results were compared
with the previous ones obtained for ionic systems, and new auxiliaries shall be proposed. A similar work has
been started, aiming the rationalization of chiral catalyst based free radical reactions. The work is still in the
beginning but we expect to get useful informations during the next months.
Application of NMR techniques and Molecular Modelling to the study of intermolecular interactions.
Molecular Interactions and CO2-Philicity in Supercritical CO2. A High-Pressure NMR and Molecular
Modelling Study of a Perfluorinated Polymer in scCO2 .The carbon and fluorine chemical shifts of mixtures
of carbon dioxide and Krytox, a carboxylic acid end-capped perfluorinated polyether used as stabilizer for the
dispersion polymerization of methyl methacrylate, have been studied using high-pressure, high resolution
nuclear magnetic resonance and molecular modelling. Both experimental 13C and 19F HP-NMR results and
molecular modeling studies support a F…CO2 site-specific Lewis acid – Lewis base interaction model. A
positive entropic variation for the formation of CO2 - fluorinated solute complex is advanced as an explanation
for the higher solubility of perfluorinated molecules when compared to the non-fluorinated analogues. (results
published in 2007)
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.
Interaction studies by NMR (STD-NMR, line broadening and 15N-HSQC) showed that CtCBM11 binds to
cellohexaose and allowed to epitope map the interaction in the ligand structure. The results are in agreement
with the average structures obtained with MD simulations.
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2007 Activities to be developed
The application of chiral piperazines as organocatalysts for other asymmetric processes will be investigated.
As a follow up to the recent research, the asymmetric addition of aldehydes or ketones to other Michael
acceptors will be investigated first. The possibility of using organocatalysts in other organic transformations
will then be tested, for example asymmetric allylic carbon-carbon bond formation, α-halogenation, epoxidations
and other oxidation reactions. The possibility of using these catalysts in processes for which there are yet no
organocatalytic versions will also be attempted.
Many compounds with different functionalities have been tested for their ability to promote the cleavage of
RNA, DNA or phosphodi(tri)ester models and a common strategy for the design of artificial nucleases is to
incorporate, into a synthetic framework, functional groups known to be present in enzyme active sites and
essential for maintaining catalytic activity. Similar approaches could be applied to extend catalytic repertoire
of PEI-based catalysts and various derivatisation reagents could be considered. It is our aim to study additional
PEI derivatisation to create microenvironments with diverse properties: introduction of positive charges
(guanidinium groups) or negative charges (carboxylate groups, after deprotection). Benzyliodide anchors
could be used to append functionality [varying R5-R7 could be used for further derivatisation (using Suzuki,
Stille and Sonogashira/Castro Stephens reactions catalysed by palladium)].
Also, we pretend to continue to explore novel methods for functionalisation of natural carbohydrates,
such as introducing vinyl ether groups based on Gassman method, and regioselective esterification under
Mitsunobu conditions. Polymerization and block copolymerization of obtained unsaturated sugar compounds
with alkene type monomers such as styrene, methyl methacrylate, terpenes, and maleic anhydride, will be
studied using advanced initiating systems, such as living cationic block-copolymerisation initiators, and
Ziegler-Natta type coordination catalysts. For thus obtained novel polymeric materials, research will be carried
out to determine their use as linkers for chemistry on solid supports, affinity chromatography, for water
soluble polymer supports or as a medium for the generation of combinatorial libraries, as well as their
biocompatibility and biodegradability as environment-friendly materials.
In a related project we propose the use of renewable feedstocks (carbohydrates) as chiral auxiliaries, to
induce the stereochemistry required in the core structure of interesting and valuable compounds. By exploiting
microwave radiation as a more efficient heating technique, combined with solventless procedures, we intend
to reach some sustainable synthesis, which can be scaled up for use in chemical industries.
Another objective will be to continue with the work of cobalt complexes, trying to introduce ligands such as
indenyl, cyclopentadienyl, alkyl, and hydride in the already synthesised Co(II) α-diimine complexes. New αdiimine 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.
We expect also to continue our work dealing with intramolecular aldol reactions based on molecular modelling,
NMR and synthesis. Our final aim is to clarify 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.
Concerning the stereoselective free radical reactions, the work for the rationalisation of chiral auxiliary
based free radical mechanisms, with or without Lewis acid catalysis, will continue.
We plan to continue the studies which explore the complementary between the information obtained from
NOE with that from PFG diffusion NMR experiments. We believe that due to the complementary of the
techniques, this combined approach will soon become a general procedure for the study of intermolecular
interactions.
In the next year we will continue to apply NMR techniques to the study of intermolecular interactions,
especially ion-pair strength in a new family of chiral ionic-liquids and also solute-solvent interactions and
chiral discrimination we will also extend these studies to chiral recognition in small bimolecular systems and
host-guest complexes.
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PHYSICAL ORGANIC CHEMISTRY / RADICAL CHEMISTRY
Head of Laboratory: Abel J. Vieira, Associate Professor
Staff Members:
João Paulo Noronha
Alexandra Moita Antunes
Assistant Professor
Ph.D. Students:
Pedro Manuel Santos, Mónica Pombinho Matos, João Adalberto Lourenço
Number of articles in scientific journals: 2 (78, 84)
2006 Activities
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.
Toxicity evaluation of the anti-HIV drug Nevirapine. Preparation of the metabolite 12-hydroxy-nevirapine,
converted into an electrophilic derivative by mesylation and used as a surrogate for the metabolically plausible
sulfate ester. Preliminary studies on the in vitro reaction of this electrophile with the amino acid cysteine and
the peptide glutathione lead to the formation of covalent adducts that were identified by HPLC-MS and, after
isolation, fully characterized by NMR.
Dtermination of the D-arabinitol/L-arabinitol ratio by GC-MS.
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CHEMICAL ENGINEERING SCIENCE
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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 Caldeira
Associate Professor
Jorge Lampreia
Assistant Professor
Cristina Costa
Assistant Professor
Anjos Macedo
Assistant Professor
Pedro Tavares
Assistant Professor
Alice Pereira
Assistant Professor
Carlos Salgueiro
Assistant Professor
José Franco
Assistant Professor
Stephane Besson
Assistant Professor
Gabriela Almeida
Assistant Professor
Sergey Bursakov
Ana Teresa Lopes
Technician
Post-Doct Fellows:
Sofia Pauleta, Miguel Pessanha, Rui Duarte, Patricia Sousa, Cristina Timóteo, Ludwig Krippahl, Krisztina
István, Gabriela Rivas, Pablo Gonzales, Celina Santos, Raquel Grazina
Ph.D. Students:
Cristina Cordas, Jorge Dias, Filipe Folgosa, Carlos Martins, Joana Raimundo, Márcia Guilherme, António
Nunes, Inês Isabel Gomes, Gonçalo Dória, Simmone dell´Acqua, Cristiano Mota, Célia Silveira, Anna Kladova
Research Students:
Américo Duarte, Miriam Sousa, Ricardo Alves, Sara Berguete, Ricardo Pais, Duarte Alves, Ana Rita Fins
Number of articles in scientific journals: 24 (2, 57-79)
A - 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).
Nitrite reductase: biosensing nitrite using the system nitrite redutase/nafion/methyl viologen
This work describes the construction and voltammetric characterization of a nitrite biosensor based on a
cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as
redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion
sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon
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must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between
the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain
diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k(m)(app).
The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout
the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in
the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear
range. For an optimized ratio enzyme/mediator the sensitivity was 445+/-8mAM(-1)cm(-2), within the linear
range 75-800muM; the lowest detected nitrite concentration was 60muM.
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.
B - STRUCTURE AND MECHANISMS IN MOLYBDENUM AND TUNGSTEN ENZYMES
Molybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a
diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W)
is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo,
hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition,
these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal
cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and
an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this
Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal
distinct aspects of these enzymes.
EPR and kinetic studies on perilplasmic Nitrate reductase from D. desulfuricans ATCC 27774 Ammonification
Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron
paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing
bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase
family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo
bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe-4S] cluster. EPR-monitored redox titrations,
carried out with and without nitrate in the potential range from 200 to -500 mV, and EPR studies of the
enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which
indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the
redox properties of the Mo active site, but not those of the [4Fe-4S] center. The possible structures and the
role in catalysis of the distinct Mo(V) species detected by EPR are discussed.
Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction
Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules
such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and
-III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of
this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The
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characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information
on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing
enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory
nitrate reductases. We review here the biological and molecular properties of these three enzymes along with
their gene organization and expression, which are necessary to understand the biological processes involved
in nitrate reduction.
Molybdenum and tungsten enzymes: the xanthine oxidase family
Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in
general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the bestcharacterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family
are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this
family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details
that complement the structural information. Altogether, these studies have provided an important amount of
information on the characteristics of the active site and the electron transfer pathways.
Biochemical and spectroscopic characterization of an aldehyde oxidoreductase isolated from
Desulfovibrio aminophilus
Aldehyde oxidoreductase (AOR) activity has been found in a number of sulfate-reducing bacteria. The enzyme
that is responsible for the conversion of aldehydes to carboxylic acids is a mononuclear molybdenum enzyme
belonging to the xanthine oxidase family. We report here the purification and characterization of AOR isolated
from the sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254, an aminolytic strain performing
thiosulfate dismutation. The enzyme is a homodimer (ca. 200 kDa), containing a molybdenum centre and two
[2Fe-2S] clusters per monomer. UV/Visible and electron paramagnetic resonance (EPR) spectra of D.
aminophilus AOR recorded in as-prepared and reduced states are similar to those obtained in AORs from
Desulfovibrio gigas, Desulfovibrio desulfuricans and Desulfovibrio alaskensis. Despite AOR from D. aminophilus
is closely related to other AORs, it presents lower activity towards aldehydes and no activity towards Nheterocyclic compounds, which suggests another possible role for this enzyme in vivo. A comparison of the
molecular and EPR properties of AORs from different Desulfovibrio species is also included.
Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase
Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio
desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR)
spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in
which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown
to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form
yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR
signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic
interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPRactive samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through
an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data
indicate that the main difference between both species is an equatorial ligand to molybdenum which was
determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the
sulfido ligand is not essential to determine the EPR properties in both Mo-As complexes is achieved through
EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately
followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer
pathway, which is essential for catalysis, is not modified upon inhibition.
C - ANTAGONISTS MO AND CU - Heterometallic clusters present on novel proteins
Orange Protein
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The orange protein structure is currently being determined by NMR and X-ray. For 3D structure of the apoprotein using NMR spectroscopy the apo-protein was heterologously expressed in E.coli using minimum
media in order to label the protein with either 15N or 15N/13C. Triple-resonance experiments were acquired,
such as 1H-15N HSQC, 1H-13C HSQC, HNHA, CACBNH, CACB(CO)NH, HNCO, HN(CA)CO for sequential
assignment and backbone assignment. For side-chain assignment (H)CCH TOCSY and was collected with
two different mixing times, and for gathering structural constraints for the structure calculation 1H-13C-HSQC
NOESY, 1H-15N HSQC – NOESY and 1H – 1H NOESY. Currently the sequential assignment is complete to
87%, and the side chains are assigned to 60%.
15N relaxation measurements were also performed. Reliable values of 15N R1 and 15N R2 and {1H}-15N NOE
were obtained for 101 of the 106 assigned backbone NH resonances. R1, R2 and NOE values were constant
throughout the molecule, with the exception of the 53 – 56 region. Average values calculated are 1.72 ± 0.06
s-1, 10.9 ± 0.4 s-1, and 0.75 ± 0.3 for R1, R2 and NOE, respectively.
For apo-orange protein X-ray crystallization conditions were optimized and crystals were obtained and preliminary
diffraction studies are currently being done. For blue protein the crystallization conditions are still being
optimized.
The OPR was heterologously expressed in E. coli as an apo-protein. The Mo-Cu cluster was reconstituted
using [MoS4]2- and CuCl2 in the presence of the over-expressed apo-protein. Biochemical characterization of
the reconstituted protein was performed including Uv-visible spectra, metal content and EPR spectroscopy
and the results were compared with the native protein. The UV-visble spectrum of the reconstituted protein is
similar to the one obtained for the native protein with maximum peak absorptions at 360 and 480 nm. The
metal cluster content ratio is the same of the native protein, 2Mo:1Cu. A 2W: 1Cu cluster was also synthesized
and the apo-orange protein was also able to incorporate this type of cluster.
A new orange protein was isolated from Desulfovibrio alaskensis and their preliminary biochemical
characterization was done. The metal content ratio determined by ICP for this protein is the same of the
D.gigas orange protein, 2Mo:1Cu. The protein is also a monomer of approximately 12.8 kDa. The N-terminal
of the D. alaskensis ORP was chemically sequenced and the NCBI alignment revealed an identity of 90 %
homology with a MTH1175-like domain family protein predicted by Desulfovibrio desulfuricans G20 complete
genome sequencing and annotation.
Blue Protein
A new blue protein was isolated from D. alaskensis. This protein contains Mo and Fe but not Cu. The molecular
weight is 260 kDa (18 subunits- 15.3 kDa). The UV/visible spectrum shows peaks at 570, 530 and 315 nm.
The complete DNA sequence was obtained and the aminoacid sequence had a 94% homology to the a zinc
resistance associated protein predicted by the annotation D desulfuricans G20 complete genome. Related to
task 7, EXAFS studies on this protein have shown that for the Mo K-edge EXAFS, the following interactions
were identified: Mo=O bond at 1.68 Å, Mo-O bond at 2.02 Å, a short Mo-S bond at 2.19 Å, a long Mo-S bond
at 2.35 Å and a Mo-Fe bond at 2.71 Å. From the Fe K- edge was possible to see that the iron is coordinated
by 3 S atoms at 2.25 Å and 1 N atom at 2.02 Å. Based on the Mo K-edge analysis the second peak has been
attributed to the Fe-Mo contribution. However, the EXAFS curve fitting resulted in significantly longer Fe-Mo
distance as compared with that obtained from the Mo K-edge analysis (2.79 Å vs. 2.71 Å). Also the respective
Debye-Waller factor was higher (0.006 Å2 vs. 0.003 Å2). This result prompted the investigation of the presence
of an additional metal-metal contribution at similar distance, which potentially could interfere destructively
with the Fe-Mo signa
Related to task 5, RT-PCR studies to address the physiological role on both ORP and blue genes were
performed for D. desulfuricans G20 (D. alaskensis) and the results are different for both proteins. The expression
of the genes coding for these proteins was followed for bacteria grown in the presence of Mo (45µM). The
results showed that the ORP and blue genes are up-regulated by about the factors 6 and 160, respectively.
For the blue protein gene, the significant increased in the amount of mRNA, together with genome analysis
strongly suggests that this protein is part of a two regulatory component system that responds to high
concentrations of Mo.
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D - CYTOCHROME C PEROXIDASE – CCP
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.
• 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) were mutated. In the first stage, the mutant pseudoazurins have being biochemical characterized
and different parameters are being determined, such as the binding affinity, electron transfer rates and a
structural characterization of the complexes is also being 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.
• Cloning and over-expression of the cytochrome c peroxidase isolated from Pseudomonas nautica.
E - ADENYLATE KINASE
Expression in E. coli was used to understand the mechanisms of metal incorporation into AK in vivo conditions.
Metal is not essential for catalysis in AK and has no direct consequence on enzyme activities, but it important
as a structural compound. Essentiality and toxicity of metals (Zn2+, Co2+, Fe2+, Ni2+, Mn2+) were determined
during bacteria growth on a rich and controlled media. Optimal conditions for the bacterial growth and
recombinant protein production with different metals were obtained. The ratio metal/protein of 1/1 was attained.
Spectroscopic characteristicsof the expressed proteins were obtained and kinetic properties (Km and Vmax) of
these forms of AK are being studied. The structure of native zinc containing AK from D. gigas was resolved
with a resolution of 2.0 Å.
F - NANOSURFACES AND NANOPARTICLES
The work under development relates to the interaction of proteins and DNA with noble metal nanoparticles and
nanostructured surfaces.
Colloidal gold has been 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. Mixed metal nanoprobes and
nanoprobes possessing a magnetic core are being developed for enhanced detection capabilities and selective
“gene fishing” in complex biological mixtures. Transmission Electron Microscopy (TEM) is being 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.
The immobilization of ferrochelatase on Self-Assembled Monolayers (SAM) of alkanethiols on gold surfaces
was studied by AFM. Alkanethiols used to build the different SAM surfaces were selected based on their
hydrophilicities and induced surface charges.
Dynamic Light Scattering (DLS) and Circular Dichroism (CD) studies of cytochrome c interaction with gold
nanoparticles already initiated ion 2006 will be continued and expanded to yeast iso-1 cytochrome that
contains a cysteine. The role of this amino acid residue in the binding to the nanoparticles will be analyzed
and characterized.
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In 2006, on the field of Surface Enhanced Resonance Raman spectroscopic investigation of heme containing
proteins 2 different works has been started. On the one hand, Resonance Raman (RR) spectra of some
ferrochelatase proteins and their mutants, namely ferrochelatase from the Baccillus subtilis bacteria and from
the mouse species, has been collected and evaluated. On the other hand, for adsorption/SERRS study of
proteins on nanostructured surfaces, a mercaptoalkaneamide compound (6-(11-Acetylsulfanylundecanoylamino)-2-(bis-carboxymethyl-amino)-hexanoic acid) (NTA) has been synthesized as a ligand for
the selective binding of His-tagged proteins.
Since in case of the wild type and variant forms of Bacillus subtilis ferrochelatase RR spectra could be
obtained when these proteins were incubated with Ni(II)-protoporhyrin but could not be obtained with protoporhyirin
IX incubation, RR studies on this system will be continued. Further RR studies are planned for new mutant
mouse ferrochelatases that are highly active for the chelation of Ni(II). The previously synthesized NTA compound
will be used to functionalize surfaces and nanoparticles and the binding of His-tagged heme-containing proteins
will be analyzed from the point of view of the enhanced RR signal.
G - DEVELOPMENT OF ELECTROCHEMICAL BIOSENSORS
In recent years, there has been a budding increase in the social and political awareness of the need for
monitoring and controlling environmental and industrial processes.
As a consequence, there is a strong need to develop improved analytical tools which allow the reliable
inspection of the chemical or biological composition of every material which interacts with consumers or
nature. In this context, biosensors technology constitutes a very important area of R&D. The electrochemical
enzyme based devices are of particular interest due to their operational simplicity, low cost fabrication,
portability and real-time monitoring ability.
Due to the growing demand for quantifying nitrates and nitrites in food, physiological and environmental
samples, we have been developing electrochemical biosensors for the reliable determination of these ions in
complex matrices. All the systems rely on solid electrodes modified with nitrite or nitrate reducing enzymes
isolated from bacterial sources. Since the enzyme immobilization strategy is a critical issue, strongly defining
the analytical behavior of the resultant biosensors, several electrode configurations have been tested, using a
variety of immobilization materials, such as non-conducting polymers, electropolymerized films functionalized
with redox groups, sol-gel glasses and modified clays. The direct electron transfer between nitrite reductase
and a pyrolytic graphite electrode have been also explored. As expected, the analytical performance (detection
limit, linear range, sensitivity and operational stability) of each system is strongly dependent on the electrode
design.
H - SIMPLE METAL SITES
Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure
determination using soft X-ray
Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the
scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was
determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using
poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 A resolution. The overall architecture is very
similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxintype Fe centre, found in other SORs, is absent. This domain conserves the beta-barrel topology with an
overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the
iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is
observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the
characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron
centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric
unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus
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SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there
was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the
TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.
Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type
proteins and superoxide reductases
In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between
rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs
from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of
classes I and II, respectively, while SOR from the syphilis spirochete Treponema pallidum (Tp) was representative
of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a
trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different
kapp values, 176.9+/-25.0 min(-1) in the case of the Tp/Tp electron transfer, 31.8+/-3.6 min(-1) for the Dg/Dg
electron transfer, and 6.9+/-1.3 min(-1) for Dv/Dv, could suggest an adaptation of the superoxide-mediated
electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another
iron-sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with
a kapp value of 108.8+/-12.0 min(-1), and was then assigned as the potential physiological electron donor in
this organism.
Superoxide reductase (2007)
• Study of the electron transfer complexes between superoxide reductase from the different classes and its
electron donors – rubredoxin-type proteins.
• NMR and molecular docking simulations will be used in order to characterize the electron transfer surface
and elucidate the mechanism and pathway of the electron transfer.
• The structure determination of D. gigas superoxide reductase and Treponema pallidum rubredoxin using
NMR spectroscopy will be attempted.
Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe-4S] cluster
Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino
acids, containing one Fe3S4 cluster per monomer. Upon studying the redox cycle of this protein, we detected
a stable intermediate (FdIIint) with four 1H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences
between FdIIox and FdIIint were attributed to conformational changes resulting from the breaking/formation of
an internal disulfide bridge. The same 1H NMR methodology used to fully assign the three cysteinyl ligands
of the [3Fe-4S] core in the oxidized state (DgFdIIox) was used here for the assignment of the same three
ligands in the intermediate state (DgFdIIint). The spin-coupling model used for the oxidized form of DgFdII
where magnetic exchange coupling constants of around 300 cm-1 and hyperfine coupling constants equal to
1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence
for the three cysteinyl cluster ligands in DgFdIIint. This study, together with the spin delocalization mechanism
proposed here for DgFdIIint, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdIIox
and DgFdIIint.
Fuscoredoxin
We are studying and revealing the unusual properties of a Fe cluster in an unusual Fe-S protein (Fuscoredoxin)
isolated from Desulfovibrio desulfuricans ATCC 27774. This protein contains two types of Fe-S clusters: a
[4Fe-4S] cubane cluster and a novel one, [4Fe-2S-2O], which can attain four redox states. EPR and Mössbauer
data reported some of the magnetic and redox properties, but they are still far from being understood. We
cloned the gene in an expression vector (pGEX-6P-1) and performed site-directed mutagenesis aiming to
correlate the biochemical properties of the aminoacid involved in the metal center coordination with the
spectroscopic data obtained for the protein, in particular the data on the iron related the atypical centre. We
also have made a mutant consisting in the deletion of the first 25 amino acids residues (from the N-terminal)
to result in the specific loss of the [4Fe-4S], simplifying the data analysis. EPR and Mössbauer studies are
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in progress to clarify the redox properties of this mutant. These studies will also be an important tool for
revealing the biological function of fuscoredoxin.
Redox chemistry of low-pH forms of tetrahemic cytochrome c3
Desulfovibrio vulgaris Hildenborough cytochrome c(3) contains four hemes in a low-spin state with bis-histidinyl
coordination. High-spin forms of cytochrome c(3) can be generated by protonation of the axial ligands in order
to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes
in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of
square wave voltammetry and UV-visible, CD, NMR and EPR spectroscopies. These processes may be used
for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity
towards small molecules.
Type I copper proteins
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.
The bands at 450 nm and 590 nm have a L-metal charge transfer character (Scys – Cu ion; 590 nm: CysS π
→ Cu x2 – y2 and 450 nm: CysS σ → 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. Interpretation and fitting of the data is being carried out in collaboration with Prof. Carlos Brondino.
• The protein was crystallized in the oxidized form as a single crystal and the structure was determined at a
resolution of 1.3 Å. I have been involved in the structure determination of pseudoazurin using X-ray in collaboration
with Prof. Maria João Romão’s group.
I - VANADIUM(V)- COMPLEXES AND CALCIUM PUMP
Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of
decameric vanadate
Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of
68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower
extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration
of “metavanadate” solution that contains ortho and metavanadate species, as observed by combining kinetic
with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable
in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8
microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not
only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition
of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic
reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the
presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the “decavanadate”
interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and
induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and
transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of
vanadium (V) effects in biological systems.
J - OXYGEN ACTIVATION AND CELLULAR DETOXIFICATION (suproxide reductases and others)
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erritin 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 aim of the ferritin project is to further understand
the kinetic and spectroscopic observations on the iron uptake mechanism of the recombinant fast ferritins,
namely 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, the resultant study will also provide more general information concerning the
chemistry of carboxylate-bridged diiron centers in proteins
The desaturase project contains two major goals: (i) elucidate the catalytic steps of oxidative desaturation; (ii)
explain the role of diiron clusters in this catalytic mechanism. A multidisciplinary approach was proposed to
complete the goals. Proteins from Arabidopsis thaliana (a known plant model) are being studied. It is of major
importance to have good overexpression systems since milligram quantities of pure protein are needed. After
accomplish successful overexpression the proteins under study are purified from cellular extracts. Then, a
biochemical and spectroscopic characterization is performed. This will serve as a starting point for the
mechanistic studies, where the use of visible stopped-flow and rapid-freeze quench techniques will be applied
to identify and characterize the intermediates of each catalytic cycle. A model will be constructed and relevant
comparisons with other biological significant diiron cluster catalysis made.
Reactive oxygen species (ROS), when in excess, are among the most deleterious species an organism can
deal with. The physiological effects of ROS include amino acid chain cleavage, DNA degradation and lipid
oxidation, among others. They can be formed in the cytoplasm in a variety of ways, including auto-oxidation
reactions (FMN and FAD-containing enzymes) and Fenton reactions due to the cytoplasmatic pool of iron
ions. Superoxide anion, despite its short half-life in solution, is particularly pernicious, since it can form other
reactive ROS, such as strong oxidant peroxynitrite, or oxidize and/or reduce cellular components. For strict
anaerobic or microaerophilic bacteria, it is of particular importance to be able to dispose of ROS in a controlled
manner, especially if these organisms are temporarily exposed to air. Superoxide reductases, SORs, can be
considered the main class of enzymes behind the oxygen detoxification pathway of anaerobic and
microaerophilic bacteria. The major goal of the superoxide reductase project it to understand the mechanism
of superoxide reduction and to establish the role of the mononuclear iron centers in this catalysis.
A series of different techniques were applied in all the work performed, namely biochemical techniques,
molecular biology methods and various spectroscopies (UV/visible, EPR and Mössbauer) in conjunction with
fast kinetic techniques (stopped-flow and rapid-freeze quench). A general approach was to use stopped-flow
absorption measurements for initial identification of the number of colored accumulating states in a specific
reaction, and the formation and decay timescale of the transient species. Based on these information rapidfreeze quench EPR and Mössbauer experiments are designed for further analysis.
A Mössbauer spectrometer setup, equipped with a Advanced 4 Kelvin Closed Cycle Refrigerator System was
our latest acquisition. This equipment will permit a more detailed and complete spectroscopic characterization.
The installation and exploit the equipment referred above has been one of the major technical advances made
in our laboratory in the past years. In many cases the equipment referred was, or still is, unique in our country
thus providing an improved capacity in the installed scientific facilities.
K - MULTIHEME ENZYMES
The bacteria Geobacter sulfurreducens (Gs) can naturally remove toxic metals from the environment, such as
U(VI) and Cr(VI). This microorganisms use a great variety of metals as final electron acceptors acquiring
energy for their metabolic process. In these methabollic pathways, that promote the use/elimination of toxic
metals, are involved some multihaem cytochromes, such as the small periplasmic trihaem cytochrome,
designated PpcA, that participates in the reduction of U(VI), Cr(VI) and Fe(III).
During 2006 it was possible to conclude the thermodynamic characterization of the several redox centers of
PpcA, using visible spectroscopy and RMN. This study enabled us to determine the individual redox potential
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of the several hemo groups, and the interactions between each one of this redox centers. Moreover, these
studies revealed that the hemo III group is extraordinarily sensitive to the oxidation state of the nearby groups.
From the thermodynamic study it was also possible to determine the pKa of the center responsible for the
redox-Bohr effect and to conclude about its proximity regarding hemo IV.
The genome analysis of Gs uncovered the existence of other four proteins homologues to PpcA that we intend
to characterize in the future together with proteins of electronic transfer already isolated and identified. This
project will continue to be developed in collaboration with the University of Chicago (Argone National Laboratory
– USA) and with the Institut of Physical-Chemistry “Rocasolano” (Madrid).
L - NON RANDOM ORIENTATION OF PROTEIN MOLECULES AND NO INTERACTION WITH BIOMOLECULES
Construction of a device to produce sample with non random orientation of protein molecules. This device is
based on the alignment of Pf1 virus in strong magnetic field (higher than 7 Tesla) and their capability to induce
alignment based on steric and electrostatic effect to protein molecules for any type of spectroscopy.
Study of the molecular mechanism of insulin chain A and B and nitric oxide in the physiological conditions of
type 2 diabetes, using combined HPLC-MS(ESI) and in vivo approaches.
Further developments of the Pf1 based aligning device for metalloproteins molecules. Application of anodic
aluminum oxide nanopores to support algned arrays of photosystem I molecules.
Study of the direct relationship of nitric oxide and intact (hexameric) insulin molecules in near physiological
conditions.
M - PROTEIN-PROTEIN INTERACTIONS
We have developed a new algorithm for (BIGGER/CHEMERA) for macromolecular interactions.
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PROTEIN CRYSTALLOGRAPHY
Head of Laboratory: Maria João Romão, Associate Professor
Staff Members:
Ana Luisa Carvalho
José Trincão
Cecília Bonifácio
Technician
Post-Doct Fellows:
Abhik Mukhopaadhyay, Shabir Najmudin, Teresa Santos Silva
Ph.D. Students:
Filipe Freire
Project Grantee:
Catarina Coelho
Aldino Viegas
Number of articles in scientific journals: 6 (58, 59, 63, 80-82)
Number of Ph. D. Thesis: 1 (10)
We have continued our work on structural and functional studies of different systems: (A) Metalloproteins; (B)
Proteins involved in cellulose degradation; (C) Proteins from the glyoxalase pathway.
One important topic of our research are molybdopterin-containing enzymes. In addition, we have pursued,
together with the Faculty of Veterinary Medicine of Lisbon, studies on several new components of the
Cellulosome assembly. More recently we established collaboration with the Faculty of Sciences of Lisbon, for
a new project on enzymes from Leishmania infantum.
A - MOLYBDOPTERIN ENZYMES
Formate dehydrogenase (Raiijmakers & Romão, JBIC, 2006)
Important achievements: Proposal of a new reaction mechanism for Formate Dehydrogenases
When analyzing the electron density maps
calculated with the deposited model for formatereduced (Boyington, Science, 1997) we found,
surprisingly, very weak electron density for an
important loop region, which includes the Mo ligand,
SeC 140, suggesting a mistracing in the original
structure! One possible reason for the mistraced loop
is that it may correspond to a poorly defined region
of the structure. After a short refinement, the electron
density revealed a very poor fit for the 138-146 loop.
Surprisingly, the Se atom was located 12 Å away
from the Mo. This implies that, SeCys140 is not a Mo
ligand and a novel mechanism was proposed.
1a
1b
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Nitrate reductases of R. eutropha and D. desulfuricans
Purification and crystallization of Re NapAB (Coelho et al, submitted)
ReNapAB was expressed and purified. Crystallization was performed at the High-Throughput Crystallization
facility. Diffraction data were collected to 1.5 Å, the highest resolution obtained for a nitrate reductase. MR
gave a clear solution yielding good phases. Rebuilding of ReNapAB model and refinement are currently under
way.
Structural studies of NapA from D. desulfuricans
Crystallization experiments with Dd Nap were performed inside an anaerobic chamber. The crystals were
cryo-cooled inside the chamber using liquid N2 although diffraction data were very anisotropic. Other
experiments included mounting the crystals in capillaries inside the chamber and cooling them directly in the
cryo stream. A highly redundant dataset at the iron edge was collected at the ESRF. The data extended
beyond 2.2 Å and anomalous data allowed the identification of sulfur atoms coordinating the Mo center. A
novel mechanism will be proposed.
B - HEME AND NON-HEME PROTEINS
The non-heme T pallidum Superoxide Reductase
(Santos-Silva, JBIC; 2006)
Superoxide reductase is responsible for the scavenging of
superoxide radicals in the cell. The crystal structure of TpSOR
was determined to 1.5 Å. In TpSOR the Dx-type Fe centre is
absent but the N-terminal domain is maintained and exhibits a
Dx-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 Pf SOR was not observed in TpSOR. X-ray
photoreduction could be affecting the oxidized form of the TpSOR
centre, preventing hexacoordination of the iron atom.
The sixteen heme cytochrome from Dgigas: a glycosylated protein in a sulphate reducing bacterium
(Santos-Silva, JMolBiol, in press)
HmcA is a High molecular weight cytochrome of 550 aa residues that harbors 16 c-type hemes. We have
solved the crystal structure of Dgigas HmcA. Crystals diffracted beyond 2.1 Å resolution. A MAD experiment
at the Fe absorption edge enabled to obtain good quality phases for structure solution and model building.
The presence of an oligosaccharide molecule covalently bound to an Asn was observed in the electron density
maps and confirmed by MALDI-TOF. It appears at the highly hydrophobic vertex, possibly acting as an anchor
of the protein to the cytoplasmic membrane.
C - THE CELLULOSOME
A dual mode of binding reveals the flexible mechanism of Cellulosome assembly
(Najmudin, JBC, 2006; Carvalho, PNAS, in press)
C thermocellum, synthesizes a plant cell wall degrading apparatus, termed the cellulosome that is an
extracellular complex of enzymatic subunits, each exhibiting specific affinities for many different cellulosederived substrates. Each enzyme, contains a type I dockerin, which binds to a cohesin. The type I dockerin,
build-up of 3 helices, contain a duplicated sequence that originates helix-1 and helix-3. These two helices
form a, almost perfect, 180º angle between them. The crystal structure of the Ct type I cohesin-dockerin
complex showed that cohesin recognition was predominantly through helix-3 of the dockerin.
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The sequence duplication is reflected in nearperfect 2-fold symmetry, suggesting that both
helices could bind to the cohesin modules in WT
proteins. Considering these observations, a helix3 disrupted mutant dockerin was prepared and
used to visualize the reverse binding. In this new
assembly of the two modules, the dockerin mutant
is rotated 180º relative to the WT. The dual binding
mode seems to confer some plasticity to the
assembly of the whole Cellulosome complex,
which purpose is the degradation of a significantly
resistant and insoluble substrate.
D - PROTEINS FROM THE GLYOXALASE PATHWAY
X-ray diffraction analysis of the glyoxalase II from Leishmania infantum
(Trincão et al, Acta Cryst D, 2006)
In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two
enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity
towards trypanothione thioester, making this enzyme an outstanding model to understand the molecular
basis of trypanothione binding. Cloned glyoxalase II from L infantum was overexpressed in E. coli, purified
and crystallized. The structure was solved by MR using the human glyoxalase II structure as a search model.
These results, together with future detailed kinetic characterization using lactoyltrypanothione, will provide
invaluable information on why trypanosomatids evolved to deploy trypanothione instead of glutathione.
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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
Ph. D. Students:
Ana Tavares dos Santos
M. Sc. Students:
Ana Madeira
Ana Martins, Patrícia Gonçalves
Number of articles in scientific journals: 1 (83)
2.1. MEMBRANE TRANSPORT
2.1.1. Detection and role of aquaporins in Debaryomyces hansenii
Debaryomyces hansenii is one of the most halotolerant species of yeast and it is considered a halophile, able
to grow in media containing a wide range of salt concentrations. Like S. cerevisiae, when D. hansenii is grown
in the presence of high salt concentrations it accumulates glycerol as the most important compatible solute.
We investigated the existence of aquaporin genes (AQY1, AQY2) in D. hansenii using online database of
Génolevures consortium. We used primers designed in order to amplify AQY1 ORF and the regulatory regions.
Yeast genomic DNA from D. hansenii was isolated after a previous treatment with liticase. Current plasmid
isolation, plasmid isolation from yeasts, agarose gel electrophoresis and restriction site mapping were performed
according to standard methods.
