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1
Sornay, Emily. "Analysis of targeted CYCD7;1 expression in seed development." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/53782/.
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D-type cyclins in plants are represented by seven conserved subgroups and play a major role in controlling cell division. Relatively little is understood of their role during seed development, although their expression pattern has been characterized and ectopic expression of CYCD3;1 has previously been shown to disrupt normal embryo development. Here the consequences of ectopic expression of CYCD7;1 using the early endosperm-specific promoter FWA in developing Arabidopsis seeds were investigated. Ectopic CYCD7;1 expression in the maternal central cell prior to fertilization, and in the endosperm from fertilization until cellularization resulted in seeds up to 45% larger. Seed enlargement was accompanied seed lethality, shown to be due to a defect of development during early and mid stages of seed development. As expected from the maternal specific expression of the imprinted FWA promoter, seed size and lethality was dependent on maternal origin of the transgene. Larger seed size was correlated to mature embryo and seed coat outgrowth, and was due to cell proliferation rather than cell elongation. In particular, embryo development was accelerated during the early stages, suggesting these may be dependent on cell division rate, whereas later stages progressed at the same rate as WT seeds. Seed-targeted CYCD7;1 expression phenocopies (1) the nucleus proliferation in the endosperm prior to fertilization observed in rbr and fis-class mutants and (2) the seed enlargement observed in paternal genome excess interploidy crosses. These suggest that CYCD7;1 may act through the RBR pathway to promote cell proliferation and modify imprinting in the endosperm, thereby influencing the parental genome balance. Mechanistically, CYCD7;1 did not interact directly with CDKA;1 but the interaction was promoted in presence of the inhibitor of CDK, ICK1/KRP1 or ICK2/KRP2 in a yeast-three-hybrid assay. However, loss of either KRP1 or KRP2 in respective mutant backgrounds did not prevent the seed enlargement phenotype.
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Baker, Laurie Louise. "Outfoxing rabies : robust vaccination designs for disease elimination." Thesis, University of Glasgow, 2019. http://theses.gla.ac.uk/41011/.
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Prediction of pathogen dynamics and the design of effective interventions to control and eliminate disease are key goals in epidemiology. While progress has been made towards the elimination of many infectious diseases, only two, smallpox and rinderpest, have been globally eradicated. Mass vaccination can greatly reduce the burden of vaccine-preventable diseases. However, there is relatively little scientific guidance on the optimal duration, frequency and placement of control interventions for achieving elimination. Such insights could greatly inform policy and practice. Rabies is a deadly and terrifying disease that exacts a heavy toll on human lives and national economies, with over 50,000 human deaths each year and many millions more requiring expensive life-saving post-exposure vaccines. Elimination of rabies is feasible through vaccination, and oral rabies vaccination (ORV) campaigns have eliminated fox rabies from Western Europe. However, scientific guidance could improve elimination efforts elsewhere, and is still needed for contingency planning to maintain rabies freedom and for emergency response to incursions. My thesis focuses on two pivotal questions in infectious disease ecology: what are the underlying determinants of disease persistence, and how can vaccination strategies be optimized to eliminate infection? To answer these questions, I analysed a rich and highly resolved spatial dataset of fox rabies cases and ORV efforts over three decades in Germany and neighbouring countries. The long-term, large-scale nature of these data provides a unique opportunity to improve our understanding of wildlife rabies dynamics in response to vaccination using novel spatial modeling techniques. In chapter 2, I create a metapopulation model of regional rabies dynamics that incorporates local transmission (within regions) and spatial coupling (between regions) using a hierarchical Bayesian state-space model. In chapter 3, I extend the model developed in chapter 2 to determine the best vaccination strategy, in terms of scale and duration of ORV efforts for three common epidemiological scenarios: {\bf endemic} circulation of rabies; {\bf high-risk} situations when rabies-free but neighbor endemic areas; and an {\bf endgame} scenario when only a single endemic foci remains. In chapter 4, I develop a space-time model of fox rabies dynamics and explore the effect of scale on estimates of transmission terms by aggregating rabies case data at different spatial resolutions. I then relate these estimates to the scaling of individual interactions to regional dynamics through population mixing. Collectively, the findings from this thesis contribute to our understanding of how infectious diseases persist and can be controlled through vaccination. The methods generated can be used to explore tradeoffs in the scale and duration of ORV efforts, and generate recommendations on the time horizon and investment required to achieve and maintain freedom from disease. The model developed in chapter 4 also presents the first steps to developing a highly resolved spatial model of local rabies dynamics. These findings have immediate application to the design of cordon sanitaires in Europe, and to strategies aiming to rapidly eliminate re-emergence in high-risk countries such as Greece and Turkey. The analytical and statistical framework developed in this thesis is also applicable to answering analogous questions for the elimination of dog-mediated rabies and for other vaccine preventable diseases.
