PARSUK Xperience 2015 – Projects

 PARSUK Xperience 2015 – Projects Contents Project 1: Accurately detect, quantify and treat early infections during acute liver failure and sepsis. .. 2 Project 2: Measurement of the reproducibility of ACTH, cortisol, DHEA and testosterone to a short
duration (30 min), high-intensity running bout in order to develop a short physical test to aid in the
diagnosis of overreaching. ..................................................................................................................... 3 Project 3: Development of extraction methods for metabolomic analysis of east cultures and
supernatants. ......................................................................................................................................... 5 Project 4: Bioinspired nanostructures for enzyme immobilisation. ........................................................ 6 Project 5: A comparative analysis of the role of “Empire” in British and Portuguese conflicts with their
American Colonies (1776 and 1822). .................................................................................................... 8 PARSUK Xperience has the institutional support of the Embassy of Portugal in London
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Project 1: Accurately detect, quantify and treat early infections during acute liver failure and sepsis. Supervisor
Institution
Field of Research
Available Period
Christophe Ruis Espirito Santo (Research associate – Post Doc)
University College of London - Institute for Liver and Digestive Health
Hepatology, Sepsis and Antimicrobial
1 July to 16 August.
Abstract
Acute liver failure often occurs where patients with no pre-existing liver disease
suffer a severe liver injury that quickly evolves into a complex multisystemic lifethreatening critical illness. After hepatic dysfunction, coagulopathy and
encephalopathy develop very rapidly leading to multiple organ failure and death in
up to half the cases. The liver plays a vital role in preventing infections, mainly from
bacteria that are able to translocate from the bowel. Kupffer cells (liver
macrophages) are responsible for bacterial defence by phagocytosis and recruiting
peripheral immune cells. If the liver is damaged this ability to fight infections is
impaired, which can be the starting point for a whole body infection. Liver impaired
patients are in a life-threatening situation, which is greatly enhanced by an infection
that might jeopardize their chance to get a transplant. Innovative ways to detect
early infections and control whole body infections are required. In this project, we
aim to develop techniques to detect early blood colonization and keep the
contamination to a minimum using an in-house designed antimicrobial therapy.
This will help doctors to make early decisions, distinguishing between viral and
bacterial infections, implementing the correct antibiotics at the right time, and also
reduce the mortality due to infections during septicemia.
Goals
We expect the student to have a steep learning curve, first acquiring knowledge in
basic microbiology techniques. This will be the groundwork of the project. Bacteria
in blood will be detected using molecular biology techniques (DNA and RNA
extraction, and quantitative real-time PCR). Simultaneously, the student will be
integrated into the new antimicrobial therapy team. Here, the student will learn a
technique used to effectively eradicate bacteria whilst identifying potential risks for
Human safety. This technique reduces bacterial contamination in plasma whilst
preserving key plasma functional constituents. Subsequently, proteomic techniques
may be employed to assess toxicity towards plasma components.
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Project 2: Measurement of the reproducibility of ACTH, cortisol, DHEA and testosterone to a short duration (30 min), high-­‐intensity running bout in order to develop a short physical test to aid in the diagnosis of overreaching. Supervisor
Institution
Field of Research
Available Period
Abstract
Diogo Luis Leal (PhD student)
Institute of Sport and Exercise Sciences (ISPAR), University of Bedfordshire
Exercise Physiology
6 July - 7 August and 17 August - 20 September
Individuals in high-demand occupations (e.g. athletes) routinely push to the limits of
their physical abilities which may lead to chronic illness if insufficient recover
occurs. Successful training programs involve progressively overloading the body in
conjunction with an adequate recovery (Jeukendrup et al., 1992; Meeusen et al.,
2010 & 2013; Hough et al., 2011). During a period of intensified training athletes
may enter into a state of functional overreaching (FOR). Here a
‘supercompensatory’ effect is expected with the athlete experiencing an
improvement in physical performance following appropriate recovery (within a
week). If FOR is not recognized and intensified training continues, the athletes can
move into a state of Non-Functional Overreaching (NFOR), leading to a stagnation
or reduction in performance, which may not resume for several weeks or even
months. Athletes in a state of NFOR are vulnerable to develop the overtraining
syndrome (OTS). The impact of the OTS on an individual is catastrophic (for
example gastro-intestinal instability, excessive weight loss, insomnia, mood
disturbances and depression) with recovery from the OTS taking several years to
no recovery at all. This being said both overreaching (both FOR and NFOR) and
the OTS are difficult to diagnose and are often diagnosed retrospectively. With
such an impact on individual’s health and wellbeing there is an urgent need to
understand the mechanisms that may contribute to overreaching and the OTS and
to uncover reliable markers of FOR/NFOR to aid in the reduction of the incidence of
the OTS.