Using this approach one fragment of 1.6 kb, from D. hansenii genomic DNA was obtained. The fragment was
cloned, characterized at the molecular level, and used to transform a deletion S. cerevisiae strain for AQY1
and AQY2. We are performing water permeability studies of the S. cerevisiae transformants using the stoppedflow light scattering spectrophotometry.
We developed a method to prepare protoplasts from D. hansenii; those protoplasts preparations were submitted
to osmotic challenges of different gradients, using the stopped-flow light scattering technique. These preparations
will be studied and compared to the S. cerevisiae transformants.
2.1.2. Inhibition of the mitochondrial pyruvate carrier by valproate and its metabolites
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. The inhibition mechanism and the kinetic parameters were
characterized.
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2.1.3. Patch-clamp studies
Three projects are in progress:
- Characterization of membrane transporters involved in pollen tube growth: two cCLC channels (Arabidopsis
thaliana) and a cATPase (Nicotiniana tabacum) were subcloned into pcDNA. Transfected HEK293 cells with
cCLC show an increase in Cl- currents, inhabitable by DPC. The protocols for the transfection of the H+ATPase are being developed. Alternatively, pollen protoplasts are obtained for comparative studies and the
first results show outward Cl- currents across the cell membrane.
(in collaboration with the Plant Development group of Prof. José Feijó from Institute Gulbenkian for Science)
- HEK293 cells as a renal sensor for diuretic drugs and hormones: Several potassium currents have been
identified in this cell line, showing different sensitivities to diuretic drugs whose molecular effects are well
documented (caffeine and acetozolamide).
- Electrophysiological studies on the nicotinic acetylcholine receptor: The effect of galantamine (an
acetylcholinesterase inhibitor) was tested on currents in response to the application of pulses of Acetylcholine,
measured from TE671LH cells (which express naturally the receptor). The results show a possible dosedependent increase of the currents.
(in collaboration with Prof. Constanta Ganea of the Biophysics Department, “Carol Davila” University of Medicine
and Pharmaceutics).
2.2. 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).
2.2.1. Tubular organ - thick ascending limb of the Henle´s loop - TAL
We concluded the simulations of the data available in the literature of the cTAL and submitted a manuscript
for publication that was accepted and is now in press in the Journal of Membrane Biology.
2.2.2. 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
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 2006 was dedicated to continue the data gathering from several hospitals (Instituto
Portugues de Oncologia, Hospital de Santa Cruz, Hospital de Setúbal) and to its statistical analysis.
2.3. STUDIES ON FOOD COMPONENTS
2.3.1. Room-temperature phosphorescence (RTP) of food colorants and other emitters
Room-temperature phosphorescence (RTP) emission spectra under continuous irradiation conditions were
collected for erythrosine and other triplet-emitters such as substituted fluoresceines and bromoanthracene
in liquid aqueous solution containing bovine serum albumine (BSA). Erythrosine/BSA solutions yield RTP as
a single broad-band centered at 680 nm with both native or denatured protein. Bromoanthracene/BSA solutions
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yield RTP as a double-band with maxima around 450-550 nm. The emission properties are similar to those
observed for such emmiters in organized media. Experiments to detect optically generated molecular triplet
states by 1H Nuclear Magnetic Resonance (400 MHz) were performed using deareated samples of
erythrosine in water. Results obtained under continuous illumination show slight variations on the chemical
shifts and bandwidths of NMR signals compared to the data obtained in the dark.
2.3.2. Behaviour of cyanidine monoglucoside isolated from the rind of Passiflora edulis (Sims) in
solution
The major anthocyanin in the rind of P edulis was confirmed to be kuromanine (cyanidine monoglucoside) by
HPLC and mass spectrometry. An investigation using UV-Visible spectroscopy was undertaken to study the
effect of media conditions on the behaviour of this pigment in solution. It was found that both the apparent
pKA and the rates of exchange between species prevailing at neutral and basic pHs are strongly dependent
on solution composition.
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DEVELOPMENT OF TRANSDUCERS
Head of Laboratory: José Luís Costa Lima, Full Professor
Staff Members:
Mª Conceição B. S. M. Montenegro
Full Professor
Cristina Maria F. Delerue Alvim Matos
Coordinator Professor
Maria do Carmo Veiga Fernandes Vaz
Alberto Nova Araújo
Associate Professor
Rui Alexandre Santos Lapa
Associate Professor
Cristina Maria C. Morais Couto
Assistant Professor
Adriana Martins Pimenta
Assistant Professor
Maria Beatriz V. N. Q. G. Junqueiro
Assistant Professor
Rita Isabel Lemos Catarino
Assistant Professor
Paula Cristina Jerónimo
Assistant Professor
João Rodrigo Silva Santos
João Pedro Almeida Lopes
Abel José Assunção Duarte
Assistant
Ph. D. Students:
Emília Maria Santos, Maria Luísa Silva, Sofia Alexandra Gomes, Célia Maria Amorim, Ana Rita Pires, Mafalda
Sofia Sarraguça, Carla Patrício Coutinho, Joana Alexandrina Carvalhido
M. Sc. Students:
Pedro Manuel Coelho, Lúcia Helena Santos, Branca Sofia Teixeira, Ana Isabel Ramos
Number of M. Sc. Thesis: 1 (1)
Different transducing schemes such as optical and electrochemical were investigated. 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 flow systems.
In more detail it can be referred the following results:
- Construction of a Nafion coated glassy carbon tubular electrode and its use coupled to a multi-commutated
flow system, for voltametric determination of acetaminophen in serum and pharmaceutical preparations. The
modification of the electrode enhance the analytical signal intensity and simultaneously, prevent the electrode
surface fouling. The multi-commutated system conferred high versatility to the manifold, allowing it to be
easily adjusted to each determination without the need to introduce any physical reconfiguration. The on line
enzymatic hydrolyses of acetaminophen, give rise to 4-aminophenol, allowed the problem of the interferences
resulting from the oxidation of the matrix serum constituents to be overcome.
- A tubular potentiometric detector sensitive to As (V) ions was been constructed and evaluated. This tubular
electrode constructed without inner reference solution comprised of a FeOOH-SiO2-graphite composite
agglutinated with an epoxy resin. For As (V) ion concentrations in the range 10-5 to 10-1 mol L-1 was been used
as detector in a flow system with a commercial Ag/AgCl reference electrode. In optimal conditions the practical
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limit of detection was 4x10-6 mol L-1 and a sampling rate of 40 samples per hour can be achieved. The
systems was applied to estimate As (V) levels in drinking water and extracts of sediments. The results
obtained by the electrochemical procedure were compared to those obtained by electrothermal atomic absorption
spectroscopy.
- A new valproate selective electrodes based on manganese (III) tetraphenylporphyrin [Mn(III)TPP-Cl], as an
inophore was been developed. The inophore was incorporated into PVC and ceramic membranes (sol-gel)
based on methyltriethoxysilane. The influence of the membrane composition and the effect of the lipophilic
cationic and anionic additives in PVC membranes were investigated concerning on the slop, response time,
selectivity and life time of the electrodes. The PVC membrane without additive and the sol-gel membrane
presented slopes and practical limits of detection of -60.8 mV dec-1 and 5x10-6 molL-1 and 60.3 mV dec-1 and
10-4 molL-1, respectively. The sol-gel membranes displayed higher selectivity for valproate when compared
with PVC membranes.
- A diclofenac selective electrodes based on Fe (III) tetraphenylporphirin-chloride ((Fe(III)TPP-Cl) and Fe(III)
tetrakis(pentafluorophenyl)porphyrin-chloride (Fe(III)TPFPP-Cl) in different mediator solvents and ionic additives
was been constructed and evaluated. The sensitivity, working range, detection limit, response mechanism,
and selectivity of the membrane sensor show a significant dependence of the type of carrier substituent and
on the pH value of the sample solution. Studies performed with different amounts of cationic additive (tetra-noctylammoniumbromide) and anionic additive (sodium tetraphenylborate) in the membranes allowed the
determination of the potentiometric mechanism of action of the used metalloporphyrins. The potentiometric
analysis of the sodium diclofenac in pharmaceutical formulations was carried out by direct potentiometry and
the obtained results compared to those provided by HPLC, presenting relative errors inferior to 1.0%.
- A tubular glassy carbon electrode was used as detector for a FIA system with electrochemical detection
dedicated to the determination of paracetamol in pharmaceutical formulations. The analytical difficulties caused
by adsorption of matrix excipients in the electrode surface were surpassed by an on-line electrochemical
regeneration of the glassy carbon tubular electrode using the carrier solution, enabling the renewal of the
electrode surface in a simple and rapid way. The flow system provided reproducibility of the sample transport
to the detector and minimized the contact time between samples and electrode surface, thus reducing its
cleaning frequency and permitting a high sampling rate to be achieved. Furthermore, the physical characteristics
of the electrochemical cell permitted its easy incorporation in the FIA manifold, offering robustness to the
system and allowed it to be generalized to the routine analysis applications.
- A tubular potassium ion selective electrode was coupled to a multi-syringe flow system for the potentiometric
determination of exchangeable potassium in soil samples. Firstly a manifold was devised to allow determination
in soil extracts prepared off-line. It was possible to analyze samples prepared in extractants with different
composition without physical or chemical modification of the manifold. A linear dynamic concentration range
of 6-391 mg L-1 was obtained allowing the direct introduction of soil extracts without dilution. A determination
frequency of 50 samples per hour was achieved, with good repeatability for 10 consecutive injections of soil
extracts (RSD less than 3.0%). The in-line preparation of the soil extract was implemented by automatic
addition of extractant solution to a previously weight portion of soil, followed by in-line filtration. Good repeatability
was attained as the variance of the extraction procedure was not significantly different from the variance
obtained in consecutive measurements of the same extract. Furthermore, results comparable to those obtained
by on-line extraction and determinations by flame emission spectrometry were attained for the two soil
samples tested. Using this procedure, a determination frequency of 13 h-1 were achieved.
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AUTOMATION AND INSTRUMENTATION
Head of Laboratory: José Luís Costa Lima, Full Professor
Staff Members:
Mª Conceição B. S. M. Montenegro
Full Professor
Alberto Nova Araújo
Associate Professor
Associate Professor
Agostinho Almiro Almeida
Assistant Professor
João Luís Machado Santos
Assistant Professor
Maria Lúcia M. F. Sousa Saraiva
Assistant Professor
Ivone Valente Oliveira
Assistant Professor
Marcela Alves Segundo
Adriano Fachini
João Alexandre Velho Prior
Assistant
Paula Cristina A.Gomes Pinto
Assistant
Ph. D. Students:
Cristina Manuela Lopes, Pedro Rodrigues Maia, Karine Lopes Marques, Marta Filipa Ribeiro, Luís Miguel
Magalhães, Eunice Raquel Rodrigues, Hugo Miguel Oliveira, Marieta Leite Passos, André Ricardo Araújo,
Ana Maria Gomes, Cristina Isabel Silvestre, Joana Patrícia Ribeiro
M. Sc. Students:
Daniel Alexandre Ribeiro, Ana Isabel Castro, Eliana Sousa e Silva
Number of Ph. D. Thesis: 2 (11, 12)
Number of M. Sc. Thesis: 2 (2, 3)
Continuous flow systems and dedicated equipment were developed and applied in automatic laboratorial
determinations or in on-line control of industrial processes. Special attention has been dedicated to procedures
based on the multicommutated flow methodologies, sequential injection analysis and multi-pumping flow
analysis.
In this theme the following activities can be stressed:
- An automatic flow procedure based on multi-syringe flow injection analysis was developed for the assessment
of the Folin-Ciocalteau reagent (FCR) reducing capacity in several types of food products using gallic acid as
standard. Different strategies for mixing of sample and reagent were tested (continuous flow of FCR, merging
zones and intercalated zones approaches); lower reagent consumptions and higher determination throughput
were attained for the merging zones approach. The application of the proposed method to compounds with
known antioxidant activity (both phenolic and nonphenolic) and to samples (wines, beers, teas soft drinks and
fruit juices) provides results similar to those obtained by conventional batch method.
- A robust, accurate and sensitive automated procedure for the determination of etodoloc, an anti-inflamatory
drug, in pharmaceutical formulas by sequential injection analysis, was been reported. The same system was
also applied to evaluated the antioxidant activity of the drug expressed as Trolox equivalent antioxidant
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capacity. The methodology is based on measuring at 734 nm the decay of the absorbance of a solution with
the radical ABTS after its reduction to etodolac. The developed methodology was applied in the analysis of
pharmaceutical preparations and the obtained results were in good agreement with those furnished by the
reference procedure with relative deviations lower than 1.4%.
- It was studied the possibility of coupling multi-pumping flow systems (MPFS) to flame atomic absorption
spectrophotometers and flame photometers. The main purpose was to study the behaviour of the solenoid
pumps used a propulsion device in the MPFS when subjected to the aspiration effect inherent to nebulizers of
the flame atomic spectrometric (FAS) detectors (Venturi effect) and to evaluate the effects of the pulsed flow
a main characteristics of the MPFS, over the efficiency of the nebulization and atomization process. Based
on this studies, several MPFS that enables the on-line dilution of samples and/or the on-line addition of
reagents (e.g. a realising agent) two sample pre-treatment steps frequently requited when using FAS detectors
in analytical work were developed.
- A sequential injection system for the automatic determination of glycerol in wine and beer was developed:
The method is based on the rate of formation of NADH from the reaction of glycerol and NAD+ catalyzed by
the enzyme glycerol dehydrogenase in solution. The determination of glycerol was performed between 0.3
and 3.0 mmolL-1 and good repeatability was attained for all samples tested. The determination rate was 54 h1
, the reagent consumption was only 0.75µmol of DNA and 5.4ng of enzyme per assay and the waste production
was 2.12 mL per assay. Results obtained for samples were in agreement with the batch enzymatic method.
- A fully automatic kinetic method for the spectrophotometric determination of buspirone in pharmaceutical
preparations was been proposed. The analytical system was implemented by using multiple solenoid micropumps for sample and reagents insertion which led to improved operational simplicity and low reagent
consumption. A three-way solenoid valve enable the establishment of two parallel reactors and the exploitation
of fixed time approach for differential kinetics. A linear calibration plots for buspirone concentrations of up to
60 mgL-1 were obtained, with a detection limit of 2.8mgL-1. The sampling rate was about 55 samples per hour.
- A flow-batch manifold coupled to a flame atomic absorption spectrophotometer was evaluated to access the
iron content by internal standard method in hydrated ethanol used as fuel in automotive industry. For this
assessment official methods require calibration procedures with matrix matching, making it difficult to obtain
accurate results for samples adulterated by the addition of water. Nickel was selected as the internal standard
since it is usually absent in samples and because it requires similar conditions of atomization. After procedure
optimization which requires about 4.25 mL of sample and standard per measurement, it was possible to get
analytical response for iron concentration between 0.12 and 1.40 mgL-1 and a detection limit of 0.04 mgL-1 .
- A novel strategy for utilization of solid reagents in flow analysis was developed. Establishment of difuse and
reproducible geometry enables the solid particles to be maintained in a constant floating, reflux and circulating
motion inside a mini-chamber. This is efficiently accomplished with pulsed flows, a characteristic of the multipumping flow systems. Drawbacks inherent in solid-phase columns, for back pressure, preferential pathways,
swelling, etc and some limitations inherent in immobilized reagents are minimised. Spectrophotometric
determination of zinc in plants was selected as an application of the technique. Dower 1-X8 anionic resin was
kept freely inside a mini-chamber. Zinc chloro-complexes were adsorbed on the moving particles and
derivatization with zircon was performed after elution.
- A tubular gas diffusion PTFE membrane is exploited for non-evasive sampling in flow analysis, aiming to
develop an improved spectrophotometric determination of ethanol in alcoholic beverages. The probe can be
immersed into the sample, allowing ethanol to diffuse through the membrane. It is collected into the acceptor
stream (acid dichromate solution), leading to formation of Cr(III), monitored at 600 nm. The analytical curve is
linear up to 50% (v/v) ethanol, baseline drift is less than 0.005 absorbance during four working-hours and
sample throughput is 30 h-1, meaning 0.6 mmol K2Cr2O7 per determination. The results obtained with the
automatic system are precise and in agreement with an official procedure.
- A fully automated multi-commutated flow system was developed for the on-line dilution of highly concentrated
rubidium solutions by employing the zone sampling approach and determination by flame atomic spectroscopy.
The developed flow methodology exhibits great versatility, allows a wide range of dilution levels (dilution
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factors between 45 and 11,500) and results in a broad working ranges. In addition, the dilution levels can be
automatically adjusted to the sample concentration via computer control without the need of manifold
reconfiguration. The proposed analytical system enables the automation of a well-established assay used to
screen drugs with potential potassium-channel modulating activity (the so-called “rubidium efflux assay”),
thus allowing high throughput screening required in drug development. The results were precise (RSD values
lower than 2.6) and accurate showing good agreement with those obtained using the manual procedure. The
sampling frequency varied from 62-111 samples per hour depending on the dilution level required.
- The coupling of short monoliths columns with sequential injection analysis (SIA) results in a new approach
to implementation of separation step to non-separation low pressure method. Our work was devoted to the
determination of ambroxol, methylparaben and benzoic acid and was developed based on a novel reversedphase sequential injection chromatography (SIC) technique with UV detection. The new SIC method was
validated and compared with HPLC. The method was found to be useful for the routine analysis of the active
compounds ambroxol and preservatives (methylparaben or benzoic acid) in several pharmaceutical syrups
and drops.
- An automatic method based on multi-syringe flow injection analysis (MSFIA) was developed for the
determination of total antioxidant capacity, measured as the cumulative capacity of the compounds present
in the samples to scavenge free radical, using the DPPH reaction. The determination is based on the colour
disappearance due to the scavenging of the DPPH by antioxidant compounds monitored at 517 nm. The
influence of DPPH concentration and sample dilution in the present methodology was studied. It was verified
that the amount of DPPH consumed by antioxidant standards (ascorbic and caffeic acid) was independent of
the initial concentration of the radical except for situations where DPPH/antioxidant molar ratio was lower
than the stoichiometric value. Furthermore, the sample dilution factor plays a important role for achieving
results comparable to those from the end-point batch method since the exhausting of the scavenging ability
of the sample should take a place during the period of absorbance measurement.
- A sequential injection analysis (SIA) system adapted to direct analysis of oil samples was been developed.
The flow system was designed by incorporation, in the SIA system, of an injection valve that was responsible
for the sample insertion. With the developed sampling strategy the sample pre-treatment outside the flow
system is avoided and also the problems associated with the viscous samples in flow systems. The developed
SIA system was applied to the determination of iron (Fe(III)) in edible oil samples and was based on the
formation of a red complex (510nm) between Fe (III) and thiocyanate in organic medium. A mixture of methanol:
chloroform (85:15) was used as carrier solution and possible refractive index associated with the
spectrophotometric detection was avoided by the introduction of a mixing chamber in the flow system.
- A fully automated flow system exploiting the advantages of the association of multi-pumping, multicommutation, binary sampling and merging zones, to accomplish the sequential determination of copper in
serum and urine by flame atomic absorption spectroscopy was been developed. The flow system allowed
multiple tasks, such as serum samples preparation (samples and standards solution viscosity adjustment),
serum copper measurement, urine copper pre-concentration and its subsequent elution and measurement to
be carried out sequentially. The implemented flow manifold presented a modular configuration consisting on
two quasi-independent modules, each one accountable for specific sample manipulation and whose combined
operation under computer control enabled the determination of copper in a wide concentration range.
- A sequential injection analysis (SIA) methodology for the fluorimetric determination of aminocaproic acid in
pharmaceutical formulations was developed. The analytical procedure is based on the derivatisation reaction
of the aminocaproic primary amine with o-phthalaldehyde and N-acetylcysteine and fluorimetric detection of
the formed product. The implementation of the SIA flow system allowed for the development of a simple, fast
and versatile automated methodology, wich exhibits evident advantages reagarding US Pharmacopeia reference
procedure. By combining the SIA time-based sample insertion with a subsequent zone sampling approach,
which permitted to select for detection of a well-defined sample zone, it was possible to implement a on-line
dilution strategy that enabled the expansion of the analytical working range of the methodology and thus its
application in dissolution studies, without manifold re-configuration.
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QUALITY CONTROL AND AUTHENTICITY OF FOOD PRODUCTS
Head of laboratory: Rosa Seabra, Full Professor / Beatriz Oliveira, Associate Professor
Staff Members:
Cristina Maria F. Delerue Alvim Matos
Manuel Rui Fernandes Azevedo Alves
Maria do Carmo Veiga Fernandes Vaz
Luísa Maria S. Vieira Peixe
Associate Professor
Olívia Maria Castro Pinho
Associate Professor
Branca Silva
Assistant Professor
Carla Alexandra Novais Oliveira e Silva
Assistant Professor
Isabel Maria Pinto L. V. Oliveira Ferreira Assistant Professor
José Oliveira Fernandes
Assistant Professor
Miguel Freire de Albuquerque Cabral
Assistant Professor
Paula Andrade
Assistant Professor
Sandra Maria Basílio Quinteira
Assistant Professor
Susana Isabel Pereira Casal Vicente
Assistant Professor
Isabel Maria Sousa Gomes Mafra
Joana Andrêa Soares Amaral
Assistant
Maria Goreti Ferreira Sales
Assistant
Maria João Ramalhosa Ferreira
Assistant
Maria de Fátima de Sá Barroso
Assistant
Maria Virgínis Teixeira da Mota
Assistant
Patrícia Valentão
Assistant
Simone Barreira Morais
Assistant
Miguel Ângelo Rodrigues Pinto de Faria Invited Assistant
Carla Sousa
Graduated Technician
Paula Celeste Baptista Paíga
Graduated Technician
Eulália Maria Bernardino Mendes
Assessor
Post-Doct Fellows:
Sónia Dópico
Ph. D. Students:
Bárbara Ribeiro, Sara Cristina Cunha, Rita Carneiro Alves, João Barreira, Patricia Antunes, Elisabete Machado
M. Sc. Students:
Alexandra Lopes, Orlanda Pereira, Ana Silva, Claúdia Martins, Sofia Almeida, Carla Barbosa, Lucia Maia,
Aida Reis, Eduarda Cabral, Ângela Barroso, Cristina Soares, Áurea Roxo, Ana Gomes, Sónia Mendes
Rosário Guimarães, Joana Lopes, Carla Sofia Silva, Rita Coelho
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Number of articles in books: 1 (3)
Number of M. Sc. Thesis: 3 (4-6)
The strong 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, carotenes,)
residues and contaminants (biogenic amines, pesticides, acrylamide, PAHs) of conventional and traditional
food products, with special emphasis to those 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 main activities under development in 2006 were:
1. Molecular biology techniques for the evaluation of food safety and food authenticity
Some activities of this line of research concerning molecular biology techniques for the evaluation of food
authenticity were focused on the evaluation and development of DNA extraction techniques applied to recalcitrant
samples, as olive oils, and the construction of a molecular database, based on SSR profiles, of regional
cultivars of Olea europaea, in order to assist the development of methodologies for the authentication of DOP
olive oils. Some regional cultivars are already analysed in a pre-graduated project.
The research will be continued in 2007, as more cultivars are being collected, to achieve a more consistent
database. We pretend to evaluate olive oil botanical authenticity using microsatellite DNA analysis having this
database as a reference.
Also, side by side with the referred activities, we will focus on the development of rt-PCR (real time PCR)
based techniques for the effective quantification of DNA in olive oils using new reference genes and the
detection and quantification of vegetable oils from several origins using rt-PCR specific probes. 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. To answer this research line a PCR methodology will be
applied to samples of hazelnut.
The development of molecular biology techniques for the adulteration detection in foods has been carried out
by identification of milk origin species in dairy products. The research work included the development of
duplex PCR techniques targeting the mitochondrial genes RNA 12S and cyt b able to simultaneously detect
bovine and caprine milks in cheeses. Several linear normalized curves between the log of bovine band intensities
vs. the log of bovine milk percentage in cheeses have been developed by means of duplex PCR. The best
models allowed the detection of 0.1% and the quantification in the range of 1 to 60% of bovine milk in caprine
cheeses. The validation of the method was performed by application of the technique to “blind” cheeses
prepared in the laboratory and in the industry. The results showed a good correlation between the predicted
cow’s milk and the real values of cheeses. The method was successfully applied to commercialized cheeses,
whose results allowed the detection of several frauds due to the addition of cow’s milk not mentioned on the
label of goat’s cheeses labelled with pure goat’s milk.
The detection of genetically modified organisms in foodstuffs has been carried out by means of DNA based
methods. Concerning the detection of GM soybean in foods, a comparative study has been performed to
evaluate 4 methods of DNA extraction of several food matrices (commercial kits NucleoSpin (MachereyNagel) and GeneSpin (GeneScan); the partial use of the commercial kit of Wizard (Promega) and the method
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based on the CTAB reagent). To evaluate the extraction methods, the DNA concentration, the purity and the
level of PCR amplification of lectin gene were determined. The amplification by real-time PCR using the SYBR
green I dye was also performe
Concerning the detection of GM maize in foods, the extraction of maize products, the development of detection
methods of maize invertase and GM maize events MON810, Bt11 and Bt176 by PCR is a research work
presently on course and planned to the near future. Real-time PCR techniques using SYBR Green I dye and
hydrolysis probes are planned to be applied for the quantification of GM events in foods. Part of this work will
be developed in a pre-graduated project.
The common bean (Phaseolus vulgaris L.) is the most consumed legume in the world. However its biodiversity
reduction it’s a current and serious problem. Nevertheless, the geographic isolation of some Portuguese
populations in Entre Douro e Minho region permitted the safeguard of a large number of populations adapted
to our edaphic and climatic conditions. 19 Portuguese accessions were analysed, experimented in two
locals: Vieira do Minho and Braga, collected by the Portuguese Genebank (BPGV). In order to characterize
the referred diversity, chemical (protein content) and molecular (microssatlellite DNA) parameters were evaluated
for these accessions. Significant differences were found among locals, accessions and locals vs accessions
interactions. The molecular characterization was achieved with 6 microsatellite loci. It was possible to
differentiate most of the accessions using these markers. Notwithstanding all the markers presented
polymorphism, some (e.g. BM210) permitted clearly a higher discriminant power. Throughout cluster analysis,
the 19 accessions were correlated in terms of genetic proximity and local of origin. The results can be useful
in future improvement programs of common bean. This work was developed in a pre-graduated project.
2. Food authenticity by means of Chemical Markers
Some coffee constituents were determined in both green and roasted coffee, in grains and beverages, and
their behaviour with roasting evaluated and discussed. The presence of heterocyclic amines correlated with
beneficial effects in neurological system was evaluated. Harman and norharman, two beta-carbolines that
might be involved in the protection for Parkinson disease, was accomplished with 17 samples analyzed in the
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. Several samples were also analyzed in
the beverage form, with similar conclusions. 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 a M.Sc. thesis as well as a Grant from IBeSa, being now under development by a doctoral
student. All these studies are being conducted in collaboration with a local coffee industry.
Several quality parameters were 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. The experimental work finished and the PhD thesis is in final righting phase.
The main source of vitamin E in Mediterranean countries is olive oil, being it consumption correlated with
reducing risk of coronary heart disease. A general improvement in the immune system has also been reported
as beneficial effect of these compounds.
The study aimed to optimize a method allowing the simultaneous determination of tocopherols and tocotrienols
in olive oils. The method was applied to several PDO Trás-os-Montes olive oils. α- tocopherol was the main
vitamin E isomer in all samples, however small amounts of β, γ and δ-tocopherols and vestigial amounts of α
and γ-tocotrienols were also found.
An HPLC/ELSD method was used to quantify the triacylglycerols in varietal virgin olive oils from Cvs. Verdeal
Transmontana, Madural, and Cobrançosa. The triacylglycerol profiles showed that Cv. Verdeal Transmontana
had the highest values for OOO (66.8%) and POO (23.4%) and the lowest for LOO (3.6%), the major
triacylglycerols in the samples studied. Using PCA statistical analysis the oils could be discriminated, showing
the potential of this method for characterizing varietal olive oils.
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A simple and accurate method based on solid-phase extraction (SPE), transesterification and gas
chromatography–mass spectrometry (GC–MS) was developed for the quantitative analysis of free and esterified
sterols of olive oil. The optimized methodology was suitable for evaluation of free and esterified sterols in
Protected Designation of Origin (PDO) olive oils and monovarietal olive oils with different maturation indices.
The prevailing phytosterols in all olive oils were b-sitosterol and campesterol. The free sterols predominated,
although they seemed to decrease with the maturation of the olive fruits.
An HPLC methodology to characterize the carotenoid profile (lutein and β-carotene) of olive oils was
implemented. This methodology was developed, validated and applied to several olive oils from all PDO
Portuguese regions and also from some Spain and Italy PDO regions. This subject will be described in a
M.Sc. thesis that waits evaluation.
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 obtained results were included in a PhD thesis. Several
papers were published and two book chapters are in press (1) and submitted (1). This work line continues with
the collaboration of an investigator from Turkey and the characterization of turkey hazelnuts samples. Also an
MsC will be developed in the field of B vitamins and the genetic characterization of the cultivars in evaluation
(walnuts)
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 farm, lamb age, breed and the
mother’s milk production per day. This research line continues with milk samples from other breedings.
Simultaneously, Portuguese protected geographical indication (PGI) sausages are under evaluation aiming to
obtain added value from 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.
3. 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 is developing. With this goal an isotope labelled
GC-MS 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.
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An improved gas chromatography-mass spectrometry (GC-MS) method to determine acrylamide (AA) in
coffee and coffee products was developed. The method was based on two main purification steps: the first
with ethanol and Carrez solutions in order to precipitate polysaccharides and proteins, respectively; and the
second with a layered solid-phase extraction (SPE) column which proved to be efficient in the elimination of
the main chromatographic interferences. The method is applicable to a wide range of coffee products. Twentysix samples of different coffee products were analysed. The levels of AA were in the range 11.4–36.2 µg/L for
‘espresso coffee’ and 200.8–229.4 µg/L for coffee blends with cereals. The results indicate that the presence
of cereals significantly increased the levels of AA. This line research resulted in a MSc Thesis, one paper and
several communications.
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. 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 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.
In the same context and similar approach Phosmet were also determined in olives after the development of
specific techniques for it extraction as well as its metabolites. The results obtained were described in a paper
in press.
Dimethoate are another pesticide in study and it determination and metabolites was performed in the Microbial
Biophysics and Residue Chemistry Research Unit - Eastern Regional Research Center (USA) with supervision
of Prof. Steven J. Lehotay. The obtained results are compiled in a submitted paper.
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
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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 was presented.
In this context another MSc. student developed an HPSEC/ELSD methodology to determine polymerized
triacylglycerols, oxidized triacylglycerides and diacylglycerides contents in the previously referred oils
correlating these contents with TPC and safety of the frying products. The results are discussed in a MsC
thesis in final preparation.
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.
Frying is a culinary process used since the beginning of times. Its simplicity, together with the organoleptic
characteristics of the fried products, justifies their increasing popularity. The main frying oils are extracted
from oleaginous seeds but, until recently, crude olive oil was also used in some countries for frying. The study
pretended to evaluate the modifications in the olive oil during frying and, simultaneously, to compare their
behaviour with a vegetable oil (alimentary oil). The study includes potatoes frying, cut in toothpicks. The
chemical characterization of the raw materials included the parameters (total polar compounds; iodine value,
refraction value, acidity, peroxide value, ñ-anisidine value, UV absorbencies, oxidative stability, composition
in tocopherols and tocotrienols (Vitamine E) and fatty acids, â-carotene and total phenolic compounds). The
quality evolution of the olive oil/oil was accompanied along frying, until a maximum of 27h, from which all of the
products in evaluation crossed the legislated maximum limit (polar compounds, 25%). It was verified that the
olive oils are able to fry during more hours than the alimentary oil (24 to 27h against 15h). In the Rancimat
test, the oils those present larger induction periods are also the extra virgin DOP olive oil and the olive oil (16h
and 15h, respectively). The obtained results showed that the composition and initial quality of the oils is a very
important parameter in their predictive behaviour along the frying process. This work was developed in a pregraduated project.
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
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
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scenery in Portuguese products, namely cookies / biscuits, a project accepted and supported by UP rectorate
was executed with the collaboration of GRAC. The objective is the implementation of environmental friendly
procedures to fat extraction (microwave). The project included 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
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. 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 exposure of Portuguese consumers
to PHAs from fish a PhD work program was drew up and will be developed. These samples will be also used
to develop a MsC Thesis in the field of heavy metals, contaminants of some health concerns.
Quantification of food additives
Global use of food additives has promoted an extensive concern in human population. Each commercialized
product must be safe and in agreement with legal requirements. Industries must therefore perform routine
analyses to their products. Simple, quick and selective methods with emission of low amounts of effluents of
small toxicity should therefore be implemented. Electrochemical techniques are explored for this purpose,
and applied to the determination of antioxidant, acidity regulators and flavour enhancers. Vitreous, carbon
paste and chemically modified electrodes are evaluated in flow set-ups of amperometric detection. Optimization
of injection volumes, flow-rates and reactor lengths were performed to ensure optimum response of the detection
device. Electrodes with good operating characteristics are applied to the analysis of commercial food samples.
This line of research is presently under development, though it was already applied to the determination of
ascorbic acid, citric acid, glutamate.
Reliable and validated analytical procedures based on microwave-assisted extraction coupled with gas
chromatography with flame ionization detector were optimized and applied for identification and quantification
of fatty acid profile in typical Portuguese cookies.
To estimate human health risks and benefits from the consumption of several types of fishes, proper sample
preparation and analytical techniques based on matrix solid-phase dispersion or microwave-assisted extraction
coupled with liquid chromatography with a fluorescence detector and liquid chromatography with positive/
negative ion mass spectrometry detection are being developed for analysis of 15 PAHs considered by EPA as
priority pollutants.
4. 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.
“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 is developing by a PhD student that obtained a Grant
from FCT. A paper that refers to the factors influencing the norharman and Harman contents in expresso
coffee is in press. The expresso coffee contents of tocopherols and tocotrienols is in evaluation.
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
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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 βlactoglobulin. 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 foodstuffs largely consumed in Portugal, some confectionery
cakes were evaluated with pre-graduated students and the results published.
Several foodstuffs largely consumed will be analysed in order to evaluated their salt contents (sodium and
chloride/ bread), their nitrate contents (vegetables) and the presence of some additives (Glutamates/soups)
applying some “green methodolies” like potenciometric and voltametric techniques.
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 30 accessions belonging to the Portuguese garlic germplasm collection held at Banco Português de
Germoplasma Vegetal, Braga, were analyzed for the allicin content by a RP-HPLC-UV method. The results
indicate a high degree of variation in the allicin content which can be very valuable for garlic breeding and
improvement of Portuguese varieties. The contents in allicin ranged from 0.28 to 6.65mg/g dry basis indicating
that some accessions can present medicinal applications. This research is part of a MSc. thesis in execution.
The antioxidant activity of foods is another field of research to develop in our group. Two PhD will work in the
antioxidant activity and radical scavenging activity in aromatized waters and in almond fruits, respectively. A
paper was already submitted with the optimization of Phenols Extraction Conditions in Almond Fruits (Prunus
dulcis) and their Radical Scavenging Activity.
5. Beer composition and beer 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).
6. 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 and several
publications.