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Lyons, Ashley. "The impacts of contrasting grazing management on biodiversity in upland calcareous grasslands." Thesis, Edge Hill University, 2017. http://repository.edgehill.ac.uk/9934/.
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Calcareous grassland, considered among the most species rich and diverse habitats in Europe, underwent wide scale loss and degradation following post 1950s agricultural intensification. Consequently, they are the focus of conservation efforts and are protected in national and international legislation (e.g. EU Habitats Directive). As elsewhere in Europe, a major cause of upland calcareous grassland loss and degradation in Britain was intensive grazing, typically with sheep. In recent years, conservation organisations have altered grazing practices in an attempt to prevent further loss and degradation by focussing management on conserving characteristic calcareous grassland vegetation. However, the impact of the contrasting grazing regimes used in this internationally important habitat on invertebrates is unknown. This study is the first to investigate the impacts of a range of established grazing regimes (low intensity sheep grazing, low intensity cattle grazing, high intensity sheep grazing and no grazing) on aspects of plant diversity and structural complexity, carabid beetle diversity, and spider diversity in upland calcareous grasslands. It also provides the first evidence based management recommendations for UK upland calcareous grasslands which incorporate both plants and invertebrates. In addition, this study is also the first to assess the biodiversity value of acid grassland and limestone heath habitat patches that occur as 3 part of the calcareous grassland matrix and are not targeted by conservation management, by examining the spider fauna in each habitat in relation to calcareous grassland. Further evidence based recommendations for the management of these non-target habitats are made for the first time.
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West, H. K. "Competing divalent cations in biological systems." Thesis, University of Worcester, 2001. http://eprints.worc.ac.uk/7188/.
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Ngandu, Mpoyi Elie. "Engineering biointerfaces to reveal collagen IV disease mechanisms." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/9032/.
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Basement Membranes (BMs) are specialised extracellular matrix (ECM) structures that underlie all endothelial and epithelial cells, and provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in the BMs major component collagen IV cause eye, kidney and cerebrovascular disease including intracerebral haemorrhaging (ICH). Haemorrhagic stroke accounts for 15% adult stroke and 50% paediatric stroke, and carries the worst prognosis and there are no therapeutic strategies. Mutations in the genes COL4A1/COL4A2 (collagen IV alpha chain 1 and 2) cause BM defects due to mutant protein incorporation in the BM or its absence by ER retention, and ER-stress due to intracellular accumulation of collagen IV. Despite this, the mechanism(s) of collagen IV mutations disease remain poorly characterised. To provide novel insights into mechanisms of collagen diseases, this study investigates the effect of defined engineered biointerfaces on cell behaviour/signalling, collagen secretion in COL4A2 mutant and wild-type cells. Atomic force microscopy and spectroscopy were employed together with confocal and biochemical analyses of cells cultured on engineered synthetic polymers, poly(ethyl acrylate) and poly(methyl acrylate), coated with ECM proteins, namely laminin, collagen IV and fibronectin. This enabled us to address the hypothesis that biomaterials may alter the behaviour of COL4A2+/G702D mutant cells by overcoming some of the defects caused by the mutation and rescuing the downstream effect of the ER stress. Of the ECM proteins that were used, only fibronectin was observed to undergo a drastic structural change depending on the substrate chemistry. On poly(ethyl acrylate), fibronectin was assembled into fibrillary networks upon adsorption, and these nanonetworks induced increased secretion of Col4a2 in COL4A2+/G702D cells than on poly(methyl acrylate) or control glass. The behaviour of the mutant cells appeared to be influenced by the underlying biointerface, increased levels of molecular chaperones and reduced ER area suggested an increased collagen IV folding capacity when the cells were cultured on the FN nanonetworks compared to the other surfaces. COL4A2+/G702D cells interacted with the adsorbed proteins and were able to mechanically translocate them. Enhanced formation of focal adhesions was also seen on FN-coated polymers, where ligand density and actin-myosin contractility accounted for the observed increase in cell adhesion strength. The stiffness of the mutant fibroblasts and of their ECMs was found to be 10 times lower than that of the wild-type cells; interestingly, mutant cells cultured on FN nanonetworks on poly(ethyl acrylate) were able to deposit a protein matrix with significantly higher Young modulus than on glass or poly(methyl acrylate). These findings suggest that biomaterials are able to influence the behaviour of these mutant cells through changes in the interfacial layer of adsorbed proteins presented to them. Collectively, these data provide an understanding of the effect of mutations on cell characteristic and a basis of concept that material may be employed to modulate effects of mutations of collagen/ECM molecules. Understanding the mechanisms through which these surfaces trigger a change in cell response will prove valuable for the development of new therapeutic approaches to address pathologies due to collagen IV mutations. In this respect, further investigation is needed to dissect the signalling pathways involved.