Resting endocrine markers of FOR/NFOR have been suggested (e.g. Mujika et al.,
1996; Robson et al., 2007), however, there are inconsistent research findings in
this area with increases, decreases and no changes being reported in resting
endocrine markers following chronic stress (intensification of training) exposure. It
has been suggested that exercise-induced responses of endocrine stress markers
may be more reliable than resting measures to diagnose FOR/NFOR with reports
of with reports of blunted cortisol and testosterone responses to an acute cycling
exercise session following intensified training (Hough et al., 2013). As yet a test
does not exist to identifying individuals at risk of suffering from OTS or track
individuals involved in periods of intensified training that utilises a running exercise
bout. This would be useful for athletes that would prefer a running exercise bout.
The originality and innovation of this project is the development of a suitable shortterm high-intensity running exercise bout that could highlight NFOR/FOR in an
attempt to prevent and avoid the occurrence of OTS. Hough et al. (2011) reported
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reproducible endocrine responses (cortisol and testosterone) to an acute bout of
cycling exercise. Further to this Hough et al. (2013 & 2015) provided first evidence
of blunted exercise-induced cortisol and testosterone responses, in individuals
following a period of intensified training. There is now a requirement to support
these findings with a running exercise bout and also to examine the precursor
hormones to cortisol (ACTH) and testosterone (DHEA) in order to provide a pattern
of the endocrine pathway that leads to the dysfunction of these steroid hormones.
Goals
After the four-week experience working in a state-of-the-art laboratory the student
will be provided with fundamental laboratory skills both in an exercise science lab
and a wet lab setting. During their time at the University of Bedfordshire the student
will have the opportunity to:
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collect and handle saliva samples;
prepare blood samples for storage and analysis;
interact with the participants in this study;
be shadowed by the PhD running this study;
learn about the importance of sample/equipment contamination/calibration
and how to avoid/confirm this;
be taught about the principles of enzyme-linked immunosorbent assays
(ELISA);
experience the use of ELISA kits to analyse plasma and salivary steroid
and peptide hormone concentrations, learning about the techniques
needed (e.g. centrifuging, sample mixing, preparing buffers, pipetting etc.);
learn about the processes and pathways related to some steroid hormones
and peptide hormones and understand their diurnal variation;
be involved in a pertinent study focusing on topics such as exercise
immunology, exercise-induced hormone responses, and/or the
Overtraining Syndrome.
The student will be part of an experienced research team working on a way to
prevent the incidence of the OTS.
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Project 3: Development of extraction methods for metabolomic analysis of east cultures and supernatants. Supervisor
Institution
Field of Research
Available Period
Alexandre Manuel Marinho Foito (Plant Biochemist – Post Doc)
The James Hutton Institute
Plant Biochemistry/Analytical Chemistry
20 July - 20 September
Abstract
Yeast are important organisms involved in many industrial processes such as
industrial-scale fermentations which are widely used in the production of alcoholic
beverages and the leavening of bread. As they are ubiquitous in a wide number of
staple foods and beverages some of these yeasts are generally regarded as safe
which has allowed these organisms to become suitable platforms for the production
of high-value chemicals. Particularly with the increasing progress in the field of
synthetic biology yeasts can now be routinely engineered with pathways present in
organisms for the production of specific natural products. To cope with this
development in synthetic biology it is necessary to develop appropriate analytical
chemistry methods to quench, and extract a range of metabolites both at the intraand extra-cellular level. There are a range of methods currently published in the
literature so the goal of this project is to test different methods for the quenching,
extraction and recovery of primary (sugars, organic acids, amino acids among
others) and secondary metabolites (with a focus on polyphenols)present in
fermenting yeast and its respective supernatant utilising some state-of-the art
analytical tools (GC-MS and LC-MS). It is expected that the student will generate
an SOP which will be applied in the future in a large number of samples generated
from a current EU project (BacHberry).