7. Evaluation of antioxidant potential: establisjment of phenolic profiles
Tronchuda cabbage
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In the continuation of a research project regarding the chemical characterization and the evaluation of the
antioxidant potential of tronchuda cabbage (Brassica oleracea L. var. costata DC), the flavonoid pattern of
larvae of cabbage white butterfly (Pieris brassicae L.; Lepidoptera: Pieridae) reared on the leaves of tronchuda
cabbage was analyzed by HPLC-DAD-MS/MS-ESI. Twenty flavonoids were identified or characterised, namely
sixteen kaempferol and four quercetin derivatives. Kaempferol 3-O-sophoroside, a minor component of tronchuda
cabbage, was found to be the main component in P. brassicae. Apart from this, only two other flavonoids
present in significant amounts in tronchuda cabbage (kaempferol 3-O-sophoroside-7-O-glucoside and kaempferol
3-O-sophoroside-7-O-sophoroside) were found in the larvae. The larvae have high amounts of quercetin derivatives,
which were present only in trace amounts in tronchuda cabbage extracts, suggesting that P. brassicae is
able to selectively sequester these flavonoids. The occurrence of a high content of flavonoids not detectable
in tronchuda cabbage extracts indicates that P. brassicae larvae are able to metabolize dietary flavonoids.
The changes in phenolic compounds and organic acids composition of tronchuda cabbage sprouts were
followed during the first 12 days of seedling development. The identified phenolic compounds include glucose,
gentiobiose and kaempferol glycoside esters of sinapic acid, as well as sinapoylcholine. The organic acids
were oxalic, aconitic, citric, pyruvic, malic, shikimic and fumaric. During germination, depletion of phenolic
compounds was observed. Kampferol, choline and glucose esters of sinapic acid showed a marked decrease
between days 2 and 6, whereas the changes in gentibiose esters of sinapic acid were smaller. The total
organic acids content increased rapidly during the first 4 days reaching. Malic acid, the major organic acid
found in sprouts, greatly contributes to this result though oxalic, pyruvic, and fumaric acids also increased in
the same manner. On the other side, aconitic, citric and shikimic acids showed a decrease between days 2
and 12 of germination.
A phytochemical study on tronchuda cabbage, cultivated under ten distinct fertilization regimes (application
of two different levels of nitrogen, three different levels of boron and of sulfur, an organic fertilizer and grown
without any fertilization) and collected at three distinct times is in course.
Edible mushroom species
Following the Ph.D. project on the chemical characterization of wild edible mushrooms species, the organic
acids and phenolics composition of nine wild species (Suillus bellini, Tricholomopsis rutilans, Hygrophorus
agathosmus, Amanita rubescens, Russula cyanoxantha, Boletus edulis, Tricholoma equestre, Suillus luteus
and Suillus granulatus) was determined. The results showed that all of the species presented a profile composed
of at least five organic acids: oxalic, citric, malic, quinic and fumaric acids. In a general way, the pair malic
plus quinic acids were the major compounds. Only very small amounts of two phenolic compounds (phydroxybenzoic acid and quercetin) were found in some of the analyzed species.
A comparative study on phenolics, organic acids and alkaloids composition of the fundamental structural
components (cap and stipe) of some species is in course.
Grapes and wine
Following the scope of a post-Doctoral project the use of Matrix Solid Phase Dispersion (MSPD) was tested
to, separately, extract phenolic compounds and organic acids from white “Vinho Verde” grapes. This method
was compared with a more conventional one previously developed, that combines solid liquid extraction (SL)
and solid phase extraction (SPE) to separate the two types of compounds. Although the results were qualitatively
similar for both techniques, the levels of extracted compounds were in general quite lower on using MSPD,
especially for organic acids. Therefore, SL-SPE method was preferred for the analysis of samples of Alvarinho,
Avesso, Asal-Branco, Batoca, Douradinha, Esganoso de Castelo Paiva, Loureiro, Pedernã, Rabigato and
Trajadura varieties, from four different locations. Principal Component Analysis (PCA) was applied separately
to establish the main sources of variability present in the data sets for phenolic compounds, organic acids
and for the global data. PCA of phenolic compounds accounted for the highest variability (77.9%) with two
PCs, enabling characterization of the varieties of samples according to their higher content in flavonol derivatives
or epicatechin. A strong effect of sample origin was observed. Stepwise Linear Discriminant Analysis (SLDA)
was used for differentiation of grapes according to the origin and variety, resulting in a correct classification of
100% and 70%, respectively.
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In another work a HPLC/UV-Vis methodology for determination of twenty free amino acids (aspartic acid,
glutamic acid, asparagine, glutamine, serine, threonine, glycine, alanine, valine, proline, arginine, isoleucine,
leucine, tryptophan, phenylalanine, cysteine, ornithine, lysine, histidine and tyrosine) in Dão red wine was
developed. This methodology was applied to the evaluation of the effect of the spoilage wine yeast Dekkera
bruxellensis. This yeast seems to influence the free amino acids composition, being aspartic and glutamic
acids, asparagine, glutamine and isoleucine the main affected compounds.
Olives
The phenolic compounds of different table olives from Portugal, namely, natural black olives “Galega”, black
ripe olive “Negrinha de Freixo”, Protected Designation of Origin (PDO) “Azeitona de Conserva Negrinha de
Freixo” olives, and “Azeitona de Conserva de Elvas e Campo Maior” Designation of Origin (DO) olives, were
determined, and seven compounds were determined: hydroxytyrosol, tyrosol, 5-O-caffeoilquinic acid,
verbascoside, luteolin 7-O-glucoside, rutin, and luteolin.
Also the phenolics composition of “alcaparra” table olives was determined in another study, being determined
luteolin 7-O-glucoside, apigenin 7-O-glucoside and luteolin. Three unidentified flavonoidic compounds and
one hydroxycinnamic derivative were also detected.
Medicinal species
Aqueous extracts of leaves of different hazel (Corylus avellana L.) cultivars (Cv. M. Bollwiller, Fertille de
Coutard and Daviana), were analysed for the definition of their phenolic composition. 3-, 4- And 5-caffeoylquinic
acids, caffeoyltartaric acid, p-coumaroyltartaric acid, myricetin-rhamnoside, quercetin 3-rhamnoside and
kaempferol 3-rhamnoside were determined. A p-coumaric acid, three myricetin and one quercetin derivatives
were also detected.
Different cultivars of walnut (Juglans regia L.) leaves (Cv. Lara, Franquette, Mayette, Marbot, Mellanaise and
Parisienne), were also investigated in what concerns their phenolic compounds. Ten compounds were
determined: 3- and 5-caffeoylquinic acids, 3- and 4-p-coumaroylquinic acids, p-coumaric acid, quercetin 3galactoside, quercetin 3-pentoside derivative, quercetin 3-arabinoside, quercetin 3-xyloside and quercetin 3rhamnoside.
8. Microbiology studies
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 are essential to assess the contribution of this
setting for antimicrobial resistance dissemination. Following, the achievements obtained in this field were:
- Salmonellosis is one of the most frequent foodborne diseases, being poultry products the most important
source of human infection. Salmonella is naturally susceptible to nitrofurans, an agent authorised in EU until
90’s for food animal production. In Portugal, during the last years, a widely illegal use of nitrofurans was
detected, affecting several types of husbandries, especially poultry ones. We report a remarkable incidence
of Salmonella isolates (65%), collected from human, food products and environment, with decreased
susceptibility to nitrofurantoin (MIC ≥ 64 mg/L), although resistance to other antimicrobial agents was also
observed. The observed results point out for another perspective related with antibiotic usage in food animal
production: the contribution of one antimicrobial agent on the emergence of particular bacteria implicated in
human infections. In fact, our findings suggest that the illegal use of nitrofurans, especially in the poultry
industry, might have contributed to the selection and prevalence of S. Enteritidis in food animals and
consequently to human salmonelosis in Portugal. Additionally, indiscriminate use of nitrofurans might also
have been implicated in the emergence of the two multiresistant nationally widespread S. Typhimurium clones
disseminated over the country.
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Class 1 integrons were detected in 78% of the sulfamethoxazole-resistant strains. Gene cassettes conferring
resistance to aminoglycosides (aadA1, aadA2 and aadA5), trimethoprim (dfrA1, dfrA12 and dfrA17), β-lactams
(blaPSE-1 and blaOXA-30) and chloramphenicol (cmlA1) were found. Other resistance genes (blaTEM, aac(3)-IV,
aphA1, catA, tetA, tetB, sul2) were identified, but not integrated as gene cassettes in integrons. Genes
blaPSE-1 and tetG were found in MDR S. Typhimurium DT104 isolates exclusively, while floR was found in
several S. Typhimurium clones.
The presence of class 1 integrons is significantly associated with multidrug resistance including to tetracycline
and chloramphenicol, which genes are not usually integrated in gene cassettes. Our results suggest that coselection and maintenance of MDR in Salmonella might be the result of the use of several agents in food
animals, namely sulfonamides, florfenicol and tetracyclines, which could difficult the reduction of antimicrobial
resistance in this zoonotic bacteria
The observation of common clones and several categories of genetic elements containing resistance genes
among human and food isolates suggest that animals for human consumption are a source of multidrug
resistant salmonella in humans.
- We analysed the presence of antibiotic resistant enterococci in faeces from Portuguese healthy humans
and the diversity of strains and genetic elements among isolates. Enterococci from poultry were included for
comparison. Antibiotic resistance was found in a variable number of samples: HLR (Highly resistant) to
streptomycin (52%), HLR to kanamycin (40%), HLR to gentamicin (11%) or VRE (5%). Co-resistance to
tetracycline, erythromycin, ciprofloxacin and quinupristin-dalfopristin were observed in most VRE, HLR to
gentamicin, streptomycin or kanamycin isolates. VRE contained vanA or vanC1. We found erm(B) in both
erythromycin resistant and non resistant isolates. No known genes were detected in some isolates resistant
to erythromycin, kanamycin or quinupristin-dalfopristin. Some HLR to gentamicin isolates from human and
poultry exhibited similar PFGE patterns. purK-1, purK-3 and purK-6 were observed among human Enterococcus
faecium isolates. Portuguese healthy people are frequently colonized with resistant enterococci. Analysis of
both PFGE and purK alleles suggest transmission of strains from animal to humans. The spread and persistence
of particular clones in the community might increase the human reservoir of enterococci of clinical significance
outside hospitals.
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ENVIRONMENTAL CONTROL AND REMEDIATION
Head of laboratory: Cristina Delerue Matos, Coordinator Professor / Helena Soares, Assistant Professor
Staff Members:
Maria do Carmo Vaz
Helena Maria Neto Ferreira de Sousa
Assistant Professor
Assistant Professor
Carla Alexandra Novais Oliveira e Silva
Assistant Professor
Assistant Professor
Maria Isabel Branco Alves Martins
Adjunct Professor
Maria Teresa de Oliva Teles Moreira
Adjunct Professor
Assistant
Florinda Figueiredo Martins
Assistant
Assistant
Assistant
Maria João Dantas Ramalhosa Ferreira Assistant
Maria Manuela Barbosa Correia
Assistant
Olga Manuela Matos de Freitas
Assistant
Salomé Sousa Teixeira
Assistant
Assistant
Sónia Adriana Figueiredo
Assistant
Valentina Maria Fernandes Domingues
Assistant
Technician
José Tomás Soares de Albergaria
Monitor
Maria Aurora Soares da Silva
Monitor
Maria Isabel Limpo de Serra
Monitor
Sérgio Alberto Monteiro de Morais
Monitor
Ph. D. Students:
Elisabete Machado, Ana Freitas, Filipa Grosso, Cristina Alves, Maria Machado
M. Sc. Students:
Joana Rocha, Joana Martins, Raquel Branquinho, Mayra Silvana, Mónica Sofia Santos
Lígia Cardoso, Ana Pedrosa
Number of articles in books: 3 (4-6)
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GRAQ:
Pollutants analysis
The pollution is one of the major problems of mankind. It is widespread in waters, soils and air. Nevertheless
the importance of a complete characterization, concerning physical, chemical and microbiological parameters,
it is of great importance to predict the possible effects of pollution over the ecosystems, by means of ecotoxicity
studies, as it was performed by our research team.
One of our researches works deals with nitrate and nitrites distribution study in rural and urban areas and
agricultural food products.
The analysis of each trace pollutant may demand a different research procedure.
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-FL, LC-MS, GC-ECD or MS, they have been
applied for the analysis food samples, air, ground and surface waters and soils. Liquid-liquid extraction (LLE),
solid-phase extraction (SPE), microextraction (SPME), stir bar sorptive extraction (SBSE) and microwaveassisted extraction (MAE) have been used to extract the analytes under study for chromatographic analysis.
MAE in combination with liquid chromatography and diode array detection has been investigated for the
determination of carbamates and ureas in tomato samples and in tomato derivatives, namely, tomato pulp
and tomato sauce used in tinned sardines and Northern bluefin tuna.
A procedure for the determination of seven indicator PCBs in soils and sediments using microwave-assisted
extraction and headspace solid-phase microextraction prior to GC-MS/MS was developed. The proposed
method can be extended to several other PCBs and used in the monitoring of soil or sediments for the
presence of PCBs.
MAE has proven to be suitable for the extraction of pesticides, over a wide range in polarity, from several
different environmental and food matrices. The merits of this extraction technique include a notable reduction
in solvent consumption and extraction time, together with good performance characteristics measured either
by the recovery or the repeatability and reproducibility of the extraction. MAE has proven to be suitable for the
extraction of pesticides, over a wide range in polarity, from several different environmental and food matrices.
The merits of this extraction technique include a notable reduction in solvent consumption and extraction
time, together with good performance characteristics measured either by the recovery or the repeatability and
reproducibility of the extraction.
Gold ultramicroelectrodes were used to study the adsorptive stripping voltammetric behavior of organophosphorus
and urea pesticides and to provide their sensitive and selective determination in soils extracts.
Waste management
Reduce the use of resources and eliminate or, if not possible, reduce the amount of waste, and also eliminate
or reduce its hazardousness are principles pursue by the waste management program (WMP). This program
supports all laboratory activities of students in a Chemical Engineering degree.
Whenever feasible, actions must be taken at the source and proceed a selective sorting of the generated
wastes; the primary steps naturally followed of suitable identification of each separated waste that will be
partially or totally constituted by used reagents, and/or expected primary reaction products or expected or
not side reactions products. Since it offers advantages in sustainable terms a reusing and/or recycling program
is preferred for each separated waste as target of treatment. This is the case of wastes of organic solvents,
recovered after distillation procedures. When not applicable, other alternative strategies may be used requiring
chemical/physical processes for a safe waste disposal. The specificity of each waste demands a detailed
study of alternative treatments. Always committed to save resources and to eliminate or to reduce toxicity of
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the present compounds by means of simple and quick operations using low cost reagents and instruments,
evaporations, filtrations, distillations, ionic exchange, neutralization, precipitation and redox reactions will be
studied. Adsorption of toxic compounds to natural materials such as cork is also exploited. The adequate
methodology for each waste may be defined by a quality control of each transformed product. Ecotoxicological
evaluations are made prior to a safe environmental disposal of detoxified wastes.
This line of research is in constant update in agreement with constant changes among in research and
educational laboratories.
Soils and groundwater remediation
The introduction of pollutants in soil, due human activities, created serious environmental problems, relating
soil and groundwater quality. The application of remediation technologies lead to the recovery of several
contaminated sites. Bioremediation and soil vapour extraction (SVE) are examples of successful soil remediation
techniques. The work developed with SVE involved two of four stages of a project that aim the construction of
a model capable to predict the remediation time and efficiency of SVE applied to a specific site. These stages
considered: i) the characterization of different soil matrices used later to prepare all types of soils (sandy,
humic or clay); ii) the elaboration of equilibrium isotherms useful to study pollutant behaviour in all soil
phases. These isotherms will be used later to calculate the amount of pollutant in each soil phase and
consequently process efficiency. Further work will involve the simulation of SVE process in all prepared soils
and the construction of the mathematical model.
SPME with GC-ECD has been used to evaluate TCE in groundwater. Batch test were carried for with four
different reagents: two strong oxidants and two reductive dehalogenators (elemental granulated iron and
nanoparticulated iron). These tests were performed in order to establish kinetics parameters.
Strains are currently used to improve degradation of trichloroethylene (TCE) and methyl tert-butyl ether (MTBE)
by soil microbial communities. The ideal conditions of incubation time and temperature were obtained and
measurements in these conditions were used as blanks. Microcosms were inoculated with the heavy metals
chosen (Cd, Hg or Pb) and with set quantities of the bacterial cultures chosen, singularly or as combinations.
In parallel, controls inoculated with sterile medium were monitored. The headspaces of the microcosms were
regularly sampled and analysed by GC-FID to determine the rates of degradation of the two pollutants.
Although the analysis of combinations is still ongoing, a number of bacterial strains and combinations there
of that positively degrade TCE or MTBE in soil in the presence of the heavy metals were discovered and tests
are being repeated for confirmation and statistical consolidation of results. At the same time, the remaining
combinations of strains/ organics/ metals are being explored. In conclusion, promising combinations of bacterial
strains for the degradation of the target pollutants (TCE and MTBE) in the presence of heavy metal contamination
was achieved and we expect to obtain even better results by fine-tuning of microcosm conditions in the
remaining months of this project.
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 maize
and peanut production. One of them explores the adsorption capacity of cork (Quercus suber L.) to remove
pyrethroids and textile dyestuffs from waters, in order to attenuate the impact of these compounds in aquatic
life.
Other study aims to explore the ability of Portuguese macro algae species for the removal of toxic metals
from aqueous solutions. A fixed-bed column was employed to evaluate the continuous biosorption performance
of macro marine algae, Pelvetia canaliculata. The objectives of present work were to investigate effects of flow
rate, the initial pH, influent concentration, ionic strength, metal solution (mono, bi and tri- component) and
electroplating industrial effluents.
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Finally, wastes from maize (Zea mais) and peanut (Arachis sp.) production have been tested successfully as
adsorbents for textile dyestuffs. Batch equilibrium and kinetic studies were performed. The experimental
results were fitted to models.
Biorremediation of wastewaters using microorganisms
As part of two going projects (POCTI/CTA/47875/2002 and UP/CGD /2005), flocculent cells of Saccharomyces
cerevisiae, a by-product from fermentative industry, were used as biosorbent for removing nickel from a real
electroplating effluent.
Chemical characterization of the electroplating effluent was performed. The efficiency of the overall system
was studied using the real electroplating effluent containing 17 ppm of nickel and two synthetic effluents. For
all three effluents, most of nickel biosorption occurred in the first stage of biosorption; the second and third (if
necessary) stages acted as polishing steps that pushed down the final concentration of nickel to values
below the legal values for water discharge (2 ppm).
Recycling of aluminium from extrusion wastes of moulds in aluminium industry
The anodising industry produces very alkaline streams containing high amounts of aluminium (about 200 g/L)
coming from extrusion wastes of moulds.
The main aim of this work was to develop a process of low cost for recycling aluminium from the extrusion
wastewaters of the moulds. For this purpose, a filtration followed by a precipitation process was developed; a
recovery of about 99% of the aluminium present in the wastewater, as aluminium hydroxide, was obtained. To
improve the purity of the aluminium salt, an optimization of the washing step was done. Therefore, an increase
of the purity of the aluminium salt up to 97% was obtained with two washing steps.
This process represents a promissory approach for reusing the residue produced by the extrusion process
with recycling of the main raw material, the aluminium.
Microbiology studies
The surveillance of the evolution of antibiotic resistance is an actual priority that includes, as fundamental
points, the investigation of the risk factors for the propagation and understanding the spreading of resistant
strains in different ecological niches, as in the environment. Wastewater, especially from hospitals, can work
as reservoirs of resistant bacteria and of transferable resistance genes in the environment. In that way,
sewage may function as an ecological niche suitable for antimicrobial resistance dissemination and wastewater
systems may contribute for environmental spreading of resistance. The screening of resistant bacteria,
characterization of antimicrobial resistance mechanisms and detection of antibiotics in different ecological
niches, namely urban sewage plants, river water, seawater and in three types of swine productions (intensive,
domestic and sustainable) is critical to control antibiotic resistance outside the hospital boundaries. Following,
the achievements obtained in this field were:
- Three hundred Entorococcus spp. isolates, 42 Pseudomonas spp. isolates and 21 Salmonella isolates were
collected from swine samples in Portuguese piggeries. Preliminary results showed that most of these isolates
presented decreased susceptibility to several groups of antimicrobial agents. Resistance to tetracycline,
erythromycin, minocyclin, streptomycin and quinupristin-dalfopristin was a common feature observed in
Entorococcus spp. isolates, which were also found to be reservoirs of several resistance genes (ermB, tetM,
tetL , tetS , aph-III and aac(6´)-aph-II). Some Pseudomonas spp. isolates (n=20) showed resistance to imipenem,
an antibiotic currently restricted to hospital setting usage. Interestingly, this resistance seems to be related
with the production of a metallo-β-lactamase as demonstrated by the positive bioassays in 9 isolates. All the
Salmonella isolates recovered were resistant to one (tetracycline) or more antimicrobial agents (streptomycin,
gentamicin, ampicillin, nalidixic acid, chloramphenicol, tetracycline, sulfonamides or trimethoprim).
Characterization of class 1 integrons revealed the presence of an array of gene cassettes (dfrA12, aadA) in
isolates of S. Typhimurium which also carried sul1, sul2 and sul3 genes and in isolates of S. Rissen, carrying
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sul1. The isolates of both serotypes were clonally related to strains previously observed in human and foodborne
isolates widely disseminated in Portugal. Interestingly, MDR isolates of the S. Rissen clone were recovered
from both piggeries studied. Other resistance genes (blaTEM, aac(3)-IV, tetA) were identified in the MDR
isolates, but not integrated as gene cassettes.
Detection of extended-spectrum beta-lactamases producers in waste water treatment plant setting, suggested,
at least, one of the contributions for their previous detection in river and marine coastal waters. Future work
will try to understand the routes of dissemination in natural environments.
Detection of extended-spectrum beta-lactamases producers in sewage system downstream hospital discharges
confirmed a method in that extended-spectrum beta-lactamases producers may function as biomarkers for
detection of antibiotic multi-resistant bacteria in the environmental compartment, making possible to track
the origin and fate of these multi-resistant pathogens or genes, in different ecological niches, providing a tool
for public health and environmental protection, helpful in the management of sustainable development.
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ANALYTICAL METHODOLOGIES
Head of laboratory: Aquiles de Barros, Associated Professor
Research Team:
Maria de Lourdes P. A. Souteiro Bastos
Full Professor
Cristina Maria Delerue-Matos
Maria do Carmo Vaz
Félix Dias Carvalho
Associate Professor
Fernando Manuel Gomes Remião
Assistant Professor
Márcia Claudia Dias de Carvalho
Assistant Professor
José António Maia Rodrigues
Assistant Professor
Paulo Joaquim Ferreira de Almeida
Assistant Professor
Maria Paula Guedes de Pinho
Maria Elisa Amorim Soares
Assessor
Helena Maria Ferreira Carmo
Assistant
Luís Guilherme de Lima Ferreira Guido
Assistant
Maria Isabel Afonso Rocha
Assessor
Assistant
Assistant
Maria de Fátima Sá Barroso
Assistant
Hendricus Petrus Antonius Nouws
Assistant
Assistant
Ph. D. Students:
Ricardo Jorge Dinis Oliveira, Vera Marisa Freitas Costa, Andreia Filipa da Silva Curto, Paulo Jorge Coimbra
Rodrigues Magalhães, João Paulo Grosso Pacheco
M. Sc. Students:
Estela Figueiredo, Paula Triunfante, Renata Silva, Paula Cristina Rodrigues Ribeiro, Susana Sabina Pires
Fernandes, Andrea Sofia Lopes Torres Saraiva, Ana Maria de Jesus Caxide Praça Pedroso
The development and validation of new analytical methodologies is essential to support the molecular and
biochemical toxicological studies and more importantly, to the identification and/or quantification of important
analytes in either human specimens, different food products, or other relevant matrices. With the collaboration
of the National Institute of Legal Medicine, we have developed studies with human post-mortem specimens for
the characterization of different intoxication agents, including drugs of clinical use such as benzodiazepines
and other toxics such as mercury.
During the period that concerns to this report the following methodologies were developed:
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Methodologies for food analysis
— 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);
— 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);
— Opening of a new line of investigation, related with the previous one, involving the qualitative and quantitative
analysis of phenolic compounds in barley and malt and the study of the contribution of the phenolics to the
antioxidant capacity of the barley and malt (in collaboration with the Department of Fermentation Chemistry
and Bioengineering of the Institute of Chemical Technology, Prague)
— 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
bioactive constituents from hop.
— Opening of a new line of investigation focused on the use of membrane based extraction techniques for the
development of analytical flow systems to be applied in food analysis.
—To address the concerns raised by the use of pesticides and environmental pollution with consequent
accumulation of metals that may cause toxicity, several metals including, aluminium, copper and lead,
spectrometric atomic absorption methods were developed and applied for their quantification in different food
products and with different pesticides treatment, namely in grapes and olive fruits.
For 2007, the objective of the group is to continue the activity of the last years, with special focus on:
— Exploitation of new possibilities of application of the equipment developed for the determination of diacetyl
on line, namely in the determination of sulphur dioxide and acetaldehyde; in this context, some modifications
are being introduced in the pervaporation module, in order to allow an easier recovery of pervaporated compounds,
with subsequent analysis using HPLC.
— Monitorization of the phenolic composition of barley and malt throughout the malting process (malting pilot
plant of Unicer); monitorization of the phenolic composition throughout the brewing process (brewing pilot
plant of Unicer).
— Qualitative and quantitative analysis of phenolic compounds in different malted barley varities; characterization
of proantocyanidin oligomers from barley and malt by LC-ESI-MS; determination of free, bound and esterified
phenolic compounds in barley and malt; study of the contribution of phenolic compounds to the antioxidant
capacity of barley and malt by using spectrophotometric tests (in collaboration with the Department of
Fermentation Chemistry and Bioengineering of the Institute of Chemical Technology, Prague).
— 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; studies
about the importance of additives for the durability of these materials.
— Continuation of the analytical, industrial and biochemical studies of some bioactive constituents from hop,
mainly xanthohumol, isoxanthohumol, ergosterol and ergocalciferol.
— 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.
Methodologies for health sciences
Determination of inorganic and organic mercury in human specimens
Mercury poisoning can occur either voluntarily or accidentally. Recently, two fatal intoxications both with a
long survival period after the inorganic mercury intake led to the study of its toxicokinetic profile in the post-
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mortem specimens of these individuals. These studies were performed under a cooperation protocol between
our research unit and the National Institute of Legal Medicine. Of particular interest was the study of the
possible formation of organic mercury specimens. To address this issue, chromatographic HPLC methodologies
with UV detection were applied to the quantification of organic mercury in human specimens, including postmortem specimens while inorganic mercury was quantified by atomic absorption spectrometry.
Determination of potentially toxic catechol metabolites in human specimens
Catechol compounds are of extreme importance for the neurotransmission process. The role of the catecholamine
neurotransmitters in stress and disease is increasingly acknowledged. These are highly reactive and their
oxidative metabolism leads to the formation of highly toxic metabolites that induce cellular damage and can
lead to cellular death. It is therefore suspected that these metabolites, that include the adducts that result
from the conjugation of the catechol compounds with important cellular nucleophyles, such as glutathione,
are involved in the pathophysiological effects related to the toxicity of the catecholamines. For their study,
analytical methodologies were developed and applied to human specimens, namely blood, which in this case
is the matrix with highest clinical relevance.
Neurotransmitters
Chromatographic methodologies with HPLC coupled to diode array and electrochemical detection were
developed for the analytical determination of glutathione adducts of different catecholamines namely, adrenaline,
noradrenaline and dopamine. Two position isomers of the glutathion-S-yl-adrenaline were synthesised by
tyrosine catalysis and characterized by its UV/Vis and mass spectra and electrochemical proprieties. The
mass analysis was performed in the Chemistry Department of Aveiro University. The adduct 5–(Glutathion-Syl)-adrenaline was subsequently synthesised and purified in the REQUIMTE unit Chemistry Department,
Faculty of Science and Technology, University Nova de Lisboa and used in this study to validate the
chromatographic methodology. A method for the extraction of these adducts from human plasma was also
developed, based on adsorption to activated alumina, which showed adequate recoveries and proved to be
crucial in removing interferences from plasma. These methodologies were then further applied and validated
for the quantification of the glutathione adducts of adrenaline, which is the main catecholamine involved in the
pathophysiological process of stress.
These developed methodologies have been applied to characterise the reactive and potentially toxic metabolites
of neurotransmitter catecholamines in humans suffering cardiovascular pathologies in patients of the Department
of Cardiothoracic Surgery of the Hospital of V. N. Gaia.
Amphetamines: “ecstasy”
The mechanisms of toxicity of the most popular amphetamine drug of abuse: 3,4methylenedioxymethamphetamine (currently known as MDMA or ecstasy) are yet to be fully elucidated.
Recent evidence point to the determinant role of oxidative metabolism in the expression of the toxic effects of
these drugs and even related with the high variability which is a common feature of the intoxications with
amphetamines. It is therefore necessary to investigate the role of these metabolites which have a catechol
structure and that can conjugate with glutathione as is the case of the catecholamine neurotransmitters. This
knowledge may prove to be crucial for the finding of efficient treatment for these intoxications that are currently
still lacking, in part because of the uncertainty concerning the toxicological pathways involved. To address
this issue we have developed and validated an extraction and chromatographic methodology with HPLC
coupled to electrochemical detection for the quantification of the major oxidative metabolite of MDMA, Nmethyl-á-methyldopamine, and applied and validated this method for the quantification of this metabolite in
human serum. Also, in collaboration with another unit of REQUIMTE (the Chemistry Department of the
Universidade Nova de Lisboa) the synthesis and full analytical characterization (including NMR and cyclic
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voltammetry methods) of the oxidative metabolites of MDMA and their glutathione adducts is performed prior
to the toxicity testing that is subsequently performed.
Currently, several other analytical methodologies are being developed and implemented. These include the
determination of metals in edible mushrooms, from different geographical areas within the country. Other
methods currently under validation for its application to human specimens, namely blood, are the quantification
of the drugs colchicine and amphotericine, which are currently used in therapy and have been implicated in
severe adverse reactions by using HPLC/UV methods. The simultaneous quantification of ethanol and its
highly toxic and unstable metabolite acetaldehyde is also currently under validation for application to different
specimens including blood and urine.
Electroanalysis
The application of square-wave adsorptive stripping voltammetry presents very special features. This has
been documented in connection with the trace analysis and characterisation of organophosphorus pesticides
using gold and mercury film ultramicroelectrodes in very small sample volumes. The use of high square-wave
frequencies decreases the interference of dissolved oxygen in the solution, enabling the inclusion of a hanging
mercury drop electrode in a flow-injection system. Based on this knowledge, methods for the analysis of the
antidepressants sertraline, fluvoxamine, paroxetine and fluoxetine in pharmaceutical products were developed.
Potentiometric devices are designed with suitable arrangements for batch and flow experiments. Sensors are
prepared with ion-exchangers or neutral carriers in a polymeric environment. Plasticizing solvents of variable
dielectric constant and of hydrophobic nature are used to provide suitable physical and chemical features to
the membrane. The potential signal is primarily due to target ions, such as inorganic and organic compounds
of environmental and pharmaceutical interest
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PHYSICOCHEMICAL CHARACTERIZATION OF FOOD PRODUCTS
Head of laboratory: Maria do Pilar Gonçalves, Associate Professor / Alberto Sereno, Associate Professor
Staff Members:
Loïc Hilliou
Post-Doct Fellows:
Marta Isabel de Glória V. M. Silva
Ph.D. Students:
Luis Mayor Lopez, Wancheng Sittikijyothin, Fabio Donato Soares Larrotonda
Number of Ph.D. Thesis: 3 (17-19)
1. COMFOOD: Controlling the Microstructure of Food Products by Rheo-Optical Methods
20% of the equipment needed to run task 1 of this project (POCI/EQU/58064/2004) could be purchased. New
funding has been transferred by the FCT in late December 2006. Despite the lack of funding, preliminary
experiments on sheared carrageenan biopolymers could be carried out, which have been presented in an
international conference, reported in the conference proceeding and in a paper to be published. Optical
microscopic imaging of sheared carrageenans has been obtained (see section C below). In addition, Atomic
Force Microscopy imaging of the same samples has been initiated.
Workplan for 2007: a demand for an extension of the project duration by 9 months has been submitted to the
FCT in order to buy, set up and validate the remaining parts of the experimental equipments.
2. Physical characterization of PHA biopolymers
An oven allowing rheological testing up to 600ºC has been installed in the laboratory in order to perform the
thermo-mechanical characterization of PHA samples.
Workplan for 2007: testing (rheology and Differential Scaning calorimetry) of commercial and newly synthesised
PHA to be released by the project partners (CQFB) in the last year of this FCT funded project (POTI/BIO/
55789/2004).
3. Characterization of biopolymer mixtures for food applications
This GRICES/DAAD cooperation allowed the characterization by means of Fourier transform rheology of
commercial gelatin, κ- and ι-carrageenan gels as well as κ-ι hybrid carrageenans extracted from Mastocarpus
stellatus seaweeds (see project POCTI/EQU/45595/2002). Results have been reported in an international
conference. Polarized optical microscopy and dielectric spectroscopy of edible films and gels were also
performed.
Workplan for 2007: study of gelatin-carrageenan mixed gels and Magnetic Resonance Imaging of food gels.
4. Extraction of added value compounds from orange peel wastes: application to mixed biodegradable
films
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The addition of mica flakes to pectin based film formulation has been tested in order to provide an alternative
reduction of the high hygroscopic properties. Preliminary results have been presented at the EUROMEMBRANE
2006 conference and reported in a scientific paper.
5. Rheological behaviour and morphology of protein-polysaccharide mixed systems
Two articles were prepared and submitted for publication. A PhD dissertation, “Rheological and microstructural
properties of β-lactoglobulin- galactomannan aqueous systems”, was presented to the Faculty of Engineering
of the University of Porto and approved.
6. Cold gelation of globular proteins
a) Results obtained by systematically varying the denaturation conditions and the protein concentration could
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. These results
were reported in a scientific paper submitted for publication.
A study on the effect of magnesium and tara gum concentrations over the texture of WPI cold-set gels was
also done and the results have been compiled in order to be submitted for publication.
b) In the frame of a CAPES/GRICES project:
i) two articles on the rheology and structure of whey proteins/hydrolysed waxy maize starch systems were
submitted and accepted for publication. A Ph.D. dissertation, “Caracterização reológica e morfológica de
sistemas mistos amido modificado/proteínas do soro do leite para aplicação em adesivos”, was presented to
IMA – UFRJ (Brazil) and approved.
ii) the rheological behavior and the texture profile of biodegradable superabsorbent polymers (SAPs) were
determined. Results were presented in an international conference and two papers were submitted to scientific
journals.
Work plan for 2007: In the frame of cold-set gelation of WPI, studies will focus on establishing relationships
between gels structure (at the mesoscopic, microscopic, and sub-microscopic scales) and mechanical
properties. Characterization of the kinetics of WPI solutions gelation after addition of salt is also envisaged.
7. Studies in Food Structure
During the last year of studies of the changes in food structure during processing by dehydration, mathematical
models were established and validated to describe the interaction relations quality-macrostructure-microstructure
of processed food materials. This was achieved by studying structural changes observed in pumpkin fruits,
used as a model food, when submitted to osmotic dehydration.
Several articles were prepared and submitted for publication. A Ph.D. dissertation, “Characterization and
modelling of structural changes in fruits and vegetable tissue submitted to dehydration processes”, was
presented to the Faculty of Engineering of the University of Porto and approved.