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Etheridge, Elizabeth C. "Aspects of the conservation biology of Coregonus lavaretus in Britain." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/1598/.
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Conservation of phenotypically variable taxa such as the European whitefish (Coregonus lavaretus) can be particularly challenging. In this thesis it is argued that the recent designation of seven native C. lavaretus populations as three endemic species (C. clupeoides, C. stigmaticus and C. pennantii) by Kottelat & Freyhof (2007) are incorrect and cannot be substantiated with the results presented here. However, evidence for important infra-specific variation between populations has been found. Two native Scottish populations of C. lavaretus show considerable variation in morphology, trophic ecology and life history. The variation in these populations warrants protection, one conservation action becoming more commonly utilised in Britain is conservation translocation. It was found that there were significant differences between source and refuge populations in Scotland. The wisdom of using this conservation measure on a phenotypically plastic organism is discussed. Nevertheless the establishment of further refuge populations are considered to be a viable conservation action. Sub-structuring within the largest native Scottish population of C. lavaretus was not found. However, evidence of residence within certain basins of Loch Lomond was found through significant differences in muscle stable isotope signatures. Investigation was also made into the trophic ecology of other fish in Loch Lomond. It was found that brown trout (Salmo trutta) in Loch Lomond have a non-typical migration pattern and invasive ruffe (Gymnocephalus cernuus) now form an important part of the trophic ecology of this site. In Britain several whitefish populations have been invaded by ruffe, a species native to Britain, but not to these sites. An experiment is conducted into the protective ability against ruffe predation on C. lavaretus ova of substrates typical on spawning grounds. It was found that pebbles and gravel form the best spawning substrate. The impact this mortality may have on the life history of Loch Lomond C. lavaretus is discussed. Using information gathered in this study, recommendations for the management of Coregonus spp. are summerised. There is the potential for these recommendations to apply to other phenotypically plastic species that vary between sites such as Arctic charr (Salvelinus alpinus) and brown trout.
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Lamb, Christopher A. "Investigation of GLUT4 sorting into the insulin responsive compartment : a role for ubiquitination and deubiquitination." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2380/.
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GLUT4 is the insulin-regulated glucose transporter found in muscle and adipose tissue. On insulin stimulation, GLUT4 translocates from a slowly recycling storage compartment (GLUT4 storage vesicles or GSVs) to the plasma membrane. This allows glucose to enter cells by diffusion down its concentration gradient, clearing glucose from the plasma. This response is defective in the disease states of insulin resistance and type 2 diabetes. The aim of this study is to understand how GLUT4 enters GSVs, which will hopefully extend our knowledge of insulin responsive tissues. Previous studies from our lab, expressing GLUT4 in yeast, have shown that GLUT4 is subject to the same nitrogen- and ubiquitin-dependent trafficking as the yeast amino acid permease Gap1p. In my thesis I have extended these studies into 3T3-L1 adipocytes, and shown that GLUT4 is ubiquitinated in this insulin responsive cell line. A ubiquitin resistant version of GLUT4 (HA-GLUT4 7K/R) has an impaired ability to enter GSVs and does not translocate in response to insulin. However GLUT4 mutants with single ubiquitination sites outwith the large intracellular loop are ubiquitinated and traffic in an identical manner to wild type GLUT4, addressing concerns that mutation of the large intracellular loop of GLUT4 in HA-GLUT4 7K/R affects its trafficking. The GGA family of clathrin adaptor proteins have previously been implicated in sorting of newly synthesised GLUT4 into GSVs. Our lab has shown previously that the two yeast Ggas are required for ubiquitin dependent trafficking of GLUT4 in yeast, as is the case for Gap1p. I have gone on to show that the ubiquitin binding function of the GGA3 GAT domain is, at least partially, required for an in vitro interaction between GLUT4 and the VHS-GAT domains of GGA3. When expressed in