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 www.parsuk.pt | registo.parsuk@gmail.com 2 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. www.parsuk.pt | registo.parsuk@gmail.com 3 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 www.parsuk.pt | registo.parsuk@gmail.com 4 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: • • • • • • • • • 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. www.parsuk.pt | registo.parsuk@gmail.com 5 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: • • • • • • 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. www.parsuk.pt | registo.parsuk@gmail.com 6 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 www.parsuk.pt | registo.parsuk@gmail.com 7 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. www.parsuk.pt | registo.parsuk@gmail.com 8 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. • • 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 www.parsuk.pt | registo.parsuk@gmail.com 9 • 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. www.parsuk.pt | registo.parsuk@gmail.com
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