8. Technology for edible films and coatings for food from low value national resources
Model composite films based on commercial pectin and κ-carrageenan were prepared and characterized in
terms of their hygroscopic and mechanical properties, and permeability to water vapour. The films were
produced using knife coating technique with an automatic film applicator. Different film formulations, using
different relative amounts of the biopolymers and several possible plasticizers were tested. Film characterisation
was achieved through the study of the dielectric properties, FTIR spectroscopy, colour and opacity. The effect
of the speed of application of the dispersion during the film formation was also assessed.
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Workplan for 2007: characterisation of starch from localy produced acorns. Determination of film permeability
to gases (oxygen and carbon dioxide). Design of films with specific permeability properties; reactive compounds
(ascorbic acid and/or calcium hydroxide to interact with oxygen and carbon dioxide, respectively) will be
added to the polymer matrix, while mica flakes or clays will be used as impermeable barriers.
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TOXICITY AND PROTECTION STUDIES
Head of Laboratory: Maria de Lourdes Bastos, Full Professor
Staff Members:
Rosa Seabra
Full Professor
Branca Silva
Assistant Professor
Paula Andrade
Assistant Professor
Felix Carvalho
Assistant Professor
Fernando Remião
Assistant Professor
Márcia Carvalho
Assistant Professor
Helena Carmo
Assistant
Patrícia Valentão
Assistant
Carla Sousa
Technician
Carla Rodrigues
Technician
Post-Doct Fellows:
Sónia Dópico
Ph. D. Students:
Bárbara Ribeiro, João Capela, Ricardo Oliveira, Helena Pontes, Vera Costa, Ema Alves, Miguel Lopes
M. Sc. Students:
Alexandra Lopes, Orlanda Pereira, Cecília Teixeira
Number of articles in scientific journals: 13 (45, 157-168)
Number of Patents: 2 (2,3)
Number of M. Sc. Thesis: 1 (13)
STUDIES OF MECHANISMS OF TOXICITY USING IN VIVO AND IN VITRO MODELS
These studies comprise the elucidation of the toxicity mechanisms through the use of in vivo and in vitro
models that allow the comprehension of the cellular and molecular events that underlie the toxicological
effects. This has been achieved through the implementation of different cellular models that include suspensions
of primary cells freshly isolated from rat and/or mouse (hepatocytes and cardiomyocytes), cultured primary
cells obtained from rat (hepatocytes and neuronal cells) and cultured immortalized cell lines that are genetically
engineered and constitute humanized models for the expression of human metabolizing enzymes.
The characterization of the mechanisms involved in the toxic effects at the cellular and molecular levels
include the evaluation of toxicity biomarkers related to oxidative stress events, cellular signalling pathways,
and mechanisms of cell death.
During the period that concerns to this report the following studies were developed:
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Studies of the toxicity mechanisms of amphetamine-like drugs of abuse
The mechanisms of toxicity of amphetamine derivatives currently used as drugs of abuse were studied with
particular emphasis on the neurotoxic, cardiotoxic and hyperthermic effects that constitute the most worrisome
in terms of acute and long-term toxicity of these drugs. Additionally, the influence of genetic variability in
human metabolism in the toxicological effects of the amphetamines has been studied.
In what concerns the neurotoxic effects of ecstasy neuronal serum free cultures from rat cortex were used.
Since MDMA intake induces hyperthermia in both animals and humans, the experiments were performed
under normal and hyperthermic conditions. The obtained results showed a dose-, time- and temperaturedependent apoptotic cell death induced by MDMA in cortical neurons. Furthermore, this study showed the
participation of free radicals in 3,4-methylenedioxymethamphetamine-induced cell death and provided for the
first time evidence that direct 3,4-methylenedioxymethamphetamine serotonin 2A-receptor stimulation leads
to neuronal cortical death.
The involvement of hepatic metabolism and increased body temperature in the neurotoxic effects of MDMA
were further studied. Metabolism of MDMA involves reactive catechols that can undergo oxidation to the
corresponding o-quinones and conjugate with GSH to form glutathionyl adducts. The neurotoxicity of MDMA
and three of its metabolites obtained by synthesis, N-Me-alpha-MeDA, alpha-MeDA, and 5-(GSH)-alphaMeDA [5-(glutathion-S-yl)-alpha-methyldopamine] was evaluated in rat cortical neuronal serum-free cultures
under normal and hyperthermic conditions. The results provided evidence that MDMA metabolites, especially
under hyperthermic conditions, contribute to MDMA-induced neurotoxicity.
The role of hyperthermia in the development of cellular toxicity has been evaluated and the mechanisms
linking heat shock, oxidative stress and cell survival/cell death were studied. The time course of hyperthermiainduced oxidative stress and cellular signalling were evaluated using freshly isolated mouse hepatocytes. The
results demonstrated hyperthermic toxic effects developed concomitantly with a putative protective process
by activation of heat shock factor 1 (HSF1) and the formation of heat shock protein 70 (HSP70).
The cardiotoxic effects of d-amphetamine and the influence of intense physical exercise that relates well to
the circumstances under which this type of drugs of abuse are often consumed were also studied. The
intraperitoneal administration of 20 mg/kg of d-amphetamine produced histological signs of cardiotoxicity,
which were not enhanced by physical exercise; however, the combined action of d-amphetamine and physical
exercise significantly aggravated protein oxidation markers.
The issue of the great interindividual variability in the susceptibility to the toxicity of amphetamine-like drugs
of abuse [MDMA and 4-methylthioamphetamine (4-MTA)] has been addressed by using in vitro assays with
genetically engineered humanized cellular models that express human metabolizing enzymes that are
polimorphically expressed and are therefore involved in the genetic variability of human metabolism. Our
studies for the first time showed that polymorphisms of the cytochrome P450 enzyme CYP2D6 influence the
cytotoxic effects of these drugs of abuse. The involved metabolites and their cytotoxic effects were characterized
for MDMA and it was proved that the polymorphic expression CYP2D6 mediates MDMA toxicity via formation
of N-methyl-alpha-methyldopamine, which raises the concern that CYP2D6 ultrarapid metabolizers with
increased capacity of producing toxic metabolites might be more susceptible to MDMA intoxications. Similar
studies that aim at characterizing the metabolites involved in the cytotoxic effects of 4-MTA are currently
under way. Additionally, the use of human liver frations are also being used to determine the relevance of these
findings to the human situation.
Studies of the toxicity mechanisms of cathecol neurotransmitters
High concentrations of circulating biogenic catecholamines are often found during the course of several
cardiovascular disorders. Additionally, coronary dysfunctions are prominent and frequently related to the
ischemic and reperfusion phenomenon (I/R) in the heart, which leads to the release of large amounts of
catecholamines, namely adrenaline, and to a sustained generation of reactive oxygen species (ROS). Thus,
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studies in freshly isolated calcium rat tolerant cardiomyocytes were developed to evaluate the toxicity biomarkers
related to the effects of the association of catecholamines with ROS at the cardiovascular level.
Studies of the toxicity mechanisms of pesticides. Investigation of possible antidotal pathways for
paraquat poisonings
The use of the process of induction of de novo synthesis of P-glycoprotein (P-gp) in the treatment of xenobioticinduced intoxications in mammals
This study concerns the process of induction of de novo synthesis of P-glycoprotein (P-gp) to be used as a
treatment of xenobiotic-induced intoxications in mammals and, in particular, of paraquat (PQ)-induced
intoxications. It was shown, for the first time that the induction of de novo synthesis of P-gp, 2 hours after PQ
exposure, led to a remarkable decrease of PQ accumulation in the lung up to 40% of the group exposed only
to PQ, and to an increase of its faecal excretion with the subsequent reversion of the toxicity, as shown by
several biochemical and histopathological biomarkers of toxicity in just 24 hours.
The use of the salicylate as an antidote for paraquat intoxications in mammals
This study concerns the use of salicylate in the treatment of mammal intoxications caused by the herbicide
paraquat (PQ). It was achieved, for the first time, 100% of survival 30 days after the administration, by
intraperitoneal route, of a PQ dose that, in the absence of treatment, is itself 100% lethal at the end of 6 days.
The administration of salicylate, two hours after PQ, reversed the PQ toxicity and extended the life of the
animals to the levels of the control group.
ECOTOXICITY STUDIES
The use of in vitro models using the crustaceans from genus Artemia have been successfully applied for the
ecotoxicity testing of environmental contaminants that include detergents and widely used pharmaceutical
drugs.
Toxicity studies in plants
Tronchuda cabbage
The ability of tronchuda cabbage to act as a scavenger of NO and ONOO- was investigated in in vitro systems.
The aqueous extracts obtained from tronchuda cabbage seeds and from its external and internal leaves
exhibited a concentration dependent scavenging capacity. The antioxidant potential observed against the two
reactive species was as follows: seeds > external leaves > internal leaves.
Studies regarding the antioxidant potential of tronchuda cabbage sprouts are in course.
Edible mushroom species
The antioxidant potential of nine wild species was evaluated. All of the species exhibited a concentrationdependent anti-radical ability. T. rutilans revealed the highest antioxidant capacity.
A comparative study on the antioxidant capacity of the fundamental structural components (cap and stipe) of
some species is in course.
Olives
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The antioxidant potential and antimicrobial activity of different table olives from Portugal, were evaluated. PDO
and DO table olives revealed a wide range of antimicrobial activity. Candida albicans was resistant to all the
analyzed extracts.
In another work “alcaparra” table olives were evaluated for their in vitro activity against several Gram positive
and Gram negative bacteria and fungi. At low concentrations “alcaparra” extract revealed significant inhibition
of both Gram positive and Gram negative bacteria growth, with exception of Pseudomonas aeruginosa.
Nevertheless, no antifungal activity was observed at the tested concentrations.
Medicinal species
Extracts of leaves of three different hazel cultivars were studied for their antioxidant and antimicrobial capacities.
The hazel leaves extracts presented high antioxidant activity in a concentration-dependent way, in general
with similar behaviour of all cultivars. Gram positive bacteria revealed to be very sensitive to the extracts.
Gram negative and the fungi displayed much lower sensitivity, being P. aeruginosa and C. albicans resistant
at the tested concentrations.
Also the antimicrobial and antioxidant properties of six walnut leaves cultivars were evaluated. Walnut leaves
selectively inhibited the growth of Gram positive bacteria, being Bacillus cereus the most susceptible one.
Gram negative bacteria and fungi were resistant to the extracts at the tested concentrations. In a general way,
all of the studied cultivars presented high antioxidant activity, being Cv. Lara cultivar the most effective one.
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BIOMIMETIC SYSTEMS AND SIMULATION
Head of Laboratory: Maria João Ramos, Associate Professor
Staff Members:
Alexandre Magalhães
Assistant Professor
André Melo
Assistant Professor
Pedro Jorge Araújo Alves da Silva
Assistant Professor
Pedro Fernandes
Invited Assistant Professor
Agostinho Antunes Pereira
Post-Doct Fellows:
Cristian Robert Munteanu, Susana Rodrigues Pereira
Ph. D. Students:
Nuno Cerqueira, Fátima Lucas, Sérgio Sousa, Ricardo Branco, Alexandra Carvalho, Irina Moreira, Zenaida
Mourão, Alexandre Carvalho, Marco Preto, Daniel Dourado, Natércia Brás, José Rui Marques, Alexandra
Marques, Bruno Tamames, Juan Tamames
M. Sc. Students:
Eva Cunha, Daniel Osório
Project Grantee:
Sónia Patrício, Marta Perez
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. Docking techniques as well as homology modelling procedures
are part of our studies too1. Just as in the previous latter years, we have also pursued our new line of
computational genomics. A more detailed description concerning our main present interests, which have
derived from year 2005 and will continue in 2007, follows:
I.
Disease Related Research
Cancer
(i)
The study of the catalytic and inhibition mechanisms of enzymes P450 1A2 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 6-10, 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
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transferase and farnesyl transferase11-13. These studies have been financed by the Fundação para a Ciência e
Tecnologia, Portugal (RNR) via project POCI/QUI/61563/2004.
A.I.D.S.
(iii)
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, continued14. 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.
Still on the AIDS theme, we have also engaged on understanding resistance by the HIV-1 virus to the
different NRTIs (Nucleoside Reverse Transcriptase Inhibitors) that are in the market, a study met with good
results15-17.
Additionally, we are now studying the catalytic mechanism of the HIV-1 Protease with a series of its substrates
with a view to understanding the way it actually works18-19.
Hypertension
(iv)
Angiotensin II (Ang II) is a peptide hormone that is involved in the mechanisms related to the regulation
of blood pressure, by activating the angiotensin type 1 (AT1) receptor. Consequently, its antagonists to AT1
receptor may find an application in the treatment of patients suffering from high blood pressure and may be of
high commercial value. It is the purpose of this project to model synthetic peptide analogues of Ang II and
evaluate their ability to act as mimetic antagonists of this hormone to AT1 receptor. These peptides have been
obtained by appropriate substitutions of original coded amino acids by non-coded ones, currently synthesized
at the laboratories of Universidade do Minho.
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 and on some of its peptidic analogues
in both environments, water and DMSO. To improve the modelation of hydophobic environments similar to the
observed at the At1 receptor, we have also performed some additional reactivity studies on small molecular
systems in non-aqueous solvents20. 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
(v)
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 spots21-23.
(vi)
Continuation of the development of new formalisms to analyse the molecular interactions in association
processes. 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.
(vii)
Molecular docking protein-ligand techniques are extremely important and widely used in molecular
modelling. We have developed a new software, a Multi staged Docking with an Automated Molecular Modelling
protocol (MADAMM), which enables flexibilisation of both the receptor and the ligand, with very important
results. The software is now being released.
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(viii)
Several host-guest complexes have been studied, namely with transition metal ions, in order to
interpret the role of non-covalent bonding in biological systems.24
III.
Computational Genomics
We have pursued our new line of research on Computational Genomics with the goal of expanding the team’s
research experience on Bioinformatics, allowing the simultaneous integration of genomic and proteomic data.
We have been supervising research on the evolution and comparative genomics of protein-coding genes with
important biological functions, namely the human HADH2/ABAD that is linked to the mitochondrial toxicity in
Alzheimer’s disease25 and the cytochromes P450 2C, which are involved in the metabolism of drugs and
carcinogenic compounds26. Additionally, we have performed molecular studies on the evolution and comparative
genomics of various vertebrate species, including fish, cat and pangolins27-31. The identification of the sequence
or function of genes in non-human organisms is an important approach for finding and elucidating the function
of human genes, its mutations, and its implications in disease progression. Part of this project is being
carried out in close collaboration with S.J.O’Brien and W.E.Johnson from the National Cancer Institute,
National Institute of Health, U.S.A. The study of the pangolin genome is being financed by the Fundação para
a Ciência e Tecnologia, Portugal, via project POCTI/BSE/47559/2002 and by the National Geographic Society,
U.S.A. (Grant 7483-03).
IV.
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] M.J.Ramos, P.A.Fernandes, Curr Comp-Aided Drug Design, 2006, 2, 57
[2] R.Fonseca; A.Melo; F.Iori; M.C.Menziani; M.J.Ramos, Med Chem, 2006, 2, 401
[3] S.Pereira; N.M.F.S.A.Cerqueira; P.A.Fernandes; M.J.Ramos, Eur Biophys J, 2006, 35, 125
[4] N.M.F.S.A.Cerqueira; P.A.Fernandes; L.A.Eriksson; M.J.Ramos, Biophys J, 2006, 90, 2109
[5] N.M.F.S.A.Cerqueira; P.A.Fernandes; M.J.Ramos, J Phys Chem B, 2006, 110, 21272
[6] M.F.Lucas; M.J.Ramos, J Phys Chem B, 2006, 110, 10550
[7] A.P.Carvalho; P.A.Fernandes; M.J.Ramos, Prog Biophys Mol Biol., 2006, 91, 229
[8] A.T.P.Carvalho; P.A.Fernandes; M.J.Ramos, J Comp Chem, 2006, 27, 966
[9] A.T.P.Carvalho; P.A.Fernandes; M.J.Ramos, J Phys Chem B, 2006, 110, 5758
[10] E.S.Henriques; M.A.C.Nascimento; M.J.Ramos, Int J Quant Chem, 2006, 106, 2107
[11] R.J.F.Branco; P.A.Fernandes; M.J.Ramos, Theoretical Chemistry Accounts, 2006, Vol. 115 (1), 27-31
[12] R.J.F.Branco; P.A.Fernandes; M.J.Ramos, Journal Phys. Chem. B, 2006, Vol 110, 16754-16762
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[13] S.F.Sousa; P.A.Fernandes; M.J.Ramos, Proteins: Structure, Function, and Bioinformatics, 2006, Vol
65, 15–26
[14] Sharma RK et al, J Med Chem, 2006, 49, 3595
[15] A.T.P.Carvalho; P.A.Fernandes; M.J.Ramos, Med Chem, 2006, 5, 491
[16] A.P.Carvalho; P.A.Fernandes; M.J.Ramos, Mini-Reviews Med Chem, 2006, 6, 549
[17] A.T.P.Carvalho; P.A.Fernandes; M.J.Ramos, Int J Quant Chem, 2006, 107, 292
[18] B.Tamanes; P.A.Fernandes; M.J.Ramos, Curr Bioact Comp, 2006, 2, 243
[19] P.A.Fernandes et tal., Letters In Drug Design & Discovery, 2006, 3, 383
[20] A.Melo; A.J.I.Alfaia; J.C.R.Reis; A.R.T.Calado, J Phys Chem B, 2006, 110, 1877
[21] I.Moreira; P.A.Fernandes; M.J.Ramos, Proteins, 2006, 63, 811
[22] I.Moreira; P.A.Fernandes; M.J.Ramos, J Phys Chem B, 2006, 110, 10962
[23] I.Moreira; P.A.Fernandes; M.J.Ramos, Int J Quant Chem, 2006, 107, 299
[24] A.Suwattanamala; D.Appelhans; M.Wenzel; K.Gloe; A.L.Magalhães; J.A.N.F.Gomes, Chem. Phys.,
2006, 320, 193
[25] A.T.Marques; A.Antunes; P.A.Fernandes; M.J.Ramos, BMC Genomics, 2006, 7, 202
[26] R.Fonseca; A.Antunes; A.Mélo; M.J.Ramos, Gene, 2006, (In press)
[27] A.Antunes; K.Gharbi; P.Alexandrino; R.Guyomard, Mol Ecol Notes, 2006, 6, 547
[28] A.Antunes; R.Faria; W.E.Johnson; R.Guyomard; P.Alexandrino, J Heredity, 2006, 97, 193
[29] H.Kim; A.Antunes; S.J.Luo; J.Menninger; W.G.Nash; S.J.O’Brien, W.E.Johnson, Gene, 2006, 366, 292
[30] W.E.Johnson; E.Eizirik; J.Pecon-Slattery; W.J.Murphy; A.Antunes; E.Teeling; S.J.O’Brien, Science,
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[31] S.Luo; Q.Cai; V.A.David; L.Zhang; P.Martelli; N.Lim; N.Ferrand; S.Chin; P.Gaubert; M.J.Ramos;
S.J.O’Brien; A.Antunes; W.E.Johnson, Mol Ecol Notes, 2006 (In press)
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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
Helena Susana da Costa M. Ferreira Assistant Professor
Catarina Mansilha
Assistant Professor
Maria José Feio
Invited Assistant Professor
Marlene Susana Dionísio Lúcio
Assistant
Post Doct Fellows:
Jerome Keldenich
Ph. D. Students
Patrícia Neves, Sofia Costa Lima, Sónia Garcia, David Costa, Christophe Siquet, Ana Maria Gomes, Marisa
Andreia Freitas, Diana Maria Gaspar
M. Sc. Students:
Carlos Miguel Azevedo, Isabel Sousa
(A) Biophysics and Biochemistry of organized media
Modifications of physical characteristics of the membrane lipid bilayer have proved to be important to elucidate
a variety of pathological processes, such as chronic inflammatory conditions, carcinogenesis and cardiovascular
diseases.
The main objective of this part of project 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.
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. The rational development of safer NSAIDs will
depend upon the understanding of the processes initiating and promoting gastric injury. 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. For this purpose, three different NSAIDs: indomethacin, acemetacin
and nimesulide were selected and their influence on the biophysical properties of 1, 2dipalmitoylphosphatidylcholine (DPPC) was studied using different and complementary techniques. DPPC is
a typical phospholipid with regard to its role in determining the physicochemical and biological properties of
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the cellular membranes. By dispersing this lipid in water, multilamellar vesicles (liposomes) are formed
spontaneously which exhibit many features of the cell membranes such as the barrier function or permeability.
Depending on the temperature aqueous dispersions of DPPC can form different mesophases. Structural
changes involved in the DPPC thermal transitions upon the interaction of the bilayers with NSAIDs were
investigated by small and wide angle X-ray scattering (SAXS/WAXS). The results of the X-ray scattering
experiments were also compared to the results obtained by differential scanning calorimetry studies (DSC).
Besides the bilayers systems, models consisting of phospholipids spread at the air–water interface as a
Langmuir monolayer and a second component (NSAIDs) dissolved in the subphase are interesting model
systems, since the bilayer can be considered as two weakly coupled monolayers. Additionally, many physicalchemical parameters of the interface as molecular density, lateral pressure, surface potential, or ionic conditions
can be changed in a well defined way and thus, the lipid monolayer 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 about the penetration mechanism of indomethacin.
We have also developed studies of the effect of NSAIDs on their interaction with membrane enzymes that are
usually involved in the inflammation processes in order to clarify the mode of action of several PLA2s on the
membrane and search for opportunities to influence this process is of great interest for the therapy of inflammation
and allergic diseases. 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 thromboxanes. 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.
We have implemented a fluorimetric to evaluate the PLA2 activity. We have also studdied the effect of the
temperature, the pH, the calcium concentration and the nature and composition of lipid matrix on the PLA2
activity using liposomes prepared with different kinds of phospholipids with or without cholesterol.
The effect of some NSAIDs on cellular membrane fluidity was evaluated by fluorescence anisotropy
measurements performed using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to investigate the
effects on membrane fluidity resulting from the interaction between non-steroidal anti-inflammatory drugs NSAID (meloxicam, lornoxicam and nimesulide) with several membrane systems (liposomes with and without
cholesterol, mouse splenocytes, mouse macrophages cell line - J774, human leukaemia monocyte cell line
- THP-1, and human granulocytes and mononuclear cells). Besides providing a mean to study biophysical
influence of the NSAID in the membrane, the fluorescent studies performed allowed to infer about the drug
location in lipid bilayers. Results obtained for the anti-inflammatory drugs studied show an increase in the
membrane fluidity in a concentration dependent manner.
We have also evaluated the effect of the phenolic compounds, considered by E.P.A. (Environmental Protection
Agency) as priority pollutants in the membrane fluidity of the cellular membrane. This work was also performed
using liposomes as biomimetic systems.
Membrane fluidity was evaluated by fluorescence using fluorescent probe molecules referred above. Some of
these probes are capable of sensing “fluidity” gradient through the bilayer leaflet. Results revealed that all the
phenolic compounds studied increase the membrane fluidity.
(B) 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.
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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), 2-styrylchromones, flavonoids and plant
extracts, as briefly explained below.
We have recently proposed that several NSAIDs may react with ROS, namely peroxyl radical (ROO.), hydroxyl
radical (HO.), superoxide radical (O2.-), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hypochlorous
acid (HOCl), and 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.
2-Styrylchromones are a small group of natural occurring chromones, vinylogues of flavones (2phenylchromones). Natural and synthetic derivatives were previously tested in different biological systems,
showing different activities with potential therapeutic applications. The antioxidant behaviour of these compounds
has been raised as a matter of particular interest. Thus, we evaluated the in vitro scavenging activities for ROS
and RNS of various 2-styrylchromone derivatives and structurally similar flavonoids. Simultaneously, five flavones
and one flavonol were tested due to the similarities of their structures with those of 2-styrylchromones. This
allowed to take advantage from the existing knowledge about flavonoids and helped to interpret the results.
The ultimate aim of this investigation was to look up for structure-activity relationships concerning the scavenging
studies and to select the most promising compounds as new antioxidant therapeutic agents. Some of the
studied 2-styrylchromones are being shown to be extremely efficient scavengers of the different ROS and
RNS, having, in some cases, IC50s under 1 µM. The hydroxylation pattern, especially in the B-ring but also
in the A ring, modulates the activity of these compounds, the catecholic derivatives being the most effective
scavengers. The styryl pattern also contributes to the outstanding antioxidant activity of these compounds.
The Euphorbiaceae Pedilanthus tithymaloides is used in Cuban traditional medicine, as an anti-inflammatory
remedy in the treatment of stomatological affections. Considering that the inflammatory response is accompanied
by the formation of ROS and RNS, a bioassay-guided methodology using the DPPH (2,2-diphenyl-1picrylhydrazyl), TEAC (Trolox Equivalent Antioxidant Capacity), and reducing power assays, as well as the
assessment of scavenging properties against O2ÿ•, H2O2, HOCl, ROOÿ,ÿNO, and ONOO• was employed to
isolate and identify the main antioxidant principles from the plant ethanol extract. The obtained results are
suggesting that the phenolic constituents kaempferol 7-O-â-d-glucopyranoside-6´´-(3´´´-hydroxy-3´´´methylglutarate), quercitrin, isoquercitrin, and scopoletin are responsible for the scavenging effects and antiinflammatory activity displayed by the tincture.
(C) Modulation of human neutrophil activation by metals
The effect of previous contact of blood with different anticoagulants (e.g. EDTA, citrate and heparin) in the
subsequent activation of neutrophil respiratory burst is yet to be known. We have been assessing the effect
of these anticoagulants on the isolation procedure and in the subsequent neutrophil calcium levels and
respiratory burst induced by phorbol myristate acetate (PMA). Isolation of human neutrophils from whole
blood has been performed by the gradient density centrifugation method. Cell viability and isolation yield is
evaluated by the Trypan blue exclusion method. PMA-induced neutrophil burst is measured by
chemiluminescence. Intracellular calcium [Ca2+]i was measured using Fluo-3 AM, a calcium-sensitive dye.
We evaluated the putative stimulation of oxidative burst in human neutrophils activated by nickel salts. Once
confirmed, the pathways leading to NADPH oxidation were evaluated, by measuring intracellular free calcium
levels and protein kinase C activation. The reactive species formed as a consequence of nickel nitrate activation
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of neutrophils were finally assessed by using an unspecific probe (luminol), as well as appropriate scavengers
and metabolizing enzymes of these species.
(D) Translocation of antibiotics through outer mambrane proteins
OmpF proteoliposomes were prepared by direct incorporation into preformed DMPC or E.Coli liposomes,
using two different techniques for detergent removal; reconstitution of OmpF protein in E.coli lipids bilayer and
DMPC liposomes using detergent adsorption on polystyrene beads (Bio-Beads SM-2, Bio-Rad) and
reconstitution of OmpF protein in the same liposomes using gel exclusion chromatography (Sephadex G50).
After reconstitution the OmpF concentration was determined on the proteoliposomes and the results show
that the encapsulation degree is independent of the techniques used for detergent removal, but are always
higher for E.Coli liposomes, with a encapsulation degree of 92% for this lipid and 85% for DMPC. Although the
reconstitution degree was high for all procedures in order to know if the protein directionality and topology is
the corrected one to have biological activity fluorescence anisotropy studies and light scattering determinations
were performed.
All the reconstitution procedures were accompanied by anisotropy studies of the probes DPH and TMA-DPH
inserted on the liposomes and proteoliposomes. These probes were used as DPH is embedded in the bilayer
while TMA-DPH is anchored at the aqueous interface with its DPH moiety intercalated between the phospholipids
acyl chains, consequently, it is possible to gather information on the integration of proteins into the core and
at the interface regions of the bilayer.
The anisotropy profile, of DPH and TMA-DPH in DMPC liposomes and proteoliposomes are clearly different.
For the liposomes the profiles are as expected with an abrupt change on the anisotropy values in the transition
temperature zone, giving a mean Tm = 24.4±0.03 ºC. For proteoliposomes the profiles are quite different with a
decrease of anisotropy below the Tm and an increase after the Tm. A closer lock also show that the anisotropy
profiles for the TMA-DPH probe are identical, within experimental error, for the two techniques used for detergent
removal, but the same is not straightforward, for the DPH probe. Changes in anisotropy values above phase
transition are, for both techniques identical, ∆r~0.05, for TMA-DPH, but for DPH this change is much higher
when the detergent was removed by Bio-Beads®, ∆r~0.09, than when it was removed by gel filtration, ∆r
~0.02. The temperature dependence of DPH and TMA-DPH anisotropy in E.Coli liposomes and proteoliposomes,
for the two techniques used for detergent removal and the results show that for the proteoliposomes prepared
by detergent removal with Bio-Beads®, there was an increase of anisotropy in all the temperature range
(∆r~0.06) and for both probes. Nevertheless, when the proteoliposomes were prepared by removing the
detergent by gel filtration the anisotropy vs temperature profile was similar for liposomes and proteoliposomes
and furthermore for the two probes.
Partition coefficients for quinolones as they are fluorescent, can be determined by steady-state anisotropy (r;
a parameter easily calculated from polarized emission, which contains information on fluorophores’ rotation
while in the excited can be used to calculate the partition coefficient of a fluorescent molecule between lipid
and aqueous phases :
r ((γ [L] − 1) + rL K pε LφL /(ε W φW )
r = w l −1
(γ l [L] − 1) + K pε LφL /(ε W φW )
−1
(φ is the fluorescence quantum yield; ideally, I should be the integrated fluorescence emission intensity but
if no significant spectral shifts occur upon increasing the lipidic concentration, [L], I may be measured at a
chosen wavelength). [L] is the total lipid concentration and γL is its molar volume. The subscripts W and L
stand for the aqueous and the lipid phase, respectively.
For nalidixic acid in E.Coli polar lipids changes in anisotropy were used to determine its partition coefficient
and a value of Kp = 1060±123 M-1 was obtained. These results support the water/octhanol partition coefficients
of the two quinolones at physiological pH and show the higher hydrophobicity of nalidixic acid.
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Ion current though single trimeric OmpF porins reconstituted into planar lipid bilayers were performed to study
translocation of nalidixic acid. 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 that nalidixic acid do not block the channel.
The values of the MICs obtained by the susceptibility tests performed for Moxifloxacin and nalidixic acid are
identical, within experimental error, to those previously reported for the ATCC standard. For the stain, BL21(BE),
the MICs are always smaller but the values obtained for these quinolones have the same profile of those
obtained for the ATCC strain. From the results it is possible to see that the nalidixic acid MIC are 100 times
greater than those for moxifloxacin, as is expected from a quinolone of 1th and 4th generation. Nevertheless,
the general trends show that porins are important for the uptake of moxifloxacin but not so much for nalidixic
acid. Concerning specifically OmpF, it is interesting to not that, the lack or inhibition of this porin increases
slightly moxifloxacin MIC but has no effect on Nald ac. MIC. Furthermore, the join deficiency of OmpF and
Lamb although increases slightly moxifloxacin MIC has the reverse effect on Nald ac. MIC, it reduces the
MIC. These results support the idea that hydrophobic quinolones do not enter bacteria by porin channels.
Growth and purification of recombinant cytochrome c6 from Phormidium laminosum and Synechocystis sp.
PCC 6803 have been attained and preliminary optimisation studies have been carried out. Aiming at isolating
cytochrome c6 from Phormidium laminosum (the thermophilic cyanobacterium), the protein was over-expressed
in E. coli. E. coli Bl21(DE3) competent cells were transformed by electroporation with [pGEMT–cyt c6] and
[pEC86], plasmids which code for the protein and the heme maturation cassette, respectively. Transformants
were identified in solid LB medium in the presence of ampicilin and chloramphenicol, antibiotics to which the
plasmids confer resistance.
E. coli cells transformed with the plasmid coding for the Synechocystis sp. PCC 6803 cytochrome c6 (the
mesophilic cyanobacterium) were prepared following the same procedure in IBVF (Instituto de Bioquímica
Vegetal y Fotosyntesis), Sevilha, Spain.
The transformed E. coli cells were grown in IBVF and both cytochromes were isolated from periplasmic
extracts of the corresponding cultures. Recombinant Synechocystis cytochrome c6 was purified according to
the method already described by the andalucian group and 12 mg of pure protein were obtained. Purity was
ascertained by UV-vis spectroscopy and MALDI-TOF. The extract containing approximately 10 mg of
recombinant P. laminosum cytochrome c6 is awaiting purification. In order to define the appropriate conditions
for the unfolding study of these proteins by a combination of UV-vis and fluorescence spectroscopies, a
commercial protein (horse heart cytochrome c, hh cyt c) was used as a model for the optimisation study of
the pH and temperature dependence. This prior optimisation of the conditions limits the consumption of the
over expressed proteins which are always scarce, expensive and whose purification is lengthy.
We have been able to conclude that hh cyt c, in agreement with the various unfolding studies published to
date with cytochromes, is a highly stable protein in the oxidised state. The protein is thermotolerant, resistant
to unfolding within a wide range of pH values and did not denature under any of the conditions used in the
study exhibiting only changes in the heme-iron coordination sphere. Only the combined action of extreme pH
and high temperature was able to induce unfolding and this process appeared reversible under all the conditions
tested.
The thermal unfolding studies in the reduced state are being hindered by the degradation of the reducing
agents available under high temperature conditions and by the fluorescence quenching effect. So, in order to
obtain direct comparison between the two redox forms of the protein, chemical unfolding induced by urea and
guanidinium chloride is being investigated. Preliminary results obtained to date seem to indicate that the
oxidised form of the protein is less resistant to unfolding than the reduced one, even though both redox forms
exhibit high tolerance to chemical unfolding when compared to other proteins.These results and their
comparison with data available in the literature suggest that cytochrome c6 may follow a similar stability
profile and have provided the conditions to conduct the comparative study of the cyanobacterial proteins
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BIOINORGANIC AND MEDICINAL INORGANIC CHEMISTRY
Head of Laboratory: Maria Rangel, Associate Professor
Staff Members:
Baltazar de Castro
Full Professor
Paula Gameiro
Assistant Professor
Ana Margarida Silva
Associate Laboratory Researcher
Ph.D. Students:
Ana Claúdia Nunes, André Neto da Silva
Project Grantee:
Andreia Daniela Leite, Mariana Andrade
Number of articles in scientific journals: 2 (242, 243)
Design of orally active insulin-mimetic drugs. In order to compare the properties of complexes formed by
multidentate and bidentate ligands we synthesized tetradentate ligands derived from 3-hydroxy-4-pyridinone
type and their corresponding metal complexes with VO(IV) Cu(II) 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. Two synthesizedf compounds have exzhibited anti-microbial
activity. The study of thebehaviour of these compounds in heterogeneous media has been initiated in ordcer
to get insight on its partition i9n biomembranes. 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 2007 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.
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COMPUTATIONAL STUDY OF METAL SURFACES
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NOVEL HETEROGENEOUS CATALYSTS
Head of Laboratory: Ana Cristina Freire, Associate Professor / Baltazar de Castro, Full Professor
Research Team:
Eulália Pereira
Assistant Professor
Ana Rosa Silva
Peter Eaton
Post-Doct Fellows:
Iwona K-Biernacka, Iwona Kiersztyn, Javier Martínez Rodríguez, Sunita Arun Salunke, Elzbieta Skiba
Ph. D. Students:
Magda Martins, Andrea Carneiro, João Tedim, Joana Fonseca, Sónia Patrício, Clara Pereira, Mário Valente,
Adelaide Miranda
M. Sc. Students:
Rosa Bessada, Tiago Borrego
Number of Ph. D thesis: 1 (25)
Number of Ms. C. thesis: 1 (17)
1 - Novel catalysts by heterogenisation of metal complexes
We pursued the preparation and characterisation of chiral manganese(III) salen complexes for immobilisation
onto carbon xerogels, new mesoporous pillared clays, clays and mesoporous silica-based materials. The
catalytic activity of the new complexes was tested in the asymmetric epoxidation of several alkenes was
evaluated using several oxygen sources. The alkene conversion, chemical selectivity of products, respective
yields and enantiomeric excesses (ee%) were determined by GC-FID.