adipocytes, a ubiquitin binding deficient mutant of myc-GGA3 reduces the proportion of GLUT4 loaded into a subcellular fraction enriched in GSVs, suggesting that GLUT4 ubiquitination is one of the signals for GGA dependent sorting into GSVs. As ubiquitination is usually thought of as a signal to direct lysosomal degradation, and only 0.1 % of total GLUT4 is ubiquitinated at any one time, there may be a role for a deubiquitination step in ubiquitin dependent GLUT4 traffic. Work by our collaborator (Nai-Wen Chi, UCSD) has demonstrated that the GSV cargo IRAP and its binding partner tankyrase-1 are required for normal insulin responsive GLUT4 traffic. An interaction between tankyrase and the deubiquitinase (DUB) USP25 has been demonstrated by yeast two hybrid analysis, and this DUB contains a putative tankyrase binding motif. USP25 may therefore be recruited to GSVs by IRAP, with tankyrase acting as a scaffold. I demonstrated that GST-USP25 binds tankyrase-1 from an adipocyte lysate, and that a version of the enzyme with a mutation in the putative tankyrase binding motif (GST-USP25 R1049A) does not. I also used siRNA to deplete USP25 from 3T3-L1 adipocytes, and found that this results in a reduction of GLUT4 levels in these cells. A concomitant reduction in the fold change of insulin-stimulated glucose transport into these cells suggests that GLUT4 is not sequestered in GSVs, but is rather directed to the lysosome. In summary, my data show that ubiquitination of GLUT4 is required for the transporter to be loaded into its insulin responsive compartment (GSVs). I also began to characterise the role of the ubiquitin binding GAT domain of GGA3 and the deubiquitinase USP25 in GLUT4 traffic, opening up two further avenues for research into the insulin regulated trafficking of GLUT4.
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Langan, Mairi A. "A contemporary history of the origins and development of UK Biobank, 1998-2005." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/104/.
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Background: This thesis examines the origins and early development of UK Biobank. This is a resource funded in 2002 by the Medical Research Council, the Wellcome Trust, the Department of Health and the Scottish Executive to gather genetic and lifestyle information from half a million participants aged 4069 years old in the UK and monitor their health for up to thirty years in order to improve the prevention, diagnosis and treatment of major diseases. UK Biobank was set up following the completion of the Human Genome Project in 2001, and was one of many established at around the same time with the goal of translating the knowledge of the human genome sequence into practical benefits for human health. (National genetic databases were also set up or proposed in Iceland, Estonia, Latvia, Sweden, Singapore, Tonga, Spain, and the United States). They, and the Human Genome Project, had raised a number of important issues about access to and ownership of genetic information. Aims: The original aim of my PhD was to examine lay and professional understandings and responses to Biobank in the light of this background. However, UK Biobank took longer than expected to reach the stage of data collection, in part because of negotiations about its organisational structure. The aim therefore changed to address the question of how and why was UK Biobank initially configured in the manner it was. Organisational structure: UK Biobank was originally set up by the funders with a ‘hub’ and ‘spoke’ model, with calls for bids from UK Universities for a central ‘hub’ charged with financial management and overall control of data and samples, and ‘spokes’ who were responsible for recruitment and data collection through primary care. The selection of both was made through the procurement rules of the EU. The hub (Manchester), six regional spokes, and the CEO (from Oxford) were all appointed simultaneously in 2003 and subsequently a Board of Directors and a number of committees were appointed. The CEO resigned in late 2004, and a new CEO and Principal Investigator was appointed in 2005, after which there were significant changes to the organisational structure. Methods: I conducted 76 oral history interviews with academic scientists directly and indirectly involved in UK Biobank, representatives of all four funding bodies, and representatives of UK Biobank Limited (the company set up to manage UK Biobank). I also conducted archival analysis of the MRC’s official documents concerning the origins and development of UK Biobank. Findings: From its beginning UK Biobank was marked by tension between academic scientists on the one hand and representatives of