Goals
The student will test different extraction and quenching processes during the fourweek placement. It is expected that within the placement period the student will
learn how to grow yeasts efficiently and will also gain significantly expertise in the
extraction of samples for MS based analysis and the ultimate goal of the student is
to contribute towards the development of a standardized procedure for the analysis
for yeasts. The more specific goals can be divided as follow:
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Establish optimal growth conditions for yeast strains tested;
Development of growth curves;
Testing of different metabolite quenching protocols;
Testing of different separation techniques for cell and supernatants;
Testing of recovery rates of primary and secondary metabolites with the
use of different extraction solvents;
In summary, with this placement the student will receive extensive training
in extraction procedures for the metabolomics analysis of yeast.
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Project 4: Bioinspired nanostructures for enzyme immobilisation. Supervisor
Institution
Field of Research
Available Period
Abstract
Ana Maria Leal Sousa (PhD Student)
University of Strathclyde
Nanoporous materials, Surface Modification/characterization, Enzyme
Immobilisation
27 July – 30 September. The project can be extended for more than 4 weeks.
This project aims to develop bioinspired, synthetic nanoporous structures that can
control biomacromolecular transport through nanopores, and hence enable a novel
strategy for enzyme encapsulation and efficient biocatalysis.
Enzymes are highly efficient, environmentally benign biocatalysts for a range of
chemical reactions. Nevertheless, enzymes are costly and they need to be
recovered after a reaction for reuse in large-scale processes. Immobilisation of
enzymes on solid material supports simplifies recovery because the proteins are
separated from the reactants in solution. However, common chemical methods of
immobilisation are harsh and lead to damaged enzymes and lowered activities. In
addition, molecular diffusion through the convoluted pores of conventional supports
such as nanoporous silica is inefficient, which leads to reduced enzyme
immobilisation and activity. A new approach to enzyme immobilization that
maintains enzyme activity and enables convenient reuse and recovery is urgently
needed. Anodic aluminium oxide (AAO) membranes can be used as a solid
support for enzyme immobilisation due to their morphological properties—the
straight, close-packed cylindrical pores through which chemical solutions can
diffuse or flow at a high flux. This nanostructure can be easily functionalised with
different chemical compounds in order to immobilise enzymes.
Polyphenol molecules have recently been proposed for forming chemically reactive
coatings on diverse material surfaces. These coatings can act as “green chemistry”
immobilisation agents under mild, aqueous conditions. In this project, we
investigate the use of polyphenol coatings for the functionalization of AAO
membranes with enzymes and other chemical compounds for improving the
efficiency of biocatalysis.
The obtained results showed that polyphenol molecules (tannic acid and pyrogallol)
coated the AAO membranes very easily – mixture of polyphenol in buffer with AAO
for one hour. The pH, concentrations of polyphenol and salt were tested to study
the influence of these parameters during the coating, so that we can control the
amount of polyphenol molecules on AAO surface.
Phosphatase and chymotrypsin were immobilised on AAO surfaces and their
activity and stability were also tested. The enzyme activity varied with the different
enzyme immobilisation method and the enzymes were stable for more than five
consecutive enzymatic reactions.
The amount of immobilised protein and the conditions during the enzyme
immobilisation method need to be improved. More conditions and a variety of
enzymes will be tested in this system in order to increase the range of applications
and to study how sensitive this system can be.