Chiral Mn(III) salen complexes were anchored using several strategies developed in our lab onto several
supports: carbon xerogels, mesoporous pillared clays (PILCs), clays and mesoporous silica materials (HMS,
MCM-41 and FSM-16). All the materials were characterised by several techniques. The catalytic properties in
the asymmetric epoxidation of several alkenes of the new catalysts were determined. For these catalysts the
leaching of the active phase and their reusabibility was also evaluated. All the catalysts showed catalytic
activity. However 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) acetylacetonates were directly immobilised onto Laponite, K10 montmorillonite
and hexagonal mesoporous silica (HMS) and after modification of the materials with the organosilane APTES.
All the materials were characterised by several techniques. Their catalytic properties in the aziridination of
styrene and epoxidation of allylic alcohols using tert-butylhydroperoxide as oxygen source, respectively, have
been evaluated. All the new heterogeneous catalysts were active and could be reused in more catalytic
cycles. In some cases there was significant leaching of the active phase.
We tried to improve the efficiency of the Jacobsen catalysts in the asymmetric epoxidation of 6-ciano-2,2dimethylcromene in RTIL, by extracting the epoxide from the reaction medium, [C4mim][PF6], by using super
critical CO2. The results showed that the extracted epoxide quantity increased with the decrease of CO2
quantity and pressure. The temperature of the extraction also influenced the extracted epoxide quantity.
However, there was always about 5 % of catalyst leaching during the extraction procedure.
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The membrane StarmemTM240 was the one chosen for the nanofiltration experiments. This membrane showed
0% of rejection towards the 6-ciano-2,2-dimethylcromene epoxide and more than 95% rejection towards the
Jacobsen catalyst. With this membrane we were able to retain/recycle the Jacobsen catalyst in the asymmetric
epoxidation of this alkene in n-hexane for 5 cycles. Nevertheless, the epoxide yields were low, probably as a
consequence of the solvent that had to be used as reaction medium. Assim, o trabalho futuro das experiências
de nanofiltração irá ser centrado no estudo de misturas de solventes que permitam elevados rendimentos e
excessos enantioméricos do produto com o objectivo de se atingir elevadas razões de TON.
FUTURE WORK
(1) Pursue the 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) Pursue the exploration of using nanofiltration as a new method for the separation of homogeneous catalysts.
(4) Anchoring/encapsulation of chiral metal complexes onto several mesoporous materials.
(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 organosilane coupling reagents for surface functionalisation of several mesoporous matrices.
(7) Use of new solvent mixtures in the epoxidation of alkenes by the Jacobsen catalysts when using nanofiltration
membranes, in order to improve the epoxide yields and ee% values.
2 - 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 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 elemental analyses,
mass spectra, EPR, FTIR and UV-Vis spectroscopies, cyclic voltammetry and DFT.
The sensing properties of the complexes were performed in several solvents using UV-vis and DFT; for the
complexes that can polymerise and give stable films recognition studies were also done by CV, UV-Vis, QCM
and electrochemical impedance.
In solution, it was only possible to determine equilibrium constants for the complexation of alkaline and
alkaline-earth cations for the metal complexes functionalised with pseudo-crown groups by Uv-vis spectroscopy.
Those functionalised with crown ethers in the diimine bridge do not show significant shifts in the electronic
bands. They are being studied by DFT.
All the polymeric films functionalised with crown ethers and pseudo crown-ethers complexes the metal cations
tested (+1, +2), although they exhibited different behaviour: those with higher change induced higher changes
in the electrochemical, UV-Vis of the films. The coordination site was unambiguously identified as the crown
ether or pseudo-crown ether by XAS of Ni and Ba K-edge. The adsorption of cations by the polymeric films
was also monitored by electrochemical quartz crystal microbalance and electroacoustic impedance. The
films functionalised with pseudo crown ethers could be reused without significant lost of their electroactivity.
The synthesis and characterisation of new nickel and copper complexes with Schiff base ligands functionalised
with polar groups was started. These complexes will be used as building blocks for the fabrication of self
assembly films using the layer-by-layer technique.
Química Verde
77
The Pd and Pt complexes were synthesised and characterised. The films were prepared by electrochemical
polymerisation and characterised by CV, in-situ UV-Vis and EQCM. They show a slightly different behaviour
when compared with the homologous of Ni and Cu.
FUTURE WORK
(1) Design, preparation and characterisation of new complexes with sensing properties for cations and anions.
Characterisation of the complexes by DFT
(2) Preparation and characterisation of their corresponding films by electrochemical polymerisation and layerby-layer assembly.
(3) Evaluation of the association constants for the interaction of cations and anions with the complexes by
experimental techniques and DFT.
(4) Study of the interactions of cations and anions with the polymeric films by new techniques by XAS and
QCM techniques
(5) Morphological characterisation of the films by SEM and AFM.
(6) Cation and anion selectivity studies and for polymeric films reutilisation studies.
3 - Molecular materials with non-linear optical properties
We pursue the synthesis and characterisation of new Schiff base complexes functionalised with donoracceptor groups, generically represented by [M(DA-salen)],with M=Ni, Cu, Pd and Pt. The complexes were
characterised by DFT, UV-Vis. 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, λ=1064
nm). The technique permits the evaluation of non linear refractive index (n2) and the non linear absorption
coefficient (β). The complexes of Pd showed mainly non linear absorption, and their polymeric films could
only be measured at the reduced as they were destroyed by the laser radiation in the oxidised state.
FUTURE WORK
(1) Design, preparation and characterisation of new Pd, Pt and Au complexes with salen and porphyrinic
ligands
(2) Preparation and characterisation of their corresponding films by electropolymerisation and layer-by-layer
self assembly technique.
(3) Evaluation of linear (UV-Vis) and 3d NLO properties for the new complexes and corresponding films.
4 - Metallosurfactants. 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 results obtained show that the morphology of the nanoparticles obtained is strongly dependent on
solvent polarity.
The compounds form also stable Langmuir-Blodgett films, that have been studied by determination of Langmuir
isotherms, and UV/vis, CV and AFM studies.
5 - Metal Nanoassemblies. Several methods have been used to prepare gold and silver nanoparticles with
controlled size/shape and different plasmon band maxima to be used as different probes in gene detection
methods and in studies of the interaction of nanoparticles with proteins. The nanoparticles (spheres, rods,
wires) have been characterized by TEM and UV/vis spectroscopy and their different spectroscopic properties
Química Verde
78
will be used to develop gene detection methods with multi-probe analysis. Functionalization with oligonucleotides
and proteins is currently under study. The synthesis of mixed gold-magnetic nanoparticles is in progress.
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.
Química Verde
ANNEXES
2006
A - 2
ANNEX I
RESEARCH TEAM
Química Verde
A - 3
A
MEMBERS OF STAFF
Química Verde
A - 4
Alberto Sundaresan Prabhakar
Ana Maria Félix Trindade Lobo
Baltazar Manuel Romão de Castro
Fernando Jorge da Silva Pina
Hugo Faria da Fonseca Gil Ferreira
Isabel Maria A. M. G. de Moura
João Paulo S. Goulão Crespo
Jorge Manuel Moreira Gonçalves
José Alberto Nunes Ferreira Gomes
José João Galhardas de Moura
José Luis Fontes Costa Lima
Manuel M. Nunes da Ponte
Maria Conceição B. S. M. Montenegro
Maria Lurdes Souteiro Bastos
Pedro Brito Correia
Rosa Maria Moreira Seabra Pinto
Professor Catedrático
Professora Catedrática
Professor Catedrático Convidado
Abel José de Sousa Costa Vieira
Alberto Manuel Carneiro Sereno
Alberto Nova Araujo
Ana Cristina Moreira Freire
Aquiles J. F.de Araújo Barros
Carlos Brondino
Duarte José da Costa Pereira
Félix Dias Carvalho
Francisco Jorge Fernandes Caldeira
Karin Tonnies Gil Ferreira
Maria Alexandra Sardinha Bernardo
Maria Ascenção Reis
Maria Beatriz Prior Pinto Oliveira
Maria Conceição S. S. Rangel
Maria João L. R. M. C. Romão
Maria João Ribeiro Nunes Ramos
Maria Natália Dias Soeiro Cordeiro
Maria Pilar Figueroa Gonçalves
Maria Salete Reis Dias Rodrigues
Maria Teresa Barros Silva
Olivia Maria de Castro Pinho
Susana Filipe Barreiros
Teresa Maria Fonseca de Moura
Professor Associado
Professor Associado
Professor Associado
Professora Associada
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Professor Associado
Cristina Maria Delerue Alvim Matos
Manuel Rui Fernandes Azevedo Alves
Maria Leonor Oliveira Madureira Pinto
Maria Carmo Veiga Fernandes Vaz
Professora Coordenadora
Professora Coordenador
Adriana Martins Pimenta
Agostinho Almiro Almeida
Alberta Paula Gameiro Santos
Alexandre Lopes Magalhães
Ana Cecília Afonso Roque
Ana I. N. M. Aguiar Ricardo
Ana Maria Ferreira Costa Lourenço
Ana Maria Martelo Ramos
André Alberto Sousa Melo
António Gil de Oliveira Santos
António Jorge Parola
Branca Maria C. Monteiro da Silva
Carla Alexandra L. A. Sousa e Silva
Carla Alexandra N.Oliveira e Silva
Carlos Alberto Gomes Salgueiro
Carmen Diniz Pereira
Catarina Isabel G. R. de Mansilha
Cristina Maria C. Morais do Couto
Eduarda Graças R. Fernandes
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
UNL
UNL
FCUP
UNL
UNL
UNL
UNL
FFUP
FCUP
UNL
FFUP
UNL
FFUP
FFUP
UNL
FFUP
UNL
FEUP
FFUP
FCUP
FCUP
UNL
FCUP
FFUP
ISCSS
UNL
ISCSS
UNL
FFUP
ICBAS-UP
UNL
FCUP
FCUP
FEUP
FFUP
UNL
FCNAUP
FFUP
UNL
UNL
ISEP
IPVC
ISEP
ISEP
UFP
FFUP
FCUP
FCUP
UNL
UNL
UNL
UNL
FCUP
UNL
UNL
UFP
UFP
UFP
UNL
FFUP
ISCSN
ISCSN
FFUP
Química Verde
A - 5
Elvira Maria M. Gaspar
Eulália Fernanda Carvalho Pereira
Eurico José da Silva Cabrita
Fernando Manuel Gomes Remião
Helena Maria Neto Ferreira
Helena Maria Vieira Monteiro Soares
Helena Susana da Costa M. Ferreira
Henrique J. R. Guedes
Isabel Borges Coutinho de Medeiro
Isabel Maria Ligeiro da Fonseca
Isabel Maria Viegas Oliveira Ferreira
Isabel Maria Rola Coelhoso
Ivone Valente Oliveira Silva Pinto
João Carlos S. B. Sotomayor
João Carlos Lima
João Luis Machado Santos
João Luís Tavares de Matos Gomes
João M. Aires de Sousa
João Paulo C. Noronha
Joaquim Silvério Marques Vital
Jorge M. A. Oliveira
Jorge M. P. Lampreia Pereira
José António Maia Rodrigues
José Augusto Caldeira Pereira
José Eduardo S. Félix Castanheiro
José Oliveira Fernandes
José Paulo Barbosa Mota
José Ricardo R. F. Tavares
Lígia Maria da Silva Rebelo Gomes
Lucília Helena de Ataíde Saraiva
Luís Guilherme L. Ferreira Guido
Luísa M. S. P. Ferreira
Márcia Claudia Dias de Carvalho
Marco Diogo R. Gomes da Silva
Maria Alice S. Pereira
Maria Beatriz Guerra Junqueiro
Maria Cristina O. Costa
Maria da Gloria C. da Silva Queiroz
Maria da Graca T. R. S. Rodrigues
Maria dos Anjos L. Macedo
Maria Gabriela Machado de Almeida
Maria Gabriela Teles Cepeda Ribeiro
Maria João Melo
Maria José Feio
Maria Lúcia Sousa Saraiva
M. Madalena A. C. S. Dionísio Andrade
Maria Manuela M. A.Pereira
Maria Margarida C. M. A. Cardoso
Maria Paulina Estorninho Neves da Mata
Maria Teresa Avilés Perea
Mário Fernando José Eusébio
Miguel Freire de A. Cabral
Paula Cristina Branquinho Andrade
Paula Cristina de Almeida Gerónimo
Paula Cristina S. Branco
Paula Maria Façanha da Cruz Fresco
Paulo Joaquim Ferreira Almeida
Pedro António Brito Tavares
Pedro Jorge Araújo Alves da Silva
Pedro Jorge Macedo Abreu
Pedro Manuel Alexandrino Fernandes
Pedro Miguel Calado Simões
Rita Isabel Lemos Catarino da Silva
Rui Manuel Freitas Oliveira
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
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
Professor Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professora Auxiliar
Professor Auxiliar
Professor 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
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar
Professor Auxiliar Convidado
Professor Auxiliar
Professora Auxiliar
Professor Auxiliar
UNL
FCUP
UNL
FEUP
FFUP
FEUP
ISCSN
UNL
UNL
UNL
FFUP
UNL
ISCSN
UNL
UNL
FFUP
UFP
UNL
UNL
UNL
FFUP
UNL
FCUP
ICBAS-UP
UE
FFUP
UNL
UNL
ISCSN
FFUP
FCUP
UNL
FFUP
UFP
UNL
UNL
FFUP
UNL
FFUP
FFUL
UNL
ISCSS
FCUP
UNL
FCUP
FFUP
UNL
UNL
UNL
UNL
UNL
UNL
FFUP
FFUP
UFP
UNL
FFUP
FCUP
UNL
UFP
UNL
FCUP
UNL
UFP
UNL
Química Verde
A - 6
Stéphane Pierre Besson
Susana Isabel Pereira Casal
Professora Auxiliar
Professor Auxiliar
Professora Auxiliar
IPSN
UL
FFUP
Maria Isabel Branco Alves Martins
Maria Teresa P. de Oliva Teles Moreira
Professora Adjunta
Professora Adjunta
ISEP
ISEP
Ana Maria Philips
Ulrich Martin Scheven
Adriano Fachini
Agostinho Pereira
Alexandra Maria Moita Antunes
Ana Isabel Bicho dos Santos
Ana Luísa Carvalho
Ana Margarida Gomes da Silva
Ana Rosa Silva
Carlos Lodeiro Espino
César António Tonicha Laia
Isabel Mafra
João Pedro Lopes
José Luiz Capelo Martinez
José Pedro da Silva Trincão
Loic Hiliou
Marcela Alves Segundo
Maria Manuel Marques
Maria Paula do A. A. Guedes de Pinho
Mário Jorge Saldanha Gomes
Marta Andrade
Miguel Ângelo da Silva Jorge
Natália de Fátima Teixeira Correia
Paulo Alexandre da Costa Lemos
Peter Jonathan Eaton
Rui Manuel de Oliveira Duarte
Serguey Bursakov
Svetlozar Velizarov
Teresa Maria Alves Casimiro Ribeiro
Investigadora Principal
Investigador Principal
Investigador Auxiliar
Investigadora Auxiliar
UNL
UNL
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
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REQUIMTE
REQUIMTE
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REQUIMTE
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REQUIMTE
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REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
Florinda F. Martins
Helder José Marques Pinheiro
Hendrikus Petrus Antonius Nouws
João Alexandre Velho Prior
Manuela Sofia Rodrigues Morato
Maria João D. Ramalhosa Ferreira
Maria Manuela Barbosa Correia
Olga Manuela Matos de Freitas
Patricia Carla Ribeiro Valentão
Paula Cristina de Azevedo Gomes Pinto
Rita Isabel Simões Ferreira
Salomé de Sousa Teixeira
Sónia Adriana R. C. Figueiredo
Valentina Maria FernandesDomingues
Ana Luísa do Vale Fonseca Claro
Helena Maria Ferreira Carmo
Micaela Margarida Ferreira de Sousa
Miguel Angelo Rodrigues Pinto de Faria
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente
Assistente Estagiária
Assistente Convidado
ISEP
ISEP
FFUP
ISEP
FFUP
FFUP
ISEP
ISEP
ISEP
ISEP
FFUP
ISEP
FFUP
FFUP
IPVC
ISEP
ISEP
ISEP
ISEP
UNL
FFUP
UNL
FFUP
Eulália Maria Bernardino Mendes
Maria Elisa A. M. Fernandes Soares
Assessora Principal
Assessora Principal
FFUP
FFUP
Química Verde
A - 7
Maria Isabel Afonso da Rocha
Assessora
FCUP
José Tomás Soares de Albergaria
Maria Aurora Soares da Silva
Maria Isabel Viana de Brito Limpo Serra
Sérgio Cruz Monteiro de Morais
Monitor
Monitora
Monitora
Monitor
ISEP
ISEP
ISEP
ISEP
Ana Teresa Lopes
Carla Maria Rodrigues
Carla Sara Ferreira de Sousa
Cecília dos Santos Bonifácio
João Emanuel Fraga
João Rodrigo da Silva Santos
Luz Catarina Fernandes
Paula Celeste Paíga
Rui Manuel Viegas
Técnica
Técnica
Técnica
Técnica
Técnico
Técnico
Técnica
Técnica
Técnico
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
REQUIMTE
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
IPSN
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)
Instituto Politécnico de Saúde do Norte
Química Verde
A - 8
B
POST-DOCTORAL FELLOWS
Química Verde
A - 9
Abhik Mukhopaadhyay
Adrian Michael Oehmen
Carla Alexandra Portugal
Celina Maria Santos
Christo Dimitrov Roussev
Cristian Robert Munteanu
Elzbieta Skiba
Ewa Bogel-Lukasik
Frederico Castelo Ferreira
Galya Ivanova
Gilda de Sousa Carvalho
Isabel Alexandra Esteves
Isabel Cristina Timóteo
Issam Oueslati
Iwona Kuzniarska-Biernacka
Javier Martínez Rodríguez
Jerome Rodolphe Keldenich
Karine Lopes Marques
Krasimira Todorova Petrova
Krisztina Istvan
Krzysztof Biernacki
Laura Rodríguez Raurell
Ludwig Krippahl
Luisa Alexandra Serafim
Lyubchik Svetlana
Maria da Graça Albuquerque
Maria Filomena Freitas
Maria Gabriela Rivas
Maria Sónia García
Marta Isabel Silva
Marta Maria Santos
Marta Sofia Carepo
Miglena Kolcheva Georgieva
Miguel Pedro Pessanha
Pablo Javier Gonzalez
Patricia Mira Videira
Pedro Miguel Rodrigues
Periyasamy Murugan
Prashant Shripad Kulkarni
Qingyou Zhang
Raquel dos Santos Fortunato
Raquel Eliana Grazina
Raquel Rial Otero
Ravi Varala
Reinaldo Molina Ruiz
Ricardo Filipe Mendonça
Rubin Gulaboski
Shabir Husein Najmudin
Snezhanka Metodieva Bakalova
Sofia Rocha Pauleta
Solange Muralha
Sunil Gupta
Sunita Arun Salunke
Susan Matthew
Susana Rodrigues Pereira
Teresa Sacadura Silva
Vesna Najdanovic - Visak
Vesselin Kostadinov Petrov
Vítor Manuel Delgado Alves
Yao Shuwen
Yuri Ivanov Binev
Química Verde
A - 10
C
Ph. D. STUDENTS
Química Verde
A - 11
Alexandra Marques
Alexandre Carvalho
Aliuska Morales Helguera
Ana Bárbara Santos
Ana Cláudia Nunes
Ana Margarida Teixeira
Ana Maria Gomes
Ana Raquel Freitas
Ana Rita Pires
Ana Sofia Martins
André Alexandre Pinheiro
André Moreira Silva
André Ricardo Araújo
Andrea Conceição Carneiro
Andreia Daniela Leite
Andreia Filipa Curto
Anna Kladova
Antonio Manuel Nunes
Bárbara Andreia Ribeiro
Bruno André Pedras
Bruno Tamames
Carina Madalena Machado
Carla Maria Ferreira
Carla Patricia Coutinho
Carlos Alberto Baptista
Carlos Eduardo Martins
Celia Maria Amorim
Célia Marisa Silveira
Christophe Raymond Siquet
Cristiano de Sousa Mota
Cristina Manuela Lopes
Cristina Maria Cordas
Cristina Maria Vicente
Cristina Maria Alves
Cristina Sofia Matos
Daniel Dourado
David Alejandro Daza
David Andrade Sousa Costa
Diana Maria Gaspar
Diogo Alexandre Latino
Elisabete Maria Machado
Ema Luís Alves
Emilia Maria Santos
Eunice Raquel Rodrigues
Fabio Donato Larotonda
Filipa Maria Grosso
Filipe Santos Folgosa
Filipe José Duarte
Filipe Miguel Freire
Francisco António Silva
Francisco Miguel Cordeiro
Gonçalo Maria Dória
Goncalo Valente Carrera
Helena de Oliveira Pontes
Hugo Miguel Oliveira
Hugo Santos
Inês Isabel Gomes
Irina Moreira
Joana Alexandrina Carvalhido
Joana Andrêa Amaral
Joana Dinis da Fonseca
Joana Lia Ferreira
Joana Patricia Ribeiro
Joana Raimundo
João Adalberto Lourenço
Joao Andre Tedim
João C. Barreira
Joao Luis Fernandes
Joao Miguel Dias
João Paulo Pacheco
Joao Paulo Capela
Jorge da Silva Dias
José Carlos Talaia
Jose Luis Santos
José Rui Marques
Juan Tamames
Letícia Conceição Giestas
Luís Filipe Pinto
Luis Mayor López
Luis Miguel Magalhães
Mafalda Sofia Sarraguça
Magda Mercês Sá
Marcia Cristiana Guilherme
Marcio Milton Temtem
Marco André Galésio
Marco Preto
Marek Kluciar
Margarida C. Moncada
Maria Adelaide Miranda
Maria Carlota Ferreira
Maria Carolina Pereira
Maria Clara Quintas
Maria Fátima Lucas
Maria Joao Morgado
Maria Luísa Silva
Maria Manuela Machado
María Teresa Plaza
Mariana Isabel Costa
Marieta Leite Passos
Mario Alberto Valente
Marta Filipa TRibeiro
Maykel Cruz Monteagudo
Miguel Ângelo Lopes
Monica Jose Matos
Natércia Brás
Nuno Cerqueira
Patricia Alexandra Raleiras
Patricia Isabel Oliveira
Patricia Sofia Antunes
Patricia Susana Neves
Paula Alexandra Rodrigues
Paula Manuela Silva
Paulo Jorge Magalhães
Pedro Manuel Santos
Pedro Manuel Antunes
Pedro Miguel Gomes
Pedro Rodrigues Maia
Rafal Bogel-Lukasik
Ricardo Branco
Ricardo Jorge Carreira
Ricardo Jorge Oliveira
Rita Carneiro Alves
Rita Gusmao Noronha
Sandra Carla Leal
Química Verde
A - 12
Sara Cristina Cunha
Sérgio Alves
Sérgio Sousa
Simone Dell´Acqua
Sofia Alexandra Barata
Sofia Alexandra Gomes
Sofia Teresa Lima
Sónia Garcia
Sónia Gonçalves Patrício
Susana Maria Gaudencio
Valdemar Jorge Figueira
Vasco Daniel Bonifácio
Vera Marisa Costa
Vitor João Rosa
Wancheng Sittikijyothin
Zenaida Mourão
Química Verde
A - 13
D
M. Sc. STUDENTS
and
other research students
Química Verde
A - 14
M. Sc. Students
Aida Reis
Alexandra Lopes
Alexandra Vitória Soares
Alexandre Manuel Jesus
Américo Jose Duarte
Ana Alexandra Lanham
Ana Catarina Silva
Ana Filipa Gomes
Ana Isabel Castro
Ana Isabel Ramos
Ana Maria Pedroso
Ana Paula Madeira
Ana Pessoa Moura
Ana Rita Ricardo
André Alexandre Pinheiro
Andrea Sofia Saraiva
Ângela Maria Barroso
Artur João Bento
Áurea Maria Roxo
Branca Sofia Teixeira
Bruno Miguel Jarrais
Carla Dulcinea Barbosa
Carlos Miguel Azevedo
Catarina Soares
Cecília Teixeira
Célia Tavares Sousa
Cláudia Daniela Nunes
Cláudia Marina Martins
Cristina Maria Soares
Cristina Paula Oliveira
Daniel Alexandre Ribeiro
Daniel Osório
David Sérgio Ribeiro
Diana Isabel Almeida
Eduarda M. Cabral
Eliana Sousa e Silva
Elisabete de Jesus Oliveira
Estela Figueiredo
Eva Cunha
Fernando Jorge Teixeira
Filipa Costa Prata
Isabel Cristina Sousa
Joana Martins
Joana Rocha
João Miguel Araújo
Joao Miguel Rosa
Lúcia Helena Santos
Lúcia Maria Maia
Luís Alexandre Branco
Luís Miguel Correia
Luisa Alexandra Neves
Mara Lília Silva
Maria Augusta Medeiros
Maria Beatriz Afonso
Maria Guadalupe Peláez
Maria Heitor
Mayra Silvana Silva
Monica Rosas da Silva
Mónica Sofia Santos
Nuno Miguel Lourenço
Orlanda Pereira
Paula Cristina Ribeiro
Paula Triunfante
Pedro Manuel Coelho
Raquel Branquinho
Raquel Filipa Fernandes
Ricardo Barata
Ricardo Manuel Salgado
Ricardo Miguel Couto
Ricardo Nuno Páscoa
Rita Marques
Rosa Bela Bessada
Rui Claudio Rodrigues
Rui Miguel Almeida
Sílvia Marisa Barros
Sofia Alexandra Almeida
Sónia Marta Mendes
Susana Sabina Fernandes
Tiago Borrego
Vera Rute Augusto
Other Research Students
Ana Paula Martins
Ana Rita Bras
Ana Rita Fins
Ana Sofia Soares
Ana Sofia Pedrosa
Anabela Ferreira Gomes
Angela Maria Machado
Carla Beatriz Vieiros
Catarina Amelia Miguel
Catarina Maria Coelho
Cristina Isabel Silvestre
Diogo Francisco Pestana
Duarte Alves
Joana Moutinho Marcelino
José Filipe Silva
Lígia Cardoso
Liliana Sara Oliveira
Maria Virgínis Mota
Marisa Andreia Freitas
Marta Andreia Perez
Miriam Sousa
Patrícia Domingos Gonçalves
Renata Sofia Silva
Ricardo Alexandre Alves
Ricardo Pais
Sara Andreia Coelho
Sónia Patrício
Vanessa Frazoa Cabral
Química Verde
A - 15
E
Profiles of the new researchers
contracted under the Associate
Laboratory Program
Química Verde
A - 16
Química Verde
A - 17
ANNEX II
PUBLICATIONS
Química Verde
A - 18
A
Scientific Articles
——
Book Chapters
——
Articles in Procedings
Química Verde
A - 19
Articles in Scientific Journals
1.
“Sequential Flow Injection Analysis System On-Line Coupled To High Intensity Focused Ultrasound:
Green Methodology For Trace Analysis Applications As Demonstrated For The Determination Of
Inorganic And Total Mercury In Waters And Urine By Cold Vapor Atomic Absorption Spectrometry”
C. Fernandez; A. C. R. L. Conceição; R. Rial-Otero; C. Vaz; J. L. Capelo
Analytical Chemistry, 2006, 78(8), 2494-2499
2.
“Sample Treatment For Protein Identification By Mass Spectrometry-Based Techniques”
D. López-Ferrer; B. Cañas; J. Vázquez; C. Lodeiro; R. Rial-Otero; I. Moura; J. L. Capelo
Trends In Analytical Chemistry, 2006, 25(10), 996-1005
3.
“Comprehensive Two-Dimensional Gas Chromatography/Mass Spectrometric Analysis Of Pepper
Volatiles”
Z. L. Cardeal; M. D. R. G Da Silva; P. J. Marriott
Rapid Communications In Mass Spectrometry, 2006, 20(19), 2823-2836
4.
“Determination Of Cd And Pb In Biological Reference Materials By Electrothermal Atomic Absorption
Spectrometry: A Comparison Of Three Ultrasonic-Based Sample Treatment Procedures”
C. Maduro; G. Vale; S. Alves; M. Galesio; M. D. R. G. Da Silva; C. Fernandez; S. Catarino; M. G.
Rivas; A. M. Mota; J. L. Capelo
Talanta, 2006, 68(4), 1156-1161
5.
“Mercury determination by FI-CV-AAS after the degradation of organomercurials with the aid of an
ultrasonic field: the important role of the hypochlorite ion”
J. L. Capelo; G. Rivas; L. G. Oliveira; C. Vilhena; A. C. Santos; T. Valada; M. Galesio; P. Oliveira;
M. D. R. Gomes da Silva, E. M. Gaspar; S. Alves; C. Fernandez; C. Vaz
Talanta, 2006, 68(3) 813-818
6.
“Characterization Of The Volatile Fraction Emitted By Phloems Of Four Pinus Species By SolidPhase Microextraction And Gas Chromatography-Mass Spectrometry”
A. M. Santos; T. Vasconcelos; E. Mateus; M. H. Farrall; M. D. R. G. Da Silva; M. R. Paiva; M.
Branco
Journal Of Chromatography A, 2006, 1105(1-2), 191-198
7.
“Evaluation Of Contaminant Elements In Portuguese Wines And Original Musts By High Intensity
Focused Ultrasound Combined With Inductively Coupled Plasma Mass Spectrometry”
S. Catarino; J. L. Capelo; A. S. Curvelo-Garcia; R. B. De Sousa
Journal International Des Sciences De La Vigne Et Du Vin, 2006, 40(2), 91-100
8.
“Simple Gas Chromatographic Method For The Stereodifferentiation Of Methyl Nilate, A Chiral AlphaMethyl-Beta-Hydroxy Ester”
E. M. S. M. Gaspar; J. G. Barroso
Journal of Chromatography A, 2006, 1108(2), 225-230
9.
“Dynamic Versus Static Ultrasonic Sample Treatment For The Solid-Liquid Pre-Concentration Of
Mercury From Human Urine”
A. Patricio; C. Fernandez; A. M. Mota; J. L. Capelo
Talanta, 2006, 69(3), 769-775
Química Verde
A - 20
10.
“Sample Treatment With Focused Ultrasound And Bath Sonication As A Powerful Tool For The
Evaluation Of Cadmium Pollution In Estuarine Waters”
R. Agapito; S. Alves; J. L. Capelo; M. L. Goncalves; A. M. Mota
Marine Chemistry, 2006, 98 (2-4), 286-294
11.
“Intramolecular Excimer Formation And Sensing Behaviour Of New Fluorimetric Probes And Their
Interactions With Metal Cations And Barbituric Acids”
C. Lodeiro; J. C. Lima; A. J. Parola; J. Seixas De Melo; J. L. Capelo; B. Covelo; A. Tamayo, A
Sens. Actuators B, 2006, 115, 276–286
12.
“Trends In Selenium Determination/Speciation By Hyphenated Techniques Based On AAS Or AFS”
J. L. Capelo; C. Fernandez; B. Pedras; P. Santos; P. Gonzalez; C. Vaz
Talanta, 2006, 68(5), 1442-1447
13.
“Specific Interaction Of Citrate With Bis(Fluorophoric) Bibrachial Lariat Aza-Crown In Comparion With
The Other Components Of The Krebs Cycle”
M. P. Clares; C. Lodeiro; D. Fernández; A. J. Parola; F. Pina; E. García-España; C. Soriano; R.
Tejero
Chem. Commm. 2006, 3824-3826
14.
“Basicity And Coordination Properties Of A New Phenanthroline-Based Bis-Macrocyclic Receptor”
C. Bazzicalupi; A. Bencini; A. Bianchi; L. Borsari; A. Danesi; C. Giorgi; C. Lodeiro; P. Mariani; F.
Pina; S. Santarelli; A. Tamayo; B. Valtancoli
Dalton Transactions, 2006, 4000-4010
15.
“Coordination Features Of A Terpyridine-Containing Polyamine Receptor”
C. Bazzicalupi; A. Bencini; A. Bianchi; L. Borsari; A. Danesi; C. Giorgi; P. Mariani; F. Pina; A.
Santarelli; B. Valtancoli
Dalton Trans., 2006, 5743-5752
16.
“Color Tuning Of A Nickel Complex With A Novel N2S2 Pyridine-Containing Macrocyclic Ligand”
A. Tamayo; J. Casabo; L. Escriche; C. Lodeiro; B. Covelo; C. D. Brondino; R. Kivekas; R. Sillampaa
Inorganic Chemistry, 2006, 45(3), 1140-1149
17.
“Spectrofluorometric Studies of a new NS3 Macrocycle Ligand Containing an Anthracene Pendantarm. Synthesis, Complexation Studies and X-Ray Structure of [PdL](BF4)2 complex”
A. Tamayo; L. Escriche; J. Casabó; B. Covelo; C. Lodeiro
Eur. J. Inorg. Chem., 2006, 2997-3004
18.
“Structural And Magnetic Properties Of A Complete Series Of Halide Complexes Of Ni(II) With A
Pyridine-Containig 14-Membered Macrocycle”
A. Tamayo; L. Escriche; C. Lodeiro; J. Ribas-Ariño; J. Ribas; B. Covelo; J. Casabó
Inorg. Chem., 2006, 45, 7621-7627
19.
“Metal Complexes With Two Tri-Aza, Tri-Oxa Pendant-Armed Macrocyclic Ligands: Synthesis,
Characterization, Crystal Structures And Fluorescence Studies”
A. Freiria; R. Bastida; L. Valencia; A. Macias; C. Lodeiro; H. Adams
Inorganica Chimica Acta, 2006, 359(8), 2383-2394
Química Verde
A - 21
20.
“Synthesis Of Four Novel Pendant Armed Macrocyclic Ligands And Their Interaction With
Lanthanide(III) Cations”
E. Bértolo; C. Lodeiro; R. Bastida
Russian Journal of Coordination Chemistry, 2006, 32, 12, 895-900
21.
“Solvent Effects On The Thermal And Photochemical Reactions Of 4’ -Iodo-8-Methoxyflavylium And
Their Consequences On The Coloring Phenomena Caused By Anthocyanins In Plants”
R. Gomes; A. J. Parola; J. C. Lima; F. Pina
Chemistry-A European Journal, 2006, 12(30), 7906-7912
22.
“Spectral and Photophysical Studies of Substituted Indigo Derivatives in their Keto Forms”
J. Seixas de Melo; R. Rondão; H. D. Burrows; M. J. Melo; S. Navaratnam; R. Edge; G. Voss
ChemPhysChem, 2006, 7, 2303-2311
23.
“Photophysics of a new indigo derivative with unique properties: Cibalackrot”
J. Seixas de Melo; R. Rondão; H. D. Burrows; M. J. Melo; S. Navaratnam; R. Edge; G. Voss
J. Phys. Chem. A, 2006, 110, 13653-13661
24.
“Anomalous “Unquenching” Of The Fluorescence Decay Times Of Beta-Lactoglobulin Induced By The
Known Quencher Acrylamide”
C. A. M. Portugal; J. G. Crespo; J. C. Lima
Journal Of Photochemistry And Photobiology B-Biology, 2006, 82(2), 117-126
25.
“Using Hydrogen Bonding Specific Interactions To Detect Water In Aprotic Solvents At Concentrations
Below 50 Ppm”
C. Pinheiro; J. C. Lima; A. J. Parola
Sens. Actuators B, 2006, 114, 978–983
26.
“Metallodendrimers Containing Both Ruthenium (Internal Layer) And Rhenium (External Layer)”
I. Angurell; J. C. Lima; L. I. Rodriguez; L. Rodriguez; O. Rossell; M. Seco
New Journal Of Chemistry, 2006, 30(7), 1004-1008
27.
“Probing The Change Of Enzymatic Activity Of Horseradish Peroxidase Induced By Membrane
Permeation Using Tryptophan Fluorescence”
C. A. M. Portugal; J. C. Lima; J. G. Crespo
Journal Of Membrane Science, 2006, 284(1-2), 180-196
28.
“Fluorescence Probing Of Structural And Functional Changes Of Proteins Induced By Ultrafiltration”
C. A. M. Portugal; J. C. Lima; J. G. Crespo
Desalination, 2006, 199(1-3), 547-549
29.
“Electron-Transfer Mechanism Of The Triplet State Quenching Of Aluminium Tetrasulfonated
Phthalocyanine By Cytochrome C”
C. A. T. Laia; S. M. B. Costa; L. F. V. Ferreira
Biophysical Chemistry, 2006, 122(2), 143-155
30.
“Catalytic Enantioselective Cross-Mannich Reaction Of Aldehydes”
M. M. B. Marques
Angewandte Chemie-International Edition, 2006, 45 (3), 348-352
Química Verde
A - 22
31.
“N-Hydroxy Indoles As Flexible Substrates In Rearrangements - A Novel Reaction With Activated
Triple Bonds”
M. P. Duarte; R. F. Mendonca; S. Prabhakar; A. M. Lobo
Tetrahedron Letters, 2006, 47(7), 1173-1176
32.
“Studies in sigmatropic rearrangements of N-prenylindole derivatives - a formal enantiomerically-pure
synthesis of tryprostatin B”
A. S. P. Cardoso; M. M. B. Marques; N. Srinivasan; S. Prabhakar; A. M. Lobo; H. S. Rzepa
Organic & Biorganic Chemistry, 2006, vol. 4, 3966-3972
33.
“Development of novel brush-type chiral stationary phases based on terpenoid selectors: HPLC
evaluation and theoretical investigation of enantioselective binding interactions”
C. Moiteiro; N. Fonseca; M. J. Curto; R. Tavares; A. M. Lobo; P. Ribeiro-Claro; V. Félix; M. G. B.
Drew
Tetrahedron Asymmetry, 2006, vol. 17, 3248-3264
34.
“Anti-Inflammatory And Antioxidant Activity Of A Medicinal Tincture From Pedilanthus Tithymaloides”
P. Abreu; S. Matthew; T. Gonzalez; D. Costa; M. A. Segundo; E. Fernandes
Life Sciences, 2006, 78(14), 1578-1585
35.
“QSAR Analysis Of Phenolic Antioxidants Using MOLMAP Descriptors Of Local Properties”
S. Gupta; S. Matthew; P. M. Abreu; J. Aires-De-Sousa
Bioorg. Med. Chem., 2006, 14(4), 1199-1206
36.
“New Macrocyclic Lathyrane Diterpenes, From Euphorbia Lagascae, As Inhibitors Of Multidrug
Resistance Of Tumour Cells”
N. Duarte; N. Gyemant; P. M. Abreu; J. Molnar; M. J. U. Ferreira
Planta Medica, 2006, 72(2), 162-168
37.
“Long Chain Alkyl And Alkenyl Phenols From The Roots Of Ozoroa Insignis”
Y. H. Liu; P. J. M. Abreu
Journal of The Brazilian Chemical Society, 2006, 17(3), 527-532
38.
“Effect Of Extraction Parameters On The Chemical Structure And Gel Properties Of Kappa/IotaHybrid Carrageenans Obtained From Mastocarpus Stellatus”
L. Hilliou; F. D. S. Larotonda; P. Abreu; A. M. Ramos; A. M. Sereno; M. P. Gonçalves
Biomolecular Engineering, 2006, 23(4), 201-208
39.
“Euphoportlandols A And B, Tetracylic Diterpene Polyesters From Euphorbia Portlandica And Their
Anti-MDR Effects In Cancer Cells”
A. M. Madureira; N. Gyemant; J. R. Ascenso; P. M. Abreu; J. Molnar; M. J. U. Ferreira
Journal of Natural Products, 2006, 69(6), 950-953
40.
“Tirucallane Triterpenes From The Roots Of Ozoroa Insignis”
Y. H. Liu; P. Abreu
Phytochemistry, 2006, 67(13), 1309-1315
41.
“Heterocyclic terpenes: Linear furano- and pyrrolo-terpenoids”
Y. Liu; P. Abreu
Nat. Prod. Rep. 2006, 23, 630-651
Química Verde
A - 23
42.
“Phytochemical Constituents From Salsola Tetrandra”
M. H. Oueslati; H. Ben Jannet; Z. Mighri; J. Chriaa; P. M. Abreu
Journal of Natural Products, 2006, 69(9), 1366-1369
43.
“Chemotaxonomy Of Portuguese Ulex: Quinolizidine Alkaloids As Taxonomical Markers”
P. A. Maximo; A. Lourenço; T. M. Wink
Phytochemistry, 2006, 67(17), 1943-1949
44.
“(-)-Agelasidine A From Agelas Clathrodes”
M. A. Medeiros; A. Lourenço; M. R. Tavares; M. J. M. Curto; S. S. Feio; J. C. Roseiro
Zeitschrift Fur Naturforschung C-A Journal of Biosciences, 2006, 61(7-8), 472-476
45.
“Neurotoxicity of Ecstasy Metabolites in Rat Cortical Neurons, and Influence of Hyperthermia”
J .P. Capela; A. Meisel; A. R. Abreu; P. S. Branco; L. M. Ferreira; A.M. Lobo; F. Remiao; M. L.
Bastos; F. Carvalho
J. Pharm. Exp. Therap., 2006, 316, 53-61
46.
“Discoloration of Architectural Photoreproductions, Causes and Prevention”
M. Vilela, L. M. Ferreira, J. Vieira
Restaurator, 2006, 27, 1-8
47.
“Physicochemical Stereodescriptors Of Atomic Chiral Centers”
Q. Y. Zhang; J. Aires-De-Sousa
Journal Of Chemical Information And Modeling, 2006, 46(6), 2278-2287
48.
“Genome-Scale Classification Of Metabolic Reactions: A Chemoinformatics Approach”
D. A. R. S. Latino; J. Aires-De-Sousa
Angewandte Chemie-International Edition, 2006, 45(13), 2066-2069
49.
“Geographical Classification Of Crude Oils By Kohonen Self-Organizing Maps”
A. M. Fonseca; J. L. Biscaya; J. Aires-De-Sousa; A. M. Lobo
Anal. Chim. Acta, 2006, 556(2), 374-382
50.
“Modern Methods for Asymmetric Hydrogenation of Ketones”
M. M. B. Marques
Org. Chem. Highlights, 2006, March 5
(URL: http://www.organic-chemistry.org/Highlights/2006/05March.shtm)
51.
“Novel Synthetic Approaches Toward Substituted Indole Scaffolds”
M. M. B. Marques
Org. Chem. Highlights, 2006, September 5
(URL: http://www.organic-chemistry.org/Highlights/2006/05September.shtm)
52.
“Synthesis Of New Chiral Amines With A Cyclic 1,2-Diacetal Skeleton Derived From (2R, 3R)-(+)Tartaric Acid”
M. T. Barros; A. M. F. Phillips
Molecules, 2006, 11(2-3), 177-196
53.
“Complexation of transition metals by 3-azidopropionitrile. An electrospray ionization mass
spectrometry study”
N. Couto; M. F. Duarte; P. Rodrigues; M. T. Barros; M. L. Costa; B. C. Cabral; M. T. Fernandez
J. Am. Soc. Mass Spectrom, 2006, 18, 453-465
Química Verde
A - 24
54.
“Synthesis, Solid State Structures And EPR Studies In Polycrystalline And Single Crystal Samples
Of a-Diimine Cobalt(II) Complexes”
V. Rosa; P. J. Gonzalez; T. Avilés; P. T. Gomes; R. Welter; A. C. Rizzi; M. C. G. Passeggi; C. D.
Brondino
Eur. J. Inorg. Chem. 2006, 4761-4769
55.
“A Theoretical Study Of The Stereoselectivities Of The Diels-Alder Addition Of Cyclopentadiene To
Ethyl-(S)-Lactyl Acrylate Catalyzed By Aluminium Chloride”
S. M. Bakalova; A. G. Santos
European Journal of Organic Chemistry, 2006, 7, 1779-1789
56.
“Deacidification Of Paper Using Dispersions Of Ca(OH)2 Nanoparticles In Isopropanol. Study Of
Efficiency”
S. Sequeira; C. Casanova; E. J. Cabrita
Journal Of Cultural Heritage, 2006, 7(4), 264-272
57.
“Modelling The Electron-Transfer Complex Between Aldehyde Oxidoreductase And Flavodoxin”
L. Krippahl; P. N. Palma; I. Moura; J. J. G. Moura
European Journal Of Inorganic Chemistry, 2006, 19, 3835-3840
58.
“Structural And Electron Paramagnetic Resonance (EPR) Studies Of Mononuclear Molybdenum
Enzymes From Sulfate-Reducing Bacteria”
C. D. Brondino; M. G. Rivas; M. J. Romão; J. J. G. Moura; I. Moura
Accounts Of Chemical Research, 2006, 39(10), 788-796
59.
“The First Crystal Structure Of Class III Superoxide Reductase From Treponema Pallidum”
T. Santos-Silva; J. Trincao; A. L. Carvalho; C. Bonifacio; F. Auchere; P. Raleiras; I. Moura; J. J. G.
Moura; M. J. Romão
Journal of Biological Inorganic Chemistry, 2006, 11(5), 548-558
60.
“EPR And Redox Properties Of Periplasmic Nitrate Reductase From Desulfovibrio Desulfuricans
ATCC 27774”
P. J. Gonzalez; M. G. Rivas; C. D. Brondino; S. A. Bursakov; I. Moura; J. J. G. Moura
61.
“Bacterial Nitrate Reductases: Molecular And Biological Aspects Of Nitrate Reduction”
P. J. Gonzalez; C. Correia; I. Moura; C. D. Brondino; J. J. G. Moura
Journal of Inorganic Biochemistry, 2006, 100(5-6), 1015-1023
62.
“Kinetics Studies Of The Superoxide-Mediated Electron Transfer Reactions Between RubredoxinType Proteins And Superoxide Reductases”
F. Auchere; S. R. Pauleta; P. Tavares; I. Moura; J. J. G. Moura
63.
“Molybdenum And Tungsten Enzymes: The Xanthine Oxidase Family”
C. D. Brondino; J. M. Romão; I. Moura; J. J. G. Moura
Current Opinion In Chemical Biology, 2006, 10(2), 109-114
64.
“Desulfovibrio Gigas Ferredoxin II: Redox Structural Modulation Of The [3Fe-4S] Cluster”
P. M. Rodrigues; A. L. Macedo; B. J. Goodfellow; I. Moura; J. J. G. Moura
Journal of Biological Inorganic Chemistry, 2006, 11, 307-315
Química Verde
A - 25
65.
“Biochemical And Spectroscopic Characterization Of An Aldehyde Oxidoreductase Isolated From
Desulfovibrio Aminophilus”
A. Thapper; M. G. Rivas; C. D. Brondino; B. Ollivier; G. Fauque; I. Moura; J. J. G. Moura
Journal Of Inorganic Biochemistry, 2006, 100(1), 44-50
66.
“Metalloenzymes Of The Denitrification Pathway”
P. Tavares; A. S. Pereira; J. J. G. Moura; I. Moura
J. Inorg. Biochem. 2006, 100, 2087-2100
67.
“Nitric Oxide Reductase: Direct Electrochemistry And Electrocatalytic Activity”
C. M. Cordas; A. S. Pereira; C. E. Martins; C. G. Timóteo; I. Moura; J. J. G. Moura; P. Tavares
Chembiochem., 2006, 7(12), 1878-81
68.
“Redox Chemistry Of Low-pH Forms Of Tetrahemic Cytochrome c3”
M. Santos; M. Correia dos Santos; M. L. S. Gonçalves; C. Costa; J. C. Romão; J. J. G. Moura
J. Inorg. Biochem., 2006, 100, 2009-2016
69.
“Vanadium Distribution, Lipid Peroxidation And Oxidative Stress Markers Upon Decavanadate In Vivo
Administration”
S. S. Soares; H. Martins; R. O. Duarte; J. J. G. Moura; J. Coucelo; C. Gutiérrez-Merino; M.
Aureliano
J. Inorg. Biochem. 2006, 101, 80-88
70.
“Decavanadate Interactions With Action: Inhibition Of g-Action Polymerization And Stabilization Of
Decameric Vanadate”
S. Ramos; M. Manuel; T. Tiago; R. Duarte; J. Martins; G. Gutierrez-Merino; J. J. G. Moura; M.
Aureliano
J. Inorg. Biochem, 2006, 100, 1734-1743
71.
“Solution structures of tetrahaem ferricytochrome c(3) from Desulfovibrio vulgaris (Hildenborough) and
its K45Q mutant: the molecular basis of cooperativity”
A. C. Messias; A. P. Aguiar; L. Brennan; C. A. Salgueiro; L. M. Saraiva; A. V. Xavier; D. L. Turner
Biochim. Biophys. Acta, 2006, 1757, 143-153
72.
“Thermodynamic Characterization Of Triheme Cytochrome Ppca From Geobacter Sulfurreducens:
Evidence For A Role Played In E(-)/H+ Energy Transduction”
M. Pessanha; L. Morgado; R. O. Louro; Y. Y. Londer; P. R. Pokkuluri; M. Schiffer; C. A. Salgueiro
Biochemistry, 2006, 45(46), 13910-13917
73.
“The Conserved Active-Site Loop Residues Of Ferrochelatase Induce Porphyrin Conformational
Changes Necessary For Catalysis”
Z. Shi; R. Franco; R. Haddad; J. A. Shelnutt; G. C. Ferreira
Biochemistry, 2006, 45(9), 2904-2912
74.
“Chelatases: Distort To Select?”
S. Al-Karadaghi; R. Franco; M. Hansson; J. A. Shelnutt; G. Isaya; G. C. Ferreira
Trends In Biochemical Sciences, 2006, 31(3), 135-142
75.
“Gold Nanoparticle Probe-Based Assay for Rapid and Direct Detection of Mycobacterium
tuberculosis DNA in Clinical Samples”
P. V. Baptista; M. Koziol-Montewka; J. Paluch-Oles; G. Doria; R. Franco
Clin. Chem., 2006, 52(7), 1433-1434
Química Verde
A - 26
76.
“Spectroelectrochemistry Of Type II Cytochrome C3 On A Glycosylated Self-Assembled Monolayer”
I. Gomes; R. E. Di Paolo; P. M. Pereira; I. A. C. Pereira; L. M. Saraiva; S. Penades; R. Franco
Langmuir, 2006, 22(24), 9809-9811
77.
“Structure and Mechanism in the Bacterial Dihaem Cytochrome c Peroxidases”
G. W. Pettigrew; A. Echalier; S. R. Pauleta
J. Inorg. Biochem., 2006, 100, 551-567
78.
“In vitro nitrosation of insulin A- and B-chains”
C. Santos; R. A. Afonso; M. P. Guarino; R. S. Patarrão; A. Fernandes; J. P. Noronha; M. P. Macedo;
J. Caldeira
Eur. J. Mass Spectrom., 2006, 12, 331-338
79.
“The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP”
J. S. Dias; A. L. Macedo; G. C. Ferreira; F. C. Peterson; B. F. Volkman; B. J. Goodfellow
J. Biol. Chem., 2006, 281, 31553 – 31561
80.
“Xyloglucan Is Recognized By Carbohydrate-Binding Modules That Interact With Beta-Glucan
Chains”
S. Najmudin; C. I. P. D. Guerreiro; A. L. Carvalho; J. A. M. Prates; M. A. S. Correia; V. D. Alves; L.
M. A. Ferreira; M. J. Romão; H. J. Gilbert; D. N. Bolam; C. M. G. A. Fontes
Journal Of Biological Chemistry, 2006, 281(13), 8815-8828
81.
“Formate-Reduced E-Coli Formate Dehydrogenase H: The Reinterpretation Of The Crystal Structure
Suggests A New Reaction Mechanism”
H. C. A. Raaijmakers; M. J. Romão
82.
“Purification, Crystallization And Preliminary X-Ray Diffraction Analysis Of The Glyoxalase II From
Leishmania Infantum”
J. Trincao; M. S. Silva; L. Barata; C. Bonifacio; S. Carvalho; A. M. Tomas; A. E. N. Ferreira; C.
Cordeiro; A. P. Freire; M. J. Romão
Acta Crystallographica Section F-Structural Biology And Crystallization Communications, 2006, 62,
805-807
83.
“Water Channels Are Important For Osmotic Adjustments Of Yeast Cells At Low Temperature”
G. Soveral; A. Veiga; M. C. Loureiro-Dias; T. H. An; P. Van Dijck; T. F. Moura
Microbiology-Sgm, 2006, 152, 1515-1521 Part 5
84.
“Dipyrone And Aminopyrine Are Effective Scavengers Of Reactive Nitrogen Species”
D. Costa; A. Vieira; E. Fernandes
Redox Report, 2006, 11(3), 136-142
85.
“Potentiometric Behaviour of Ion Selective Electrodes Based on Iron Porphyrins: The Influence of
Porphyrin Substituents on the Response Properties and Analytical Determination of Diclofenac in
Pharmaceutical Formulations”
E.M.G. Santos; A.N. Araújo; C.M.C.M. Couto; M.C.B.S.M. Montenegro
J. Pharm. Biom. Anal., 2006, 42, 535-542
Química Verde
A - 27
86.
“Flow System with Electrochemical Detection for Determination of Paracetamol in Pharmaceutical
Preparations”
M.L.S. Silva; M.B.Q. Garcia; J.L.F.C. Lima; E. Barrado
Port. Electrochim. Acta, 2006, 24, 261-271
87.
“Modified Tubular Electrode in a Multi-Commutated Flow System Determination of Acetaminophen in
Blood Serum and Pharmaceutical Formulations”
M.L.S. Silva; M.B.Q. Garcia; J.L.F.C. Lima; E. Barrado
Anal. Chim. Acta., 2006, 573-574, 383-390
88.
“Potentiometric Multi-Syringe Flow Injection System for Determination of Exchangeable Potassium in
Soils with in – Line Extration”
M.I.G.S. Almeida; M.A. Segundo; J.L.F.C. Lima, A.O.S.S. Rangel
MicroChem. J., 2006, 83, 75-80
89.
“Tubular Potentiometric Detector Used to Determine As(V) in Sediment Extrats by Flow Injection”
E. Barrado; J.A. Rodríguez; M.B. Quinaz, J.L.F.C. Lima
Intern. J. Environ. Anal. Chem., 2006, 86, 563-572
90.
“Construction and Evalution of PVC and Sol-Gel Sensor Membranes Based on Mn(III)TPP-CI.
Application to Valproate Determination in Pharmaceutical Preparations”
E.M.G. Santos; A.N. Araújo; C.M.C.M. Couto; M.C.B.S.M. Montenegro
Anal. Bioanal. Chem., 2006, 384, 867-875
91.
“Automatic Method for the Determination of Folin-Ciocalteu Reducing Capacity in Food Products”
L.M. Magalhães; M.A. Segundo; S. Reis; J.L.F.C. Lima; A.O.S.S. Rangel
J. Agric. Food Chem., 2006, 54, 5241-5246
92.
“Determination and Antioxidant Activity Evaluation of Etodolac, an Anti-Inflammatory Drug, by
Sequential Injection Analysis”
J.B. Garcia; M.L.M.F.S. Saraiva; J.L.F.C. Lima
Anal. Chim. Acta., 2006, 573-574, 371-375
93.
“Coupling of Multi-Pumping Flows Systems to Flame Atomic Spectrometric Detectors: Preliminary
Studies and Analytical Applications in the Analysis of Biological Samples”
C.M.P.V. Lopes; A.A. Almeida; J.L.M. Santos; J.L.F.C. Lima
Atom. Spectroscopy, 2006, 27, 156-164
94.
“Kinetic Enzymatic Determination of Glycerol in Wine and Beer Using a Sequential Injection System
with Spectrophotometric Detection”
H.M. Oliveira; M.A. Segundo; J.L.F.C. Lima; V. Grassi; E.A.G. Zagatto
J. Agric. Food Chem., 2006, 54, 4136-4140
95.
“Fully Automated Spectrophotometric Method for the Determination of Buspirone in Pharmaceutical
Preparations”
M.F.T. Ribeiro; J.L.M. Santos; J.L.F.C. Lima; A.C.B. Dias; E.A.G. Zagatto
Anal. Lett., 2006, 39, 2243-2253
96.
“A Flow-Batch Internal Standard Procedure for Iron Determination in Hydrated Ethanol Fuel by Flame
Atomic Absorption Spectrometry”
J.E. Silva; F.A. Silva; M.F. Pimentel; R.S. Honorato; V.L. Silva; M.C.B.S.M. Montenegro; A. Araújo
Talanta, 2006, 70, 522-526
Química Verde
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97.
“Fluidized Beds in Flow Analysis: use with Ion-Exchange Separation for Spectrophotometric
Determination of Zinc in Plant Digests”
M.F.T. Ribeiro; A.C.B. Dias; J.L.M. Santos; J.L.F.C. Lima; E.A.G. Zagatto
98.
“Exploiting Gas Diffusion for Non-Invasive Sampling in Flow Analysis: Determination of Ethanol in
Alcoholic Beverages”
S. Vicente; E.A.G. Zagatto; P.C.A.G. Pinto; M.L.M.E.S. Saraiva; J.L.F.C. Lima; E.P. Borges
An. Acad. Bras. Cienc., 2006, 78, 23-29
99.
“Automated Multicommutated Flow System for Flame Atomic Spectroscopy Determination of
Rubidium at High Concentrations”
Atom. Spectroscopy, 2006, 27, 13-18
100. “Determination of Ambroxol Hydrochloride, Methylparaben and Benzoic Acid in Pharmaceutical
Preparations Based on Sequential Injection Technique Coupled with Monolithic Column”
D. ‘atínský; J. Huclová; R.L.C. Ferreira; M.C.B.S.M. Montenegro; P. Solich
J. Pharm. Biom. Anal., 2006, 40, 287-293
101. “Automatic Method for Determination of Total Antioxidant Capacity Using 2,2-Diphenyl-1Picrylhydrazyl Assay”
L. M. Magalhães; M.A. Segundo; S. Reis; J.L.F.C. Lima
Anal. Chim. Acta, 2006, 558, 310-318
102. “A Flow Sampling Strategy for the Analysis of Oil Samples Without Pre-Treatment in a Sequential
Injection Analysis System”
P.C.A.G. Pinto; M.L.M.F.S. Saraiva; J.L.F.C. Lima
Anal. Chim. Acta, 2006, 555, 377-383
103. “Automatic Flow System for the Sequential Determination of Copper in Serum and Urine by Flame
Atomic Absorption Spectrometry”
Anal. Chim. Acta, 2006, 555, 370-376
104. “Fluorimetric Determination of Aminocaproic Acid in Pharmaceutical Formulations Using a
Sequencial Injection Analysis System”
P.C.A.G. Pinto; M.L.M.F.S. Saraiva; J.L.M. Santos; J.L.F.C. Lima
Talanta, 2006, 68, 857-862
105. “Table Olives from Portugal: Phenolic Compounds, Antioxidant Potential and Antimicrobial Activity”
J. A. Pereira; A P. G. Pereira; I. C. F. R. Ferreira; P. Valentão; P. B. Andrade; R. Seabra; L.
Estevinho; A. Bento
J. Agric. Food Chem., 2006, 54, 8425-8431
106. “Contents of Carboxylic Acids, Two Phenolics and Antioxidant Activity of Dried Portuguese Wild
Edible Mushrooms”
B. Ribeiro; J. Rangel; P. Valentão; P. Baptista; R. M. Seabra; P. B. Andrade
J. Agric. Food Chem., 2006, 54, 8530-8537
Química Verde
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107. “Phenolics and Antimicrobial Activity of Traditional Stoned Table Olives ‘Alcaparra’”
A. Sousa; I. C. F. R. Ferreira; R. Calhelha; P. B. Andrade; P. Valentão; R. Seabra; L. Estevinho; A.
Bento; J. A. Pereira
Bioorg. Med. Chem., 2006, 14, 8533-8538
108. “Principal Component Analysis as a Tool of Characterization of Quince (Cydonia oblonga Miller) Jam”
B. Silva; P. Andrade; R. C. Martins; R. Seabra, M Ferreira
Food Chemistry, 2006, 94, 504-512
109. “Chemical Composition and Antioxidant Activity of Tronchuda Cabbage Internal Leaves”
F. Ferreres; C. Sousa; V. Vrchovská; P. Valentão; J. A Pereira; R. Seabra,P. Andrade
Eur Food Res Technol, 2006, 222, 88-98
110. “Effects of Roasting on Hazelnut Lipids”
J. Amaral; S. Casal; R. Seabra; B.P. Oliveira
J. Agric Food Chem, 2006, 54, 1315-1321
111. “Tocopherol and Tocotrienol Content of Hazelnut Cultivars Grown in Portugal”
J. Amaral; S. Casal; R. Alves; R. Seabra; B. Oliveira
J. Agric Food Chem, 2006, 54, 1329-1336
112. “Characterization of Several Hazelnut (Corylus avellana L.) Cultivars Based in Chemical, Fatty, and
Sterol Composition”
J. Amaral; S. Casal; I. Citová; A. Santos; R. Seabra; B. Oliveira
Eur Food Res Technol, 2006, 222, 274-280
113. “Antioxidative Properties of Tronchuda Cabbage (Brassica oleraceae L. var costata DC) External
Leaves Against DPPH, Superoxide Radical, Hydroxyl Radical and Hypochlorous Acid”
V. Vrchovská; C. Sousa; P. Valentão; F. Ferreres; J. Pereira; R. Seabra; P Andrade
Food Chemistry, 2006, 98, 416-425
114. “Influence of Cultivar and Environmental Conditions on the Triacylglycerol Profile of Hazelnut (Corylus
avellana L.)”
J. Amaral; S. C. Cunha; A. Santos; ?M. Alves; R. Seabra; B P. P. Oliveira
J. Agric Food Chem, 2006, 54, 449-456
115. “Discrimination of vegetable oils by triacylglycerols evaluation of profile using HPLC/ELSD”
S. C. Cunha; M.B.P.P. Oliveira
Food Chemistry, 2006, 95, 518-524
116. “Monitorization of consumer and naif panels in the sensory evaluation of two types of potato chips by
predictive biplots applied to generalized procrustes and three-way Tucker-1 analysis”
M. Rui Alves; M.B.P.P. Oliveira
J. of Chemometrics, 2006, 20, 1-11
117. “Quantification of free and esterified sterols in Portuguese olive oils by solid-phase extraction and
gas-chromatography-mass spectrometry”
Sara S. Cunha; J.O. Fernandes; M.B.P.P. Oliveira
J. of Chromatography A, 2006, 1128, 220-227
Química Verde
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118. “Quantification of Tocopherols and Tocotrienols in Portuguese Olive Oils using HPLC with Three
Different Detection Systems”
S. Cunha; J. Amaral; J.O. Fernandes; M. Beatriz P.P. Oliveira
J. Agric. Food Chem., 2006, 54, 3351-3356
119. “Determination of acrylamide in coffee & coffee products by GCMS using an improved SPE clean-up”
C. Soares; S. Cunha; J. Fernandes
Food Additives and Contaminants, 2006, 23, 1276-1282
120. “Biogenic Amines in Portuguese Traditional Foods and Wines: a review”
I.M.P.L.V.O. Ferreira; O. Pinho
J. Food Protection, 2006, 69, 2293-2303
121. “Method Optimization by Solid-Phase Microextraction in Combination with GC/MS for Analysis of
Beer Volatile Fraction”
O. Pinho; I.M.P.L.V.O. Ferreira; L. H.M.L.M. Santos
J. Chrom. A, 2006, 1121, 145-153
122. “Casein breakdown in Terrincho ovine cheese: Comparison with bovine cheese and with bovine/ovine
cheeses”
I.M.P.L.V.O. Ferreira; C. Veiros; O. Pinho; A.C.A. Veloso; A.M. Peres; A. Mendonça
J. Dairy Sci, 2006, 89, 2397-2407
123. “Headspace solid-phase microextraction (SPME) in combination with GC/MS for quantification of
volatile compounds in food matrices”
I.M.P.L.V.O. Ferreira; Olívia Pinho
Trends in chromatography, 2006, 2, 31-41
124. “Tripsin hydrolysis of whey protein concentrates: characterization using multivariate data analysis”
M.V.T. Mota; I.M.P.L.V.O. Ferreira; M.B.P. Oliveira; C. Rocha; J.A.Teixeira; D. Torres; M.P.
Gonçalves
Food Chemistry, 2006, 94, 278-286
125. “Polypeptides and proteins with influence on beer foam quality and analytical methods used in its
study”.
F. Silva; I.M.P.L.V.O. Ferreira; N. Teixeira
Quim. Nova 2006, 29, 1326-1331
126. “Characterization of antimicrobial resistance and class1 and 2 integrons in Salmonella enterica
isolates from different sources in Portugal”
P. Antunes; J. Machado; L. Peixe
J. Antimicrob. Chemother., 2006, 58, 297-304
127. “Dissemination and Persistence of blaCTX-M-9 Are Linked to Class 1 Integrons Containing CR1
Associated with Defective Transposon Derivatives from Tn402 Located in Early Antibiotic Resistance
Plasmid of IncHI2, IncP1-a, and IncFI Groups”
A. Novais; R. Cantón; A. Valcerde; E. Machado; J. Galán; L. Peixe; A. Carattoli; F. Baquero; T.
Coque
Antimicrob. Agents Chemother., 2006, 50, 2741-2750
Química Verde
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128. “Antimicrobial resistance among faecal enterococci from healthy individuals in Portugal”
C. Novais; T. M. Coque; J. C. Sousa; L. V. Peixe
Clin Microbiol Infect., 2006, 12, 1131-1134
129. “Illegal use of nitrofurans in food animals: contribution to human salmonellosis”.
P. Antunes; J. Machado; L. Peixe
Clin Microbiol Infect., 2006, 12, 1047-1049
130. “Analysis of PCBs in soils and sediments by microwave-assisted extration, headspace-SPME and
high resolution gas chromatography with ion-trap tandem mass spectrometry”
Paulo Herbert; Simone Morais; Paula Paíga; Arminda Alves; Lúcia Santos
Intern. J. Environ. Anal. Chem., 2006, 86(6), 391-400
131. “Development and validation of a novel method for the analysis of chlorinated pesticides in soils using
microwave-assisted extration-headspace solid phase microextraction and gas chromatographytandem mass spectrometry”
Paulo Herbert; Simone Morais; Paula Paíga; Arminda Alves; Lúcia Santos
132. “A waste management school approach towards sustainability”
M. G. F. Sales; C. Delerue-Matos; I. B. Martins; I. Serra; M. R. Silva; S. Morais
Resources, Conservation and Recycling, 2006, 48, 197-207
133. “Soil remediation time to achieve clean-up goals I: Influence of soil water content”
Maria da Conceição M. Alvim-Ferraz; José Tomás Albergaria; Cristina Delerue-Matos
Chemosphere, 2006, 62, 853-860
134. “Soil remediation time to achieve clean-up goals II: Influence of natural organic matter and water
content”
Maria da Conceição M. Alvim-Ferraz; José Tomás Albergaria; Cristina Delerue-Matos
Chemosphere, 2006, 64, 817-825
135. “Remediation efficiency of vapour estraction of sandy soils contaminated with cyclohexane: Influence
of air flow rate, water and natural organic matter content”
José Tomás Albergaria; Maria da Conceição M. Alvim-Ferraz; Cristina Delerue-Matos
Environmental Pollution, 2006, 143, 146-152
136. “Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-Producing Enterobacteriaceae Strains
Containing the aac(6’)-Ib-cr Gene, Which Encodes an Aminoglycoside-and FluoroquinoloneModifying Enzyme”
E. Machado; T. M. Coque; R. Cantón; F. Baquero; J. C. Sousa; L. Peixe; The Portuguese
Resistance Study Group
Antimicrob. Agents Chemother., 2006, 50, 3220-3221
137. “Beta-Nitrostyrene derivatives as potential antibacterial agents: A structure-property-activity
relationship study”
N. Milhazes; R. Calheiros; M. P. Marques; J. Garrido; M. N. Cordeiro; C. Rodrigues; S. Quinteira; C.
Novais; L. Peixe; F. Borges
Bioorganic & Medicinal Chemistry, 2006, 14, 4078-4088
Química Verde
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138. “Multiniche Screening Reveals the Clinically Relevant Metallo-b-Lactamase VIM-2 in Pseudomonas
aeruginosa Far from the Hospital Setting: an Ongoing Dispersion Process?”