Further investigations in this project also include: i) the application of this strategy
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in a microfluidic where the enzyme is immobilised on anodic alumina oxide
membranes and a substrate solution pass through the membrane, and ii) prototype
system construction, using AAO membrane as an ultrafiltration membrane.
Goals
This is an interdisciplinary project spanning the fields of nanomaterials, physical
chemistry, and enzyme biotechnology, and the student will learn and work with all
these domains. Specific research training will focus on:
• Functionalization of alumina oxide surfaces;
• Preparation of buffer, protein and chemical solutions;
• Enzyme immobilisation using different approaches (one- and two-step
procedures);
• UV-vis absorption to characterize enzyme, substrate and product
concentrations;
• Analyse and discuss data related with the enzymatic reactions;
• Improve the enzyme immobilisation method
• Specific non-research training will focus on:
• Presentation skill: The student will have to present the work during and at
the end of the placement showing the results, conclusions and the future
work.
• Project management: In the first two weeks the student will have
supervision and will know the research aims and objectives. The daily
meetings the student will develop project management skills through
discussion and showing his/her progress and plans.
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Project 5: A comparative analysis of the role of “Empire” in British and Portuguese conflicts with their American Colonies (1776 and 1822). Supervisor
Institution
Field of Research
Available Period
Paulo Henrique de Magalhaes Arruda (PhD Student)
King's College London
History
1-30 September
Abstract
A relevant current scholarship has suggested that diverging concepts of empire
were at the centre of the dispute between Britain and its colonies in North
America(1776), where conflicting discursive strategies sought to inform not only the
relation between the parts, but the very existence of the whole. If both parties
seemed to draw on Edmund Burke's interpretation of empire as an “aggregate of
several states under the rule of a common head”, defining that 'common head'
proved to be an entirely contentious matter. Across the Atlantic, colonists owed
their allegiance to king and crown, but refused to submit to a metropolitan
parliament. If the various uprisings that followed Westminster's increasing attempts
to levy higher taxes shared the motto of “no taxation without representation”, the
emphasis was never on attaining the right to a delegation in London, but rather on
securing the right to self-government and self-determination via representative
assemblies established in the colonies. In Britain, however, parliament maintained
its prerogative to legislate on matters pertaining to the whole of empire, where the
various parts were hierarchically arranged and must necessarily defer to
Westminster. These divergences would ultimately lead to armed conflict and
American Independence from Britain. This passage lends to a very fruitful
comparative analysis of Portugal-Brazil relations in 1822. Just how relevant were
similar discussions, or, put differently, how central was the concept of empire to the
dispute leading to the loss of Portugal's dependencies in South America? This
investigation aims to dialogue primarily with a literature that has highlighted the
significance of the idea of Luso-Brazilian empire, of dom Rodrigo de Sousa
Coutinho and a generation of men of state, particularly Brazilians, who sought to
redefine relations within the Portuguese empire. An elite who would later hold key
positions in the government of the new independent Brazil, these formerly loyal
subjects of the Portuguese crown had held on to the hope of a united empire until
this project was clearly no longer feasible. A more accurate historical interpretation
of such multi-faceted events must account, therefore, for how differing concepts of
empire may have informed increasingly hostile debates on the future of the union
of Portuguese subjects on both sides of the Atlantic. A comparative analysis of
British-American relations has much to add.
Goals
Research activities will comprise a total of four weeks and will be divided into two
key stages.
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Stage 1: the student is expected to undertake a critical review of the
existing historiography on the British-American and Luso-Brazilian cases.
This is to be done especially with an eye to identifying possible
intersections between the two, themes that may facilitate a comparative
analysis.
Stage 2: the student will have the opportunity to conduct archival work and
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explore the various sources that lend to a better understanding of the
analysis of British-American relations. It is key here that the student
complement an appreciation of the literature with relevant archival research
in London. The student will also need to account for a similar appreciation
of corresponding materials in Portuguese archives for future research.
Finally, upon successful completion of the activities described above, the
student will be encouraged to submit a research report or final paper.
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