S. Quinteira; L. Peixe
Applied and Environmental Microbiology, 2006, 72, 3743-3745
139. “Interpretation of non–Nernstian slopes in graphic analysis of data collected in pH–range close to
deprotonation of a ligand. Part I. A glass electrode potentiometric and polarographic study of Cd–
(TAPSO)x–(OH)y and Zn–(TAPSO)x–(OH)y systems”
C.M.M. Machado; I. Cukrowski; H.M.V.M. Soares
Talanta, 2006, 68, 819-830
140. “Complex formation in the region of metal hydrolysis and M(OH)2 precipitation. A glass electrode
potentiometric and polarographic study of Cd–(AMPSO)x–(OH)y and Zn–(AMPSO)x–(OH)y systems”
C.M.M. Machado; I. Cukrowski; H.M.V.M. Soares
Electroanalysis, 2006, 18, 719-729
141. “Electrochemical determination of citalopram by adsorptive stripping voltammetry- determination in
pharmaceutical products”
Henri P. A. Nouws; Cristina Delerue-Matos; Aquiles A. Barros
Analytical Leters, 2006, 39, 1907-1915
142. “Electroanalytical Determination of Paroxetine in Pharmaceuticals”
Henri P. A. Nouws; Cristina Delerue-Matos; Aquiles A. Barros; José A. Rodrigues
Journal of Pharmaceutical and Biomedical Analysis, 2006, 42(3), 341-346
143. “Flow injection potentiometric determination of chlorpromazine”
M. Goreti F. Sales; José F. C. Tomás; Sandra R. Lavandeira
Journal of Pharmaceutical and Biomedical Analysis, 2006, 41, 1280-1286
144. “Electroanalytical study of the pesticide ethiofencarb”
M. Fátima Barroso; M. Carmo V. F. Vaz; M. Goreti F. Sales; Paula Paíga; Cristina Delerue-Matos
Analytical Letters, 2006, 39, 2387-2403
145. “Voltammetric quantification of fluoxetine: application to quality control and quality assurance
processes”
Rui P. Lencastre; Cristina D. Matos; Jorge Garrido; Fernanda Borges; E. Manuela Garrido
Journal of Food and Drug Analysis, 2006, 14(3), 242-246
146. “The periodic table: contest and exhibition”
Aurora Silva; M. Fátima Barroso; Olga Freitas; Salomé Teixeira; Simone Morais; Cristina DelerueMatos
Journal of Chemical Education, 2006, 83(4), 557-560
147. “Validation of an electrothermal atomization atomic absorption spectrometry method for the
determination of aluminum, copper, and lead in grapes”
L. Correia; M. E. Soares; M. L. Bastos
J Agric Food Chem, 2006, 54, 9312-9316
148. “Validation of a method to quantify copper and other metals in olive fruit by ETAAS. Application to the
residual metal control after olive tree treatments with different copper formulations”
M. E. Soares; J. A. Pereira; M. L. Bastos
J Agric Food Chem, 2006, 54, 3923-3928
Química Verde
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149. “The impact of sulphur dioxide and oxygen on the behaviour of 2-furaldehyde in beer: an industrial
approach”
J. R. Carneiro; L. F. Guido; P. J. Almeida; J. A. Rodrigues; A. A. Barros
International Journal of Food Science and Technology, 2006, 41, 545-552
150. “Determination of beta-damascenone in alcoholic beverages by reversed-phase liquid chromatography
with ultraviolet detection”
J.R. Carneiro; J. A. Ferreira; L. F. Guido; P. J. Almeida; J. A. Rodrigues; A. A. Barros
Food Chemistry, 2006, 99, 51-56
151. “Electroanálise de derivados acetaldeído com hidrazina e determinação em amostras de álcool
combustível por voltametria de onda quadrada”
Vanessa S. Rodgher; Nelson R. Stradiotto; Maria Valnice B. Zanoni; Aquiles A. Barros
Química Nova, 2006, 29, 662-665
152. “Analysis of xanthohumol and isoxanthohumol in different hop products by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry”
Paulo J. Magalhães; Luís F. Guido; José M. Cruz; Aquiles A. Barros
Journal of Chromatography A, 2006, 1150, 295-301
153. “Thermal and viscoelastic properties of kappa/iota-hybrid carrageenan gels obtained from
Mastocarpus stellatus Portuguese seaweeds”
L. Hilliou; F.D.S Larotonda; A.M Sereno ; M.P. Gonçalves
Journal of Agricultural and Food Chemistry, 2006, 54, 7870-7878
154. “Rheological and morphological characterisations of heat-set whey protein concentrate in the
presence of hydrolysed waxy maize starch and urea at pH 7.5”
G.K. Lopes; D.S. Alviano; D.Torres; M.P. Gonçalves; C.T. Andrade
Colloid and Polymer Science, 2006, 285, 203-210
155. “Design of biodegradable composite films for food packaging”
V. Alves; N. Costa; L. Hilliou; F. Larotonda; M.P. Gonçalves; A. M. Sereno; I. Coelhoso
Desalination, 2006, 199, 331-333
156. “Kinetics of osmotic dehydration of pumpkin with sodium chloride solutions”
L. Mayor; R. Moreira; F. Chenlo; A.M. Sereno
Journal of Food Engineering, 2006, 74(2), 253-262
157. “Use of the genus Artemia in ecotoxicity testing”
B. S. Nunes; F. D. Carvalho; L. M. Guilhermino; G. Van Stappen
Environ Pollut, 2006, 144, 453-462.
158. “Effects of widely used pharmaceuticals and a detergent on oxidative stress biomarkers of the
crustacean Artemia parthenogenetica”
B. Nunes; F. Carvalho; L. Guilhermino
Chemosphere, 2006, 62, 581-594
159. “Cytotoxicity and cell signalling induced by continuous mild hyperthermia in freshly isolated mouse
hepatocytes”
M. J. Santos-Marques; F. Carvalho; C. Sousa; F. Remiao; R. Vitorino; F. Amado; R. Ferreira; J. A.
Duarte; M. L. Bastos
Toxicology, 2006, 224, 210-218
Química Verde
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160. “Ecstasy-induced cell death in cortical neuronal cultures is serotonin 2A-receptor-dependent and
potentiated under hyperthermia”
J. P. Capela; K. Ruscher; M. Lautenschlager; D. Freyer; U. Dirnagl; A. R. Gaio; M. L. Bastos; A.
Meisel; F. Carvalho
Neuroscience, 2006, 139, 1069-1081
161. “Influence of CYP2D6 polymorphism on 3,4-methylenedioxymethamphetamine (‘Ecstasy’)
cytotoxicity”
H. Carmo; M. Brulport; M. Hermes; F. Oesch; R. Silva; L. M. Ferreira; P. S. Branco; D. D. Boer; F.
Remiao; F. Carvalho; M. R. Schon; N. Krebsfaenger; J. Doehmer; M. D. Bastos; J. G. Hengstler
Pharmacogenet Genomics, 2006, 16, 789-799
162. “P-glycoprotein induction: an antidotal pathway for paraquat-induced lung toxicity”
R. J. Dinis-Oliveira; F. Remiao; J. A. Duarte; R. Ferreira; A. S. Navarro; M. L. Bastos; F. Carvalho
Free Radic Biol Med, 2006, 41, 1213-1224
163. “Single high dose dexamethasone treatment decreases the pathological score and increases the
survival rate of paraquat-intoxicated rats”
R. J. Dinis-Oliveira; J. A. Duarte; F. Remiao; A. S. Navarro; M. L. Bastos; F. Carvalho
Toxicology, 2006, 227, 73-85
164. “Kinetics of paraquat in the isolated rat lung: Influence of sodium depletion”
R. J. Dinis-Oliveira; M. J. Valle; M. L. Bastos; F. Carvalho; A. S. Navarro
Xenobiotica, 2006, 36, 724-737
165. “Acute paraquat poisoning: report of a survival case following intake of a potential lethal dose”
R. J. Dinis-Oliveira; A. Sarmento; P. Reis; A. Amaro; F. Remiao; M. L. Bastos; F. Carvalho
Pediatr Emerg Care, 2006, 22, 537-540
166. “Paraquat exposure as an etiological factor of Parkinson’s disease”
R. J. Dinis-Oliveira; F. Remiao; H. Carmo; J. A. Duarte; A. S. Navarro; M. L. Bastos; F. Carvalho
Neurotoxicology, 2006, 27, 1110-1122
167. “The role of foetal red blood cells in protecting cultured lymphocytes against diepoxybutane-induced
chromosome breaks”
B. Porto; R. J. Oliveira; C. Sousa; J. Gaspar; J. Rueff; F. Carvalho; I. Malheiro
Mutat Res, 2006, 603, 41-47
168. “Cholinesterase from the common prawn (Palaemon serratus) eyes: catalytic properties and
sensitivity to organophosphate and carbamate compounds”
M. F. Frasco; D. Fournier; F. Carvalho; L. Guilhermino
Aquat Toxicol, 2006, 77, 412-421
169. “QM/QM Study of the Coverage Effects on the Adsorption of Amino-Cyclopentene at the Si(100)
Surface”
H. R. R. Santos; G. Ujaque; M. J. Ramos; J. A. N. F. Gomes
Journal of Computational Chemistry, 2006, 27(15), 1892-1897
170. ”Comparative Study on the Transition Metal Complexes of a Novel Thiacalix[4]Arene Derivative”
A.Suwattanamala; D. Appelhans; M. Wenzel; K. Gloe; A. L. Magalhães; J. A. N. F. Gomes
Chemical Physics, 2006, 320(2-3), 193-206
Química Verde
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171. “Molecular Interactions Between Human Cytochrome P 450 1A2 and Flavone Derivatives”
R. Fonseca; A. Melo; F. Iori; M. C. Menziani; M. J. Ramos
Medicinal Chemistry, 2006, 2, 401-406
172. “Atomic-Level Rational Drug Design”
M. J. Ramos; P. A. Fernandes
Curr Comp-Aided Drug Design, 2006, 2, 57
173. “Biological evaluation, chelation, and molecular modeling studies of novel metal-chelating inhibitors of
NF-kappa B-DNA binding: Structure activity relationships”
R. K. Sharma; S. Chopra; S. D. Sharma; V. Pande; M. J. Ramos; K. Meguro; J. I. Inoue; M. Otsuka
J Med Chem, 2006, 49, 3595-3601
174. “Computational Studies on Class I Ribonucleotide Reductase:Understanding the Mechanisms of
Action and Inhibition of a Cornerstone Enzyme for the Treatment of Cancer”
S. Pereira; N. M. F. S. A. Cerqueira; P. A. Fernandes; M. J. Ramos
Eur Biophys Journal, 2006, 35, 125-135
175. “Dehydration of Ribonucleotides Catalyzed by Ribonucleotide Reductase: The Role of the Enzyme”
N. M. F. S. A. Cerqueira; P. A. Fernandes; L. A. Eriksson; M. J. Ramos
Biophysical Journal, 2006, 90(6), 2109-2119
176. ”Enzyme Ribonucleotide Reductase: Unraveling an Enigmatic Paradigm of Enzyme Inhibition by
Furanone Derivatives”
N. M. F. S. A. Cerqueira; P. A. Fernandes; M. J. Ramos
Journal of Physical Chemistry B, 2006, 110(42), 21272-21281
177. “Unusual Solvent Effect on a S(N)2 Reaction. A Quantum-Mechanical and Kinetic Study of the
Menshutkin Reaction Between 2-Amino-1-Methylbenzimidazole and Iodomethane in the Gas Phase
and in Acetonitrile”
A. Melo; A. J. I. Alfaia; J. C. R. Reis; A. R. T. Calado
Journal of Physical Chemistry B, 2006, 110, 1877-1888
178. “Detailed Microscopic Study of the Full Zipa: Ftsz Interface”
I. Moreira; P. A. Fernandes; M. J. Ramos
Proteins Structure Function and Bioinformatics, 2006, 63, 811-821
179. “Unraveling the Importance of Protein-Protein Interaction: Application of a Computational AlanineScanning Mutagenesis to the Study of the Igg1 Streptococcal Protein G (C2 Fragment) Complex“
180. “Hot Spot Computational Identification: Application to the Complex Formed Between the Hen Egg
White Lysozyme (HEL) and the Antibody Hyhel-10”
International Journal of Quantum Chemistry, 2006, 107(2), 299-310
181 “Mechanism of a Soluble Fumarate Reductase from Shewanella Frigidimarina: A Theoretical Study”
M. F. Lucas; M. J. Ramos
Química Verde
A - 36
182. “Computational Study of Some Benzamidine-Based Inhibitors of Thrombin-Like Snake Venom
Proteinases”
E. S. Henriques; M. A. C. Nascimento; M. J. Ramos
International Journal of Quantum Chemistry, 2006, 106, 2107-2121
183. “Similarities and Differences in the Thioredoxin Superfamily”
A. P. Carvalho; P. A. Fernandes; M. J. Ramos
Prog Biophys Mol Biol., 2006, 91(3), 229-248
184. “Molecular Dynamics Model of Unliganded HIV-1 Reverse Transcriptase”
A. T. P. Carvalho; P. A. Fernandes; M. J. Ramos
Medicinal Chemistry, 2006, 2(5), 491-498
185. ”Molecular Insights into the Mechanisms of Hiv-1 Reverse Transcriptase Resistance to Nucleoside
Analogs”
A. P. Carvalho; P. A. Fernandes; M. J. Ramos
Mini-Reviews in Medicinal Chemistry, 2006, 6(5), 549-555
186. ”Determination of the Delta Pka Between the Active Site Cysteines of Thioredoxin and Dsba”
Journal of Computational Chemistry, 2006, 27, 966-97
187. ”Theoretical Study of the Unusual Protonation Properties of the Active Site Cysteines In Thioredoxin”
188. “Parameterization of AZT- A Widely Used Nucleoside Inhibitor Of HIV-1 Reverse Transcriptase”
International Journal of Quantum Chemistry, 2006, 107(2), 292-298
189. ”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, 2006, 115(1), 27-31
190. “Molecular Dynamics Simulations of the Enzyme Cu, Zn Superoxide Dismutase”
R. J. F. Branco; P. A. Fernandes; M. J. Ramos
Journal Phys. Chem. B, 2006, 110, 16754-16762
191. “Protein–Ligand Docking: Current Status and Future Challenges”
S. F. Sousa; P. A. Fernandes; M. J. Ramos
Proteins: Structure, Function, and Bioinformatics, 2006, 65, 15-26
192. “Comparative Evolutionary Genomics of the HADH2 Gene Encoding A²-Binding Alcohol
Dehydrogenase/17²-Hydroxysteroid Dehydrogenase Type 10 (ABAD/HSD10)”
A. T Marques; A. Antunes; P. A Fernandes; M. J Ramos
BMC Genomics, 2006, 7, 202
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193. “Lead Optimisation: Improving the Affinity of the Antiretrovirals Nelfinavir and Amprenavir for HIV-1
Protease”
P. A. Fernandes; M. L. A. C. Araújo; A. J. M. Barbosa; C. N. Alves; Z. Q. Ferreira; C. Gonzalez; C.
F. R. A. Lima; C. I. E. Loureiro; J. M. T. Magalhães; F. C. Maia; S. A. Moura; E. M. M. Peredo; M.
A. S. Perez; A. Q. Rodrigues; A. M. Pessoa; C. A. M. Sil
Letters In Drug Design & Discovery, 2006, 3(6), 383-389
194. “Particle in a Box: Software for Computer-Assisted Learning in Introductory Quantum Mechanics
Courses”
A. L. Magalhães; V. P. S. Vasconcelos
European Journal of Physics, 2006, 27(6), 1425-1435
195. “Evolutionary Analysis of a Large Mtdna Translocation (Numt) Into the Nuclear Genome of the
Panthera Genus Species”
H. Kim; A. Antunes; S. J. Luo; J. Menninger; W. G. Nash; S. J. O’Brien, W. E. Johnson
Gene, 2006, 366(2), 292-302
196. “Life on the Edge: The Long-Term Persistence and Contrasting Spatial Genetic Structure of Distinct
Brown Trout Life Histories at Their Ecological Limits”
A. Antunes; R. Faria; W. E. Johnson; R. Guyomard; P. Alexandrino
Journal of Heredity, 2006, 97(3), 193-205
197. “Characterization of Transferrin-Linked Microsatellites in Brown Trout (Salmo Trutta) and Atlantic
Salmon (Salmo Salar)”
A. Antunes; K. Gharbi; P. Alexandrino; R. Guyomard
Molecular Ecology Notes, 2006, 6(2), 547-549
198. “Interactions Between Oxicams and Membrane Bilayers: an Explanation for Their Different COX
Selectivity”
M.Lúcio; H. Ferreira; J.L.F.C. Lima; S. Reis
Med.Chem., 2006, 2, 447-456
199. “Antioxidant Activity and Inhibition of Human Neutrophil Oxidative Burst Mediated by Arylpropionic
Acid Non-Steroidal Anti-Inflammatory Drugs”
D. Costa; L. Moutinho; J.L.F.C. Lima; E. Fernandes
Biol. Pharm. Bull., 2006, 29, 1659-1670
200. “Antioxidant Activity of b-Blockers: An Effect Mediated by Scavenging Reactive Oxygen and Nitrogen
Species?”
A. Gomes; D. Costa; J.L.F.C. Lima; E. Fernandes
Bioorg. Med. Chem., 2006, 14, 4568-4577
201. “Extraction and Recovery of Chromium From Electroplating Sludge”
P.T.S. Silva; N.T. Mello; M.M.M. Duarte; M.C.B.S.M. Montenegro; A.N. Araújo; B.B. Neto; V.L. Silva
J. Hazard. Mater., 2006, B128, 39-43
202. “Use of Fluorescence Probes for Detection of Reactive Nitrogen Species: A Review”
A. Gomes; E. Fernandes; J.L.F.C. Lima
J. Fluorescence., 2006, 16, 119-139
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203. “Antioxidant Profile of Dihydroxy-And Trihydroxyphenolic Acids-A Structure-Activity Relationship
Study”
C. Siquet; F. Paiva-Martins; J.L.F.C. Lima; S. Reis; F. Borges
Free Rad. Research, 2006, 40, 433-442
204. “Inhibition of Human Neutrophil Oxidative Burst by Pyrazolone Derivatives”
D. Costa; A.P. Marques; R.L. Reis; J.L.F.C. Lima; E. Fernandes
Free Rad. Biol. Med., 2006, 40, 632-640
205. “2,2,4,4-Tetra-Tert-Butyl-1,3,5,2,4-Benzotrioxadisilepine-7-Carbaldehyde”
L.C.R. Andrade; J.A. Paixão; M.J.M. Almeida; C. Siquet; F. Borges
Acta Cryst., 2006, C62, 95-97
206. “Application of Sequential Injection Analysis to Pharmaceutical Analysis”
A.M. Pimenta; M.C.B.S.M. Montenegro; A.N. Araújo; J.M. Calatyud
J.Pharm. Biom. Anal., 2006, 40, 16-34
207. “Multisyringe Flow Injection Analysis: State-Of-The-Art and Perspectives”
M.A. Segundo; L.M. Magalhães
Anal. Sci., 2006, 22, 3-8
208. “Thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis sp. 6803:
comparison with its thermophilic counterpart from Phormidium laminosum”
M.J.Feio; A. Diaz- Quintana; J.A. Navarro; M.A. de la Rosa
Biochemistry, 2006, 45, 4900-4906
209. “Mossbauer effect studies on the formation of iron oxide phases synthesized via microwavehydrothermal route”
P. P. Bakare; S. K. Date; Y. B. Khollam; S. B. Deshpande; H. S. Potdar. S. Salunke-Gawali; F.
Varret F; E. Pereira
Hyperfine Interactions, 2006, 168(1-3), 1127-1132
210. “Gold and Gold-Iron Oxide Magnetic Glyconanoparticles: Synthesis, Characterization and Magnetic
Properties”
J. M. de la Fuente; D. Alcantara; P. Eaton; P. Crespo; T. C. Rojas; A. Fernadez; A. Hernando; S.
Penadés
211. “Covalent attachment of chiral manganese(III) salen complexes onto functionalised hexagonal
mesoporous silica and application to the asymmetric epoxidation of alkenes”
A. R. Silva; K. Wilson; J. H. Clark; C. Freire
Microp. Mesoporous Mat., 2006, 91, 128-138
212. “Copper acetylacetonate anchored onto functionalised hexagonal mesoporous silica as a
heterogeneous catalyst for the aziridination of styrene”
A. R. Silva; A. C. Whitwood; K. Wilson; J. H. Clark; C. Freire
Eur. J. Inorg. Chem, 2006, 1275-1283
213. “Encapsulation of chiral Mn(III) salen complexes into aluminium pillared clays: application as
heterogeneous catalysts in the epoxidation of styrene”
P. Das; I. Kuzniarska-Biernacka; A. R. Silva; A. P. Carvalho; J. Pires; C. Freire
J. Mol. Cat. A, 2006, 248, 135-143
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214. “Third-order non-linear optical properties of DA salen type nickel(II) and copper(II) complexes”
J. Tedim; S. Patrício; R. Bessada; R. Morais; C. Sousa; M. Marques; C.Freire
Eur. J. Inorg. Chem., 2006, 3425-3433
215. “Styrene oxidation by manganese Schiff base complexes in zeolite structures”
M. Silva; C. Freire; B. de Castro; J. L. Figueiredo
J. Molec. Cat. A., 2006, 258, 327-333
216. “Ring slippage vs. charge transfer in the reductive chemistry of [IndMo(CO)2(±- diimine)]+ cations”
Cláudia C. L. Pereira; Paulo J. Costa; Maria José Calhorda; Cristina Freire; Sandra S. Rodrigues;
Eberhardt Herdtweck; Carlos C. Romão
Organometalics, 2006, 25, 5223-523
217. “Pré- And Postjunctional Effects Of Angiotensin II In Hypertension Due To Adenosine Receptor
Blockade”.
M. Morato; D. Pinho; T. Sousa; S. Guimarães; D. Moura; A. Albino-Teixeira
Eur. J. Pharmacol., 2006, 531, 209-216
218. “The Inhibition Of Nociceptive Responses Of Spinal dorsal Horn Neurones During The Hypertension
Induced By Adenosine Receptor Blockade Involves The Spinal GABAergic System And A Pain
Modulatory Centre Located At The Caudal Ventrolateral Medulla”
M. Morato; D. Pinho; T. Sousa; I. Tavares; A. Albino-Teixeira
J. Neurosci. Res., 2006, 83, 647-655
219. “Lesion Of The Caudal Ventrolateral Medulla Prevents The Induction Of Hypertension By Adenosine
Receptor Blockade In Rats”
D. Pinho; M. Morato; T. Sousa; I. Tavares; A. Albino-Teixeira
Brain Res., 2006, 1073-1074, 374-382
220. “Involvement of G²³ Subunits on The Influence of Inhibitory ±2-autoreceptors on The Angiotensin AT1receptor modulation of Noradrenaline release in the Rat Vas Deferens”
C. Talaia; G. Queiroz; H. Pinheiro; D. Moura; J. Gonçalves
Neurochem. Int., 2006, 49, 698-707
221. “Mitochondrial-Dependent Ca2+ Handling in Huntington’s Disease Striatal Cells: Effect of Histone
Deacetylase Inhibitors”
J.M.A. Oliveira; S. Chen; S. Almeida; R. Riley; J. Gonçalves; C.R. Oliveira; M.R. Hayden; D.G.
Nicholls; L.M. Ellerby; A.C. Rego
J. Neurosci., 2006, 26, 11174-11186
222. “New Insights On The Anticancer Properties Of Dietary Phenols”
P. Fresco; F. Borges; C. Diniz; M.P.M. Marques
Med. Res. Rev., 2006, 26, 747-766
223. “Semiautomated Computer-assisted Image Analysis To Quantify 3,3'-Diaminobenzidine
tetrahydrochloride-immunostained Small Tissues.”
S. Leal; C. Diniz; C. Sá; J. Gonçalves; A.S. Soares; C. Rocha-Pereira; P. Fresco
Anal. Biochem., 2006, 357, 137-143
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224. “Cytotoxic and Cox-2 Inhibition Properties Of Hydroxycinnamic Derivatives.”
M.P.M Marques; F. Borges; J. Sousa; R. Calheiros; J. Garrido; A. Gaspar; F. Antunes; C. Diniz; P.
Fresco
Letters In Drug Design & Discovery, 2006, 3, 316-320
225. “Specific Modulation of Apoptosis and Bcl-xL Phosphorylation in Yeast by Distinct Mammalian
Protein Kinase C Isoforms”
L. Saraiva; R. Silva; G. Pereira; J. Gonçalves; M. Côrte-Real
J. Cell Science, 2006, 119, 3171-3181
226. “A Comparative Study of the Anion Transfer Kinetics Across a Water/Nitrobenzene Interface by
Means of Electrochemical Impedance Spectroscopy and Square-Wave Voltammetry at Thin Organic
Film-Modified Electrodes”
R. Gulaboski; V. Mirceski; C. M. Pereira; M. N. D. S. Cordeiro; A. F. Silva; F. Quentel; M. L’Her; M.
Lovric
Langmuir, 2006, 22(7), 3404-3412
227. “Molecular Dynamics Studyo 2-Nitrophenyl Octyl Ether and Nitrobenzene”
M. Jorge; R. Gulaboski; C. M. Pereira; M. N. D. S. Cordeiro
228. “Application of the Replacement Method as a Novel Variable Selection Strategy in QSAR. 1.
Carcinogenic Potential”
A. Morales Helguera; P. R. Duchowicz; M. A. C. Perez; E. A. Castro; M. N. D. S. Cordeiro; M. P.
Gonzalez
Chemometrics and Intelligent Laboratory Systems, 2006, 81(2), 180-187
229. “Simple Electrochemical Method for Deposition and Voltammetric Inspection of Silver Particles at the
Liquid-Liquid Interface of a Thin-Film Electrode”
V. Mirski; R. Gulaboski
Journal of Physical Chemistry B, 2006, Vol. 110(6), 2812-2820
230. “Study of Nitrobenzene and 2-Nitrophenyloctyl Ether Saturated with Water by Molecular Dynamics”
M. Jorge; R. Gulaboski; C. M. Pereira; M. N. D. S. Cordeiro
Mol. Phys., 2006, 104, 3627
231. “Modelling the Thermal Stability of Precursor Nanoparticles in Zeolite Synthesis”
M. Jorge; S.M. Auerbach; P.A. Monson
Mol. Phys., 2006, 104, 3513
232. “Ab Initio and Density Functional Study of a Caffeic Acid Amide”
D. A. R. Daza.; E. E. Daza C.; M. D. S. Cordeiro
J. Mol. Struct. THEOCHEM, 2006, 804, 57
233. “Modeling of Farnesyltransferase Inhibition by some Thiol and Non-Thiol Peptidomimetic Inhibitors
Using Genetic Neural Networks and RDF Approaches”
M. P. González; J. Caballero; A. Tundidor-Camba; A. M. Helguer; M. Fernández
Bioorganic Medicinal Chemistry, 2006, 14, 1, 200-213
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234. “Radial DistributionFunction Descriptors: an Alternative for Predicting A2A Adenosine Receptors
Agonists”
M. P. González; C. Terán; M. Teijeira; A M. Helguera
European Journal of Medicinal Chemistry, 2006, 41, 1, 56-62
235. “Quantitative Structure Activity Relationship for the Computational Prediction of Nitrocompounds
Carcinogenicity”
A M. Helguera; M. A Cabrera; R. D. Combes RD; M. P. González
Toxicology, 2006, 220, 51-62
236. “A Topological Substructural Molecular Design to Predict Soil Sorption Coefficients for Pesticides”
M. P. González; A . M. Helguera; I. G. Collado
Molecular Diversity, 2006, 10, 109-118
237. “A Radial Distribution Function Approach to Predict Rodent Carcinogenicity”
A. M. Helguera; M. A. Cabrera; M. P. González
Journal of Molecular Modeling, 2006, 19, 1-12
238. “Novel 2D Maps and Coupling Numbers for Protein Sequences. The fFirst QSAR Study of
Polygalacturonases; Isolation and Prediction of a Novel Sequence from Psidium Guajava L.”
G. Aguero-Chapin; H. Gonzalez-Diaz; R. Molina; J. Varona-Santos; E. Uriarte; Y. Gonzalez-Diaz
FEBS Lett., 2006, 580(3), 723-30
239. “Quantitative Structure Activity Relationships as Useful Tools for the Design of New Adenosine
Receptor Ligands. 1. Agonist”
M. P. Gonzále; C. Terá; M. Teijeira; A . M. Helguera
Current Medicinal Chemistry, 2006, 13, 2253-2266
240. “Simple stochastic fingerprints towards mathematical modeling in biology and medicine 2. Unifying
Markov model for drugs side effects”
M. Cruz-Monteagudo; H. Gonzalez-Diaz; E. Uriarte
Bulletin of Mathematical Biology, 2006, 68(7), 1527-1554
241. “SimpleStochastic Fingerprints Towards Mathematical Modeling in Biology and Medicine. 3. Ocular
Irritability Classification Model”
M. Cruz-Monteagudo; H. Gonzalez-Diaz; H. F. Borges; Y Gonzalez-Diaz
Bulletin of Mathematical Biology, 2006, 68(7), 1555-1572
242. “Spectroscopic and potentiometric characterization of oxovanadium(IV) complexes formed by 3hydroxy-4-pyridinones. Rationalization of the influence of basicity and electronic structure of the
ligand on the properties of (VO)-O-IV species in aqueous solution”
M. Rangel; A. Leite; M. João Amorim; E. Garriba; G. Micera; E. Lodyga-Chruscinska
Inorg. Chem., 2006, 45, 8086-8097
243. “New Lipophilic 3-hydroxy-4-pyridinone iron(III) complexes: synthesis and EXAFS structural
characterization”
W. Schlindwein; E. Waltham; J. Burgess; N. Binsted; A. Nunes; A. Leite; M. Rangel
Dalton Trans., 2006, 1313-132
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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, vol. 2, Ed. A. M Boldi, Combinatorial
Chemistry Series, CRC Press, Chapt 11, 257-352, 2006
2.
A. M. Lourenço, Luciane Cruz López and Arturo San Feliciano Martín
“Extracción, Fraccionamento y Aislamiento de Compuestos Activos”
in Manual de Técnicas Experimentales Utilizadas en el Estudio Preclínico de Fármacos con
Actividad Gastrointestinal, Ed. Maria José Martín Calero and Bettina Berenguer Fröehner, 33-56,
2006
3.
S. Casal; J Amaral; MBPP Oliveira
“Effects of Food Thermal Processing on Vitamin E Contents”
in Vitamin E, New Research, ed. M.H. Braunstein, Nova Science Publishers, 39-67, 2006
4.
Cristina Matos; Florinda Martins; Goreti Sales Isabel Martins; Mónica Rosas e Sérgio Morais
“Guia de segurança para laboratórios”
ALABE (eds), 2006
5.
José Miguel Loureiro; Ana Mafalda Ribeiro and Sónia Adriana Figueiredo
“Focusing Materials Research through Process Modeling”
in NATO Advanced Research Workshop on Combined and Hybrid Adsorbents: Fundamentals and
Applications, J.M. Loureiro and M.T. Kartel (eds), Springer, Netherlands, 327-338; 2006
6.
O. M. Freitas; C. D. Matos; R. R. Boaventura,
“Adsorption study of lead by Ascophyllum nodosum using a factorial experimental design”
in Combined and Hybrid Adsorbents: Fundamentals and Applications, J. M. Loureiro and M. T. Kartel
(eds), Springer, 261-266, 2006
7.
Rosa M. Seabra, Paula B. Andrade, Patrícia Valentão, Eduarda Fernandes, Félix Carvalho, Maria de
Lourdes Bastos
“Antioxidant compounds extracted from several plant materials”
in Biomaterials from Aquatic and terrestrial Organisms, eds. Miltion Fingerman and Rachakonda
Nagabhushanam, Science Publishers Inc, 115-174, 2006
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Articles in Proceedings
1.
“Bioassay-guided isolation of antioxidant and anti-inflammatory compounds from Pedilanthus
tithymaloides”
T. L. González; P. Abreu; S. Matthew; D. Costa; M. Segundo; E. Fernandes
FAPRONATURA, First Symposia about Pharmacology of Natural Products, Rev. Cubana Farm, 2006
2.
“Analysis of the chemical composition of Pinus spp needles using comprehensive two-dimensional
techniques: GCxGC and GCxMS”
E. P. Mateus; M. D. R. Gomes da Silva; J. Zrostlíková; P. J. Marriott; M. R. Paiva
22nd ISCE Annual Meeting, Barcelona, Espanha, Julho, 2006
3.
“Characterisation of Creosote treated railway wood sleeper components by GCxGC-NPD and
GCxGC/TOFMS”
M. D. R. Gomes da Silva; E. P. Mateus; P. J. Marriott; A. B. Ribeiro
29th International Symposium on Capillary Chromatography, Riva del Garda, Itália, Maio/Junho, 2006
4.
Comprehensive two-dimensional separation and analysis using one dimensional gas
chromatography and mass spectrometry - gcxms - of complex samples
Gomes da Silva, MDR; Mateus, EP; Marriott, PJ; Ribeiro, AB; Paiva, MR
5.
“Aroma Compounds which differentiates, fruity, bitter, fusty and mouldy sensorial attributes in olive
oil samples”
L. H. Ribeiro; A. M. Costa Freitas; J. M. B. Gouveia; M. D. R. Gomes da Silva
6.
“Screening of organic polluants in municipal solid waste incinerator (MSWI) fly ashes from Portugal
by GC/TOFMS”
M. D. R. Gomes da Silva; E. P. Mateus; A. F. Rodrigues; L. M. Ottosen; P. J. Marriott; A. B. Ribeiro
7.
“Cytochromes of Shewanella respiratory pathways”
R. O. Louro; C. A. Salgueiro
Metal Ions in Biology and Medicine: vol. 9 (M.C. Alpoim, P.V. Morais, M.A. Santos, A.J. Cristovão,
J.A. Centeno, P. Collery) pp 236-241John Libbey Eurotext, Paris, 2006
8.
“Influence of CYP2D6 polymorphism on 3,4-methylenedioxymethamphetamine (“ecstasy”)
cytotoxicity”
H. Carmo, M. Brulport, M. Hermes, F. Oesch, R. Silva, L.M. Ferreira, P. Branco, D. de Boer, F.
Remião, F. Carvalho, M.R. Schon, N. Krebsfaenger, J. Doehmer, M.L. Bastos, J.G. Hengstler
43rd Congress of the European Societies of Toxicology & 6th Congress of Toxicology in Developing
Countries. Cavtat,(Croatia)20-24 September 2006. Toxicology Letters 164S: S118
9.
“Neurotoxicity of ecstasy metabolites in rat cortical neurons, and influence of hyperthermia”
J. P. Capela, A. Meisel, A. R. Abreu, P. S. Branco, L. M. Ferreira, A. M. Lobo, F. Remião, M. L.
Bastos, F. Carvalho
Countries. Cavtat,(Croatia)20-24,September2006. Toxicology Letters 164S: S295
10.
“Validation of a HPLC-ECD method for the quantification of the highly reactive metabolite of ecstasy,
N-methyl-alpha-methyldopamine, in human serum”
G. Mergen; R. Silva; L. M. Ferreira; P. S. Branco; F. Remião; F. Carvalho; M. L. Bastos; T.
Soylemezoglu; H. Carmo
Countries. Cavtat,(Croatia) 20-24 September 2006. Toxicology Letters 164S: S309
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11.
“Organic Metabolites in Exhaled Human Breath: A SPME and GC-TOF-MS Study”
E. M. S. M. Gaspar; M. M. Gonves
29th International Symposium on Capillary Chromatography, Riva del Garda, Italy, CD ROM, T.
Sandra and P. Sandra (Ed.), 2006
12.
“Simultaneous determination of chlorinated organic compounds-Dioxins, Furans, Dioxin-like PCBs
and Hexachlorobenzene in atmospheric air and stationary source emissions”
P. Antunes; P. Viana; T. Vinhas; E. Gaspar; J. L. Capelo-Martinez
Proceedings of 26th International symposium on halogenated persistent organic pollutants. Oslo,
august 2006, Norway
13.
“Recombinant metal-chelated forms of adenylate kinase”
S. A. Bursakov; R. Alves; A. Kladova; O. Y.Gavel; I. Moura; J. J. G. Moura
Proceedings of 8th European Biological Inorganic Chemistry Conference (EUROBIC8), Aveiro,
Portugal, p.329, 2006
14.
“Physicochemical characterization of peroxidase from royal palm tree Roystonea regia”
M. G. Roig; L. S. Zamorano; J. Calvete; S. A. Bursakov; I. Polikarpov; G. G. Zhadan; V. L. Shnyrov
Proceedings of Peroxidase 2006, satellite meeting of 8th European Biological Inorganic Chemistry
Conference (EUROBIC8) July 7-9, 2006, Aveiro, Portugal, p.74, 2006
15.
“Genome-Scale Classification Of Metabolic Reactions Without Assignment Of Reaction Centers”
J. Aires-De-Sousa; D. A. R. S. Latino
Abstracts Of Papers Of The American Chemical Society, 231: - 78-Cinf, 2006
16.
“In Silico Prediction Of Mutagenicity Using Molecular Maps Of Atom-Level Properties (Molmaps) And
Empirical Physicochemical Descriptors”
J. Aires-De-Sous; Q. Y. Zhang
17.
“Estimating 1H NMR Coupling Constants With ANN Models For Chemical Shifts: Spectra Simulation
In The SPINUS System”
J. Aires-De-Sousa; Y. Binev
18.
“Desulfovibrio gigas: toxicity of copper and molybdenum”
A. R. Lino; C. R. Farinha; S. Pereira; S. A. Bursakov
Metal ions in biology and medicine: V.9, Eds.: MC.Alpoim, PV.Morais, MA.Santos, AJ.Cristovão,
JA.Centeno, P.Collery, John Libbey Eurotext, Paris, 2006, 231-235
19.
“Jacobsen catalyst anchored onto modified carbon xerogel as enantioselective heterogeneous
catalysts for alkene epoxidation”
F. Maia; N. Nahata; A. R. Silva; M. F. R. Pereira; C. Freire; J. L. Figueiredo
Proceedings of the International Conference on Carbon, Aberdeen, Scotland, 2006
20.
“Gelling properties of a kappa/iota-hybrid carrageenan: effect of salt, concentration and steady
shear”.in Fischer P., Erni, P., Windhab E.J., editors.
L.Hilliou; M.P. Gonçalves
Proceedings of the 4th International Symposium on Food Rheology and Structure. Lappersdorf:
Kerschensteiner Verlag; pp 363-367 (2006).
21.
Antioxidant Compounds Extracted From Several Plant Materials
R.M. Seabra; P.B. Andrade; P. Valentão; E. Fernandes; F. Carvalho; M.L. Bastos
in Biomaterials From Aquactic and Terrestrial Organisms, ed. M. Fingerman; R. Nagabhushanam,
Science Publishers, 115-174 (2006)
22.
“A new and vital antidotal pathway for paraquat poisonings more than 60 years later: Induction of
lung P-glycoprotein”
R. J. Dinis-Oliveira; F. Remiao; J. A. Duarte; R. Ferreira; A. S. Navarro; M. L. Bastos; F. Carvalho
Toxicology Letters, 2006, Vol. 164, S75-S75.
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23.
“Effect of adrenaline and oxygen free radicals on calcium tolerant cardiomyocytes: Formation of
glutathione adducts”
V. Costa; M. Carvalho; F. Carvalho; M. L. Bastos; R. Carvalho; F. Remiao
24.
“Validation of a HPLC-ECD method for the detection of adrenaline-GSH adducts in biological
samples”
R. Silva; V. M. Costa; S. Boldt; H. Carmo; F. Carvalho; M. Carvalho; M. L. Bastos; F. Remiao
25.
“Hyperthermia-induced toxicity in freshly isolated mouse hepatocytes: Involvement of oxidative
stress and cellular signalling”
M. J. Santos-Marques; C. Sousa; R. Vitorino; R. Ferreira; F. Remiao; F. Amado; J. A. Duarte; F.
Carvalho; M. L. Bastos
26.
“Ethanol and ecstasy: Allied enemies of freshly isolated mouse hepatocytes”
H. Pontes; M. J. Marques; F. Remiao; F. Carvalho, M. L. Bastos
27.
“Dexamethasone treatment decreases the pathological effects and increases the survival rate of
paraquat-intoxicated rats”
R. J. Dinis-Oliveira; F. Remiao; J. A. Duarte; A. S. Navarro; M. L. Bastos; F. Carvalho
28.
“Ecstasy-induced cell death in cortical neuronal cultures is 5-HT2A-receptor-dependent and
potentiated under hyperthermia”
J. P. Capela; K. Ruscher; M. Lautenschlager; D. Freyer; U. Dirnagl; A. R. Gaio; M. L. Bastos, A.
Meisel; F. Carvalho
Química Verde
A - 47
B
Dissertations
Química Verde
A - 48
Ph. D. Dissertations
1.
“Dispositivos Moleculares Fluorescentes Contendo Receptores Poliamínicos”
Sérgio Paulo do Carmo Alves
Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, July 2006
Supervisor: Fernando Pina
2.
“Estudos em Derivados do 2-Fenil-1-benzopirílio”
Margarida Maria de Mesquita Cabral de Moncada
Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, July 2006
Supervisor: Fernando Pina
3.
“Synthetic molecules approach to biology”
Rita Gusmão de Noronha
FCT-UNL, Lisboa, 2006
Supervisor: K. Nicolau with M. M. Pereira
4.
“Novos Sistemas para Rearranjos 3-aza-Cope”
P. M. C. Glória
Supervisor: S. Prabhakar, co-supervisor: A. M. Lobo
5.
“Síntese Assimétrica de N-Sulfinil Aziridinas”
V. D. B. Bonifácio
Supervisor: S. Prabhakar, co-supervisor: A. M. Lobo
6.
“Síntese de Alcalóides Indolocarbazólicos: Obtenção da staurosporinona (K-252)”
S. Gaudêncio
Supervisor: A. M. Lobo, co-supervisor: S. Prabhakar
7.
“Novel Metalloproteins from Sulphare-Reducing Bacteria”
Olga Yurievna Gavel
UNL April 2006
Supervisor: I. Moura
8.
“Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel
cofactors and revealing new features of old enzymes”
Maria Gabriela Rivas
UNL December 2006
Supervisor: I. Moura e C. Brondino
9.
“Periplasmic Nitrate Reductases: Structural and Spectroscopic Studies”
Pablo Gonzalez
UNL December 2006
Supervisor: J. J. G. Moura and C. Brondino
Química Verde
A - 49
10.
“Crystallographic Studies Of Two Iron-Containing Proteins: Superoxide Reductase And Sixteen-Heme
Cytochrome”
Teresa Santos-Silva
UNL, Dez. 2006
Supervisor: Maria João Romão and Robert Huber
11.
“A Análise por Injecção Sequencial com Base de Sistemas Automáticos em Fluxo Contínuo”
Paula Cristina de Azevedo Gomes Pinto
UP, Fev de 2006
Supervisor: Lúcia Saraiva and José Luís Costa Lima
12.
“Novos Sistemas de Fluxo Contínuo para a Automatização da Análise Quimiluminométrica de
Produtos Farmacêuticos”
Karine Lopes Marques
UP, Nov de 2006
Supervisor: João Luís Santos and José Luís Costa Lima
13.
“Contribuição para a Caracterização Química das Folhas e Sementes de Aveleira (Corylus avellana
L.) e de Nogueira (Juglans regia L.)”
Joana Andrêa Soares Amaral
UP, Set de 2006
Supervisor: R. M. Seabra and B. P. P. Oliveira
14.
“Qualidade e autenticidade de produtos. Uso da quimiometria no estudo de parâmetros químicos,
reológicos e sensoriais”
Manuel Rui Azevedo Alves
FFUP, Abr 2006
Supervisor: Beatriz Oliveira
15.
“Eco-epidemiologia de bacilos de Gram negativo produtores de carbapenemases com impacto
clínico”
UP, July 2006
Supervisor: L. V. Peixe
16.
“Interaction of biological pH buffers, used in biological and environmental studies, with metal ions”
Carina Madalena Martins Machado
FEUP, May 2006
Supervisor: Helena M.V.M. Soares
17.
“Caracterização reológica e morfológica de sistemas mistos amido modificado/proteínas do soro do
leite para aplicação em adesivos”
Gisela Kloc Lopes
UFRJ (Brazil), August 2006
Supervisor: Cristina Tristão de Andrade, Maria do Pilar F. Gonçalves
18.
“Rheological and microstructural properties of b-lactoglobulin- galactomannan aqueous systems”
Wancheng Sittikijyothin
FEUP, November 2006
Supervisor: Maria do Pilar F. Gonçalves
Química Verde
A - 50
19.
“Characterization and modelling of structural changes in fruits and vegetable tissue submitted to
dehydration processes”
Luis Mayor Lopez
FEUP, December 2006
Supervisor: Alberto Manuel Carneiro Sereno
20.
“Molecular Modeling on Inhibitors of Nuclear Factor-Kappa B”
Vineet Pande
FCUP, Fev de 2006
Supervisor: M. J. Ramos
21.
“Computational Studies on Cytochromes P450”
Rute Andreia Rodrigues da Fonseca
FCUP, Nov de 2006
Supervisor: M. J. Ramos e A. Melo
22.
“Mecanismo de Inibição de Enzimas Radicalares”
Susana Rodrigues Pereira
FCUP, Nov de 2006
Supervisor: M. J. Ramos e P. Fernandes
Ana Pessoa Moura
23.
“Estudos de Anti-Inflamatórios não Esteróides em Modelos Membranares”
UP, Nov de 2006
Supervisor: Salette Reis e José Luís Costa Lima
24.
“Utilização de Lipossomas como Modelos de Biomembranas na Avaliação e Quantificação da
Actividade de Anti-Inflamatórios”
Helena Susana da Costa Machado Ferreira
UP, Dez de 2006
Supervisor: Salette Reis e José Luís Costa Lima
25.
“Filmes poliméricos do tipo [M(salen)] funcionalizados para o reconhecimento de catiões
representativos”
Andrea da Conceição Ferreira Carneiro
UP, Mar. de 2006
Supervisor: Cristina Freire
26.
“Mitochondrial Function and Ca2+ Homeostasis in Experimental Models of Huntington’s Disease:
Understanding Disease Pathogenesis and Pharmacological Interventions”
Jorge Miguel de Ascenção Oliveira
Universidade do Porto, Dez 2006
Supervisor: Jorge Manuel Moreira Gonçalves, Ana Cristina Carvalho Rego
27.
“Computational Study on Thiacalix (4)Arenes Complexes”
Akapong Suwattanamala
FCUP, Mar de 2006
Supervisor: J.A.N.F. Gomes e A. Magalhães
Química Verde
A - 51
M. Sc. Dissertations
1.
“Monitorização In-Situ de Processos de Tratamento de Águas Residuais com Espectroscopia de
Infra-Vermelho Próximo”
Ricardo Nuno Mendes de Jorge Páscoa
UP, Dez de 2006
Supervisor: João Lopes
2.
“Metodologias Automáticas para Controlo de Qualidade de Formulações com Alopurinol Baseadas
em Biocatalisadores”
Cristina Isabel da Cunha Silvestre
UP, Dez de 2006
Supervisor: M. Lúcia Saraiva e Marcela Segundo
3.
“Avaliação do Uso de Fibras Minerais Naturais na Remoção do Octilfenol Polietóxido Etanol de
Soluções Aquosas”
Daniel Alexandre de Marçal Ribeiro
UP, Dez de 2006
Supervisor: Adriano Fachini
4.
“Aplicação da técnica de reacção em cadeia da polimerase na avaliação da autenticidade do queijo
de cabra Transmontano”
Á. Roxo
FFUP, Abr 2006
Supervisor: I. Mafra and I.M.PLV.O. Ferreira
5.
“Controlo de qualidade de óleos de fritura em restauração colectiva. Validação do plano HACCP”
S. Mendes
FFUP, Jul 2006
Supervisor: Beatriz Oliveira and Susana Casal
6.
“Caracterização do teor de norarmana e harmana em cafés verdes e torrados”
A. F. Gomes
FFUP, Out 2006
Supervisor: Beatriz Oliveira and Susana Casal
7.
“Desenvolvimento de uma metodologia por HPLC para a detecção de aductos das catecolaminas
com a GSH em amostras biológicas”
Renata Sofia Araújo da Silva
Departamento de Biologia da Universidade de Aveiro, Novembro de 2006
Supervisor: Fernando Manuel Gomes Remião e Maria Paula Polónia Gonçalves
8.
“Influência da administração de “ecstasy” e do exercício físico agudo na taxa de produção de
peróxido de hidrogénio “in vivo” no músculo esquelético de ratinho”
Filipe Soares Ferreira
Faculdade de Ciências do Desporto e Educação Física da Universidade do Porto, Nov 2006
Supervisor: José Alberto Ramos Duarte e Félix Dias Carvalho
9.
“Explicações de Química – Percepções dos Alunos”, Mestrado em Química para o Ensino
Andreia de Sousa Guimarães
FCUP, Mar 2006
Supervisor: Maria Gabriela Ribeiro e Aquiles A. Barros
Química Verde
A - 52
10.
“Ensino/Aprendizagem da Ligação Covalente no 10º Ano da Disciplina de Física e Química”
Dominique Azevedo da Costa
FCUP, Mai 2006
Supervisor: Maria Gabriela Ribeiro e Aquiles A. Barros.
11.
“Desenvolvimento de uma Metodologia Analítica para a Determinação de Xanto-Humol e IsoxantoHumol no Lúpulo e na Cerveja”,
Paulo Jorge Coimbra Rodrigues Magalhães
FCUP, Jul 2006
Supervisor: Luís Guilherme L. F. Guido.
12.
“Determinação de Alfa-Dicetonas Vicinais em Cerveja por HPLC-UV, após Extracção Contínua por
Difusão Gasosa”
João Paulo Grosso Pacheco
FCUP, Set 2006
Supervisor: José António Maia Rodrigues.
13.
“Estabilidade de benzodiazepinas em amostras post-mortem armazenadas a diferentes
temperaturas”
Paula Isabel Landeiro e Melo
Instituto Nacional de Medicina Legal-Porto / Serviço de Toxicologia da FFUP, Abril 2006
Supervisor: Maria de Lourdes Bastos
14.
“Estudo da Activação de Neutrófilos Humanos pelo Nitrato de Níquel”
Marisa Andreia Carvalho de Freitas
UP, Nov de 2006
Supervisor: Eduarda Fernandes e José Luís Costa Lima
15.
“Avaliação do Efeito de Compostos Fenólicos na Fluidez da Membrana Celular”
Cláudia Daniela Oliveira de Lacerda Nunes
UP, Dez de 2006
Supervisor: Salette Reis e Anabela Silva
16.
“Avaliação do Efeito de Alguns Fármacos na Fluidez da Membrana Celular”
Célia Tavares de Sousa
UP, Dez de 2006
Supervisor: Salette Reis e Anabela Silva
17.
‘Reconhecimento de catiões alcalinos por filmes poliméricos de Ni(II) funcionalizados com grupos
éter coroa’
Rosa Bela Bessada
UP, Dez. de 2006
Orientadores: Cristina Freire e Cosme Moura (CIQ-UP)
Química Verde
A - 53
C
Patents
Química Verde
A - 54
Patents
1.
PCT Patent Pending - PCT/IB2006/052314 (2006)
Selective acceleration of fragmentation through joint application of enzymes and ultrasound
José L. Capelo
2.
National Patent Request: Nº 103420 (2006)
Utilização do processo de indução da síntese de novo da glicoproteína P (gp-p) no tratamento das
intoxicações dos mamíferos por xenobióticos
Authors: Ricardo Jorge Dinis-Oliveira, Fernando Remião, José Alberto Duarte, Amparo Sánchez
Navarro, Maria de Lourdes Bastos, Félix Carvalho
3.
National Patent Request: Nº: 103480 (2006)
Utilização de salicilato como antídoto nas intoxicações dos mamíferos pelo paraquato
Authors: Ricardo Jorge Dinis-Oliveira, Fernando Remião, José Alberto Duarte, Amparo Sánchez
Navarro, Maria de Lourdes Bastos, Félix Carvalho
Química Verde
A - 55
ANNEX III
RESEARCH PROJECTS
Química Verde
A - 56
International funding
EU-Marie Curie Network
MRTN-CT-2005-019335
“Molecular Basis of Antibiotic Translocation”
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)
Project PICS Nº 1392
“Analyse des interactions protéines-protéines par arrimage moléculaire sous contraintes RMN”
Centre National de la Recherche Scientifique / ICCTI
Coordinator: José J.G.Moura
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
“Towards New Supramolecular Devices: from new synthetic methods to multifunctional applications”
POCI/QUI/55519/2004
Coordinator: Carlos Lodeiro Espiño
“Chemical and Sensorial Characterization of Malolactic Fermentation impact in Wines”
POCTI/AGR/55432/2004
Coordinator: Ana Costa Freitas, REQUIMTE participation: M.D.R. Gomes da Silva
“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: Luís Fonseca
“Chemosensors Based on Fluorescent Poliamine Receptors”
POCTI/QUI/47357/2002
Coordinator: Fernando Jorge da Silva Pina
“Photochromism of Flavylium Compounds in Water/Ionic Liquid Biphasic Systems”
Coordinator: Fernando Jorge da Silva Pina
“A cor na iluminura portuguesa: uma abordagem interdisciplinar / An interdisciplinar approach to the study
of color in Portuguese manuscript illuminations”
POCTI/EAT/33782/2000
Coordinator: Maria João Seixas de Melo
Química Verde
A - 57
“As Moléculas da Cor na Arte: um Estudo Fotoquímico / The Molecules of Colour in Art: a Photochemical
Study”
POCI/QUI/55672/2004
Coordinator: João Sérgio Seixas de Melo
“Natural algicides against harmful microalgae”
POCI/AMB/60351/2004
Coordinator: A. M. Lobo (collaboration with INSA)
“New Synthetic methodology towards chiral gamma-amino acids useful in the design of new drugs”
Coordinator: M. Manuela Pereira
“Quinolizidine alkaloids as chiral synthons”
Coordinator: A. M. Lourenço
“Volatile phenol yeast producers: a great concern in wine industry to be understood”
Coordinator: Prof. M. Malfeito Ferreira, Collaboration: Prof. J. Aires de Sousa
“Quiral chelators agents for using in metal remediation, industrial and domestic applications: selective
synthesis, complexation and biodegradation studies”
POCI/QUI/57891/2004
Coordinator: Helena Soares
“New Molecular devises: from new synthetic methods to multifuntional applications”
“Tailored Synthesis of Biopolymers by mixed microbial cultures from molallse”
POCI/BIO/55789/2004
“Saccharose as a Water Soluble Chiral Auxiliary and a Linker for Solid Phase Chemistry”
“Bacterial cytochrome c peroxidases- Activation, Enzymatic Mechanism And Structure”
Coordinator: Isabel Moura
“Enzimas Chave da Reduçâo do Nitrato. Redutase do Óxido Nítrico e Redutase do Óxido Nitroso-As Duas
Enzimas Terminais”
POCTI/BME/42265/2001
“High Pressure NMR spectroscopy of polymers and biopolymers in CO2 emulsions”
Coordinator: Maria dos Anjos Macedo
“Estudos Electroquímicos Dinâmicos em Proteínas de Transferência Electrónica”
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”
Coordinator: Francisco Jorge Caldeira
Química Verde
A - 58
“Structural and mechanistic studies of fatty acid desaturases. Looking for reactivity of diiron clusters”
Coordinator: Pedro Tavares
“Mechanistic and Structural Studies of Iron Oxidation and Storage by Fast Ferritins”
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, Margarida Santos
“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”
Coordinator: M. Gabriela Almeida
“New molybdo and copper cofactors in proteins isolated from sulphate reducers”
“Cobalt in metabolism of sulfate reducers. Noncorrin Co/Zn ATPS, AK, and a new Co-corrinoid protein”
Coordinator: Sergey Bursakov
“(Per)chlorate enzymatic reduction”
Coordinator: Cristina Costa
“Spectroscopy of Oriented Proteins on Conductive Polymers”
Coordinator: Francisco Caldeira
“Studying kinetics and mechanism of superoxide reduction”
Coordinator: Alice Pereira
“Análise estrutural e funcional de um novo citocromo multihémico: um modelo para redutases do fumarato”
Coordinator: C. Salgueiro
“Caracterização Bioquímica de Proteínas com Actividade Bioremediativa”
POCI/QUI/60060/2004
“Caracterização de CymA: uma proteína chave na respiração anaeróbia de Shewanella”
POCI/BIA-PROQUI/58722/2004
Química Verde
A - 59
“Desenvolvimento de biossensores enzimáticos para controlo e monitorização multiparamétrica de
amostras ambientais”
POCI/QUI/58026/2004
Coordinator: M. Gabriela Almeida
“Nanostructures of noble metals in organized media: towards technological applications”
POCTI/QUI/45141/2002, 2004-2007
Coordinator: Ricardo Franco
“Structural and Functional Characterization of Nitrate Reductases and Formate Dehydrogenases”
Coordinator: Maria João Romão
“Molecular determinants of ligand specificity in carbohydrate-binding modules and cohesin-dockerin
complexes”
POCTI/BIA-PRO/59118/2004
Coordinator: Carlos Fontes, Co-proposer: Maria João Romão
“Freezing and dehydration of yeasts and grape berries: a role for mediated water fluxes”
“Biological Effects of dietetic and supplemented conjugated linoleic acid (CLA) isomers in the portuguese
population”
POCTI/2002/44750
“Natural Vegetal Antioxidants”
Coordinator: Abel José de Sousa Costa Vieira
“Assessing the dietary exposure of Portuguese consumers to acrylamide. improvement, development and
validation of rapid screening procedures and confirmatory analytical methodologies”
Coordinator: José Fernandes e João L. M. Santos
“Síntese de cromonas e avaliação in vitro das propriedades antioxidantes e anti-inflamatórias”
Coordinator: José L.F.C. Lima
“Desenvolvimento de biossensores enzimáticos para a determinação de nitratos e nitritos em amostras
complexas”
Coordinator: M. Conceição Montenegro
“Phytochemical and antioxidant characterization of Brassica oleracea var. costata (tronchuda cabbage) in
in vivo and in vitro cultures”
POCI/AGR/57399/2004
“Technologies for the valorization of Salvia sp bioproducts”
Coordinator: Manuel Ferreira
“Phylogenetic Study of Fabaceae in Portugal”
POCI/AGR/56696/2004
“Avaliação da exposição de consumidores portugueses à acrilamida. Optimização, desenvolvimento e
validação de métodos rápidos de despistagem e de metodologias analíticas de confirmação”
Coordinator: José Fernandes, João L. M. Santos
Química Verde
A - 60
“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
“Biological Markers of contamination by hospital wastewater”
POCTI/ESP/39885/2001
Coordinator: Helena Maria Neto Ferreira
“Metodologias para controlo de solos contaminados com pesticidas”
Coordinator: Simone Barreira Morais
“Bioremediation of organic pollution in soil by augmentation with robust methylotrophic bacteria”
POCI/AMB/57353/2004
Coordinator: Paolo De Marco
“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 Emissions from
Tailings Disposals”
POCI/AMB/61315/2004
Coordinator: Maria da Conceição M. Alvim Ferraz
“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”
Coordinator: Loic Hilliou
“Tailored synthesis of biopolymers by mixed microbial cultures from molasses”
POCTI/BIO/55789/2004
“Development of a clean technology for heavy metals removal and recovery from industrial effluents”
POCTI/CTA/47875/2002
“New potentially biodegradable chelating agents for industrial and domestic applications environmentfriendly”
POCI/QUI/57891/2004
“Oxidative stress in heart disease: role of catecholamines”
POCI/SAU-OBS/55849/2004
Coordinator: Fernando Manuel Gomes Remião
Química Verde
A - 61
“Toxicokinetic and toxicological consequences of the interactions between ethanol and pharmaceutical
drugs used in association with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). Evaluating the
risks of polydrug intake”
POCI/SAU-FCF/57187/2004
Coordinator: Maria de Lourdes P. A. S. Bastos
“Avaliação da estrutura e funções da proteína membranar OmpF na acumulação de fluoroquinolonas e
derivados”
POCI/SAU-FCF/56003/2004
“Nanoestruturas de metais nobres em meios organizados: aplicações tecnológicas”
“Desenvolvimento de membranas catalíticas poliméricas para a epoxidação de olefinas terpénicas
(Development of polymeric membranes for the epoxidation of terpenic olefins)”
POCTI/CTM/45449/2002
“Catalisadores de carbono nanoestruturado (Nanostructured carbon catalysts)”
“Fabricação de materiais nanoporosos baseados em argilas por intercalação com sílica funcionalizada
com complexos de metais de transição: aplicação em catálise enantioselectiva (Engineering the structure
of nanoporous clays by intercalation of silica functionlised with transition metal complexes: application to
enantioselective catalysis)”
POCTI/CTM/561927/2004
“Propriedades bioactivas de cogumelos silvestres comestíveis (Bioactive properties of wild edible
mushrooms)”
“Development of sustainable chemical catalytic processes by integration of nanofiltration and aqueous
media”
Bilateral cooperations
Portugal-England 2006 Nº B-16/06 and Renovation 2007 Nº B-61/07
“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
Portugal-Spain HP2006-0119
“Rapid bacterial identification based on mass spectrometry techniques”
Portuguese Coordinator: Elvira Gaspar
Spanish Coordinatior: Benito Canhas
Department of Analytical Chemistry
Universidad Complutense de Madrid
2005-2007
Portugal-England
“Learning facilitated and supported through the use of information and communication technologies”
Coodinator: Prof. Emília Bértolo Pardo, Canterbury Christ Church University.
Project partnet: Dr. Carlos Lodeiro. Participation as Portuguese member
Química Verde
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Portugal-Spain, Action 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
Portugal-Spain, AI/E-59/2006
“Compostos orgânicos azotados: desenho de moléculas com actividade biológica baseado em sugestões
da natureza (actividade e biotransformações)”
Coordinator: Ana M. Lobo
Partner: Universidade de Barcelona
Portugal-France 2006, Action Nº F-27/06
“Synthesis of new cobalt complexes: application in olefin polymerization”
Portugal-Spain, Action Nº E-62/05
“Resonance Raman and AFM study of membrane heme-containing proteins adsorbed to glicosylated
SAMs”
CRUP
2005-2006
Portugal-Spain 2006, Action N0 E-62/06
“Thermodynamic Structural Studies of Temperature Induced Unfolding of Plant Thermophylic and Bacterial
Mesophylc Enzymes”
CRUP
Coordinator: Sergy Bursakov
Portugal-Spain, Action Nº 52/05
“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”
CRUP
Collaborators: Carla Novais, Teresa Coque
Portugal-Brasil
“Desenvolvimento de equipamentos automáticos e metodologias expeditas para controlo ambiental na
perspectiva de um desenvolvimento sustentado”
FCT/GRICES/CNPq
Coordinator: José L.F.C. Lima
Portugal-Brasil
“Estudo e aplicabilidade de sensores contendo ciclodextrina na monitorização de entidades químicas de
interesse biológico”
FCT/GRICES/CAPES
Coordinator: Alberto N. Araujo
Portugal-Brasil
“Estratégias para a implementação de processos de controlo empregando conceitos de Química-Verde”
FCT/GRICES/CAPES
Coordinator: M. Lúcia Saraiva
Portugal-Germany, Action Nº A-6/06
“Influence of NSAIDs on the activity of enzymes at interfaces”
CRUP
Coordinator: Salette Reis
Química Verde
A - 63
CZ/06/BF/PP-168025
“ICT as a gateway to scientific education of blind people and people with visual disabilities”
European Union - Leonardo da Vinci/2006
Partners: Portugal, Check Republic, Slovakia, Norway
Researchers: A. M. Lobo (in charge of Portuguese team), P. Mata, J. Aires de Sousa
X-10, CYTED (PIGASTRIN)
“Iberoamerican project for the search and evaluation of new active agents in digestive disorders”
Coordinator: A. M. Lourenço
Portugal-Brazil
“Frutas tropicais de alta humidade: processamento, embalagem sob atmosfera modificada e avaliação da
qualidade”
GRICES/CAPES
CYTED project XI.20
“Tecnologia de películas biodegradaveis para alimentos en ibero-américa” “Technology for food grade
biodegradable films in Ibero-America”
International Co-ordinator: Dr. Paulo José do Amaral Sobral (Brazil)
Portugal-Germany
Characterization of biopolymer mixtures for food applications
GRICES/DAAD
2005/2006
Portugal-Germany, Action Nº 5/06
“Membrane Transporters”
CRUP
Portugal-Spain
“Estudocomparativo da termoestabilidade do citocromo c6 de cianobactérias termófilas e mesófilas”
GRICES-CSIC, projecto bilateral com o IBVF, Sevilla
2006-2007
Project from the Luso-American Foundation (FLAD)
“FLAD Nanotechnology Network”
2003-2006
“SciencEduc”
http://www.cienciaviva.pt/projectos/scienceduc/
Project coordinated by the Ecole Normale Supérieure (Paris) in partnership: 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)
P. Mata as project advisor of the portuguese partner
Project “Pollen – Seed Cities for Science – A Community Approach for a Sustainable Growth of Science
Education in Europe”
Commission of the European Communities - SAS6 contract number 518399
École Normale Supérieure (France), Université Libre de Bruxelles (Belgium), University of Tartu (Estonia),
Freie Universität Berlin (Germany), Consortium Innovation Training Educational Inquiry (Italy), Universiteit
van Amsterdam (Netherlands), Ciência Viva-Agencia Nacional para a Cultura Científica e Tecnológica
(Portugal), P.A.U. Education (Spain), Royal Swedish Academy of Sciences (Sweden), University of
Leicester (United Kingdom), Apor Vilmos Catholic College (Hungary), Státny Pedagogick Ústav - National
Institute for Education(Slovenia).
Collaboration: P. Mata
Química Verde
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Funded by REQUIMTE / Others
REQUIMTE/CQFB/CEQUP/2004
“Anthocyanin blues: A green approach towards food colorants and new pigments based on anthocyanines
and synthetic flavylium salts”
Coordinator: João Carlos Lima
1/9/2004 - 31/8/2006
REQUIMTE Project 2004
“Bioassay-guided isolation and identification of anti-inflammatory and antioxidant compounds, from a
medicinal extract of Pedilanthus tithymaloides”
Coordinator: P. M. Abreu
“Quiral chelators agents for using in metal remediation, industrial and domestic applications: selective
synthesis, complexation and biodegradation studies”, financed by the associated lab – REQUIMTE
“Molecular determinants of ligand Specificity in family 11 Carbohydrate binding modules: an X-ray, NMR
and computational chemistry combined approach”, financed by the associated lab – REQUIMTE
“Searching for bioactive peptides. Valorisation of cheese whey protein fraction by separation and
characterization of bioactive peptides to be applied in food and pharmaceutical industries”
Alice Pereira and Pedro Tavares
REQUIMTE, CQFB and CEQUP
“Development of Electrochemical Biosensors for On-Line Determination of Nitrite and Nitrate in Complex
Samples”
M. Gabriela Almeida
REQUIMTE, CQFB and CEQUP
“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
“Molecular Determinants of Ligand Specificity in Family 11 Carbohydrate Binding”
Modules: An X-ray Crystallography, NMR and Computational Chemistry combined approach
Coordinator: Eurico Cabrita (CQFB, FCT/UNL)
Collaboratos: Ana Luísa Carvalho (REQUIMTE), Maria dos Anjos Macedo (CQFB, FCT/UNL), Maria João
Ramos (CEQUP)
REQUIMTE - “Evaluation of the in vitro antioxidant activity of non-steroidal anti-inflammatory drugs
(NSAIDs)”
Coordinator: Eduarda Fernandes
Protocolo INVESTIGACIÓN DE SINIESTROS S.L./FCT-UNL - “Arson Analysis “
Coordinator: J.P. Noronha
“Development of electrochemical biosensors for on-line determination of nitrite and nitrate in complex
samples”
REQUIMTE (REQELE)
Coordinator: M. Conceição Montenegro
Química Verde
A - 65
“Integrated Process for prodution of drug-loaded micro/mano sized polymers using supercritical fluid
technology”
REQUIMTE (REQINT)
Coordinator: Rui A. S. Lapa
“Bioassay-Guided isolation and identification of anti-inflammatory drugs and antioxidant compounds from a
medical exctract of PEDILANTHUS TITHYMALOIDS “
REQUIMTE (REQBIO)
Coordinator: Marcela Alves Segundo
“Evaluation of in vitro antioxidant activity of non.steroidal anti-inflammatory drugs (NSAIDS)”
REQUIMTE (REQEVA)
“Avaliação da actividade anti-oxidante de anti-inflamatórios não esteroides em neutrófilos humanos”
Reitoria da UP/Caixa Geral de Depósitos (REIT-EF)
“Contribuição para o estudo do impacto no organismo humano do aumento dos níveis ambientais de
elementos do grupo da platina (PGEs) através da análise post mortem de tecidos”
Reitoria da UP/Caixa Geral de Depósitos (REIT-AA)
Coordinator: Agostinho Almeida
“O envolvimento dos elementos vestigiais no processo aterosclerótico: evidencias da análise post mortem
de vasos lesados”
Reitoria da UP/Caixa Geral de Depósitos (REIT-TSAA)
Coordinator: Agostinho Almeida
Projecto Agro 800
“Rede Nacional para a Conservação e Utilização de Plantas Aromáticas e Medicinais”
Projecto Agro 482
“Protecção contra pragas do olival numa óptica de defesa do ambiente e do consumidor”
Universidade do Porto - Programa Investigação Científica na pré-graduação
“Ácidos Gordos Trans em Bolachas: Contribuição para a Ingestão Total de Gorduras Trans da População
Portuguesa e Consequente Risco Cardiovascular”
Susana Isabel Pereira Casal Vicente (FFUP)
Simone Morais e C. Delerue-Matos (ISEP)
Instituto de Biologia Molecular e Celular (IBMC/UP)
“Extraction of added value compounds from orange peel wastes: application to mixed biodegradable films”
M. P. Gonçalves.
A. M. Sereno.
L. Hilliou
“Chiral chelators for industrial and domestic applications: synthetic, complexation and biodegradation
studies”
REQCHI, 2004
Helena Soares
“Uso integrado de uma abordagem química e biológica na remoção de metais pesados de efluentes
industriais”
Universidade do Porto/Caixa Geral de Depósitos, 2005
Helena Soares
“Síntese e avaliação neurotóxica da metilenedioxiamfetamina (MDMA; ecstasy) e seus metabolitos em
neurónios corticais de rato”
Finnanced by REQUIMTE
Coordinator: Félix Dias Carvalho.
Química Verde
A - 66
“Development of sustainable chemical catalytic processes by integration of nanofiltration and nonconventional solvents”
financiado pelo REQUIMTE, Laboratório Associado em Química Verde, Tecnologias e Processos Limpos.
Janeiro de 2005 a Dezembro de 2006.
“Imobilização de Complexos Metálicos em Materiais de Carbono Nanoestruturados para Aplicação em
Catálise Enantioselectiva”
financiado pela Reitoria da Universidade do Porto e Caixa Geral de Depósitos, projecto de Investigação
Científica na Pré-graduação. Outubro de 2005 a Setembro de 2006.
“Metal ion uptake in sensing films’, Experiment Proposal SRS (EXAFS)”
Cristina Freire (REQUIMTE/Departamento de Química, Faculdade de Ciências, Universidade do Porto,
Portugal), A. R. Hillman (Department of Chemsitry, University of Leicester, Reino Unido);
Ref 43238, Outubro de 2005 a Março de 2006.
“Metal ion uptake in sensing films’, Experiment Proposal SRS (EXAFS)”
Cristina Freire (REQUIMTE/Departamento de Química, Faculdade de Ciências, Universidade do Porto,
Portugal), A. R. Hillman (Department of Chemsitry, University of Leicester, Reino Unido);
Ref 57406, Outubro de 2006 a Março de 2007.
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 “Ciência Viva”
CV VI - ID 821
“Cientistas de Palmo e Meio” (Young Scientists – Science in Kindergarten and Primary School)
Coordinator: P. Mata
CV VI - ID 1122
“Da Cozinha para a Escola: Um Cheirinho de Ciência” (From the Kitchen to School: A Taste of Science)”
Coordinator: M. C. Loureiro Dias (ISA), Partner: P. Mata
CV VI - ID 1293
“Cit- Júnior” (Experimental Science in Primary School)
Coordinator : Dina Teresa Rodrigues Formigo Guimarães (Externato Champagnat), Partner: P. Mata
CV VI - ID 1639
“Espaço de Dinamização de Ciência” (Promoting Science in School)
Coordinator: Maria Luisa dos Anjos Lima (Agrupamento de Escolas “O Rouxinol”), Partner: P. Mata
Química Verde

report 2006

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March 9 , 2015 TO WHOM IT MAY CONCERN This letter is to

Abstract

ICEx Prof. Dr. José Danilo Ayala Sala 254

Annual Report 2003

Bulletin July 12

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Juramento de Maimônides

report 2004

report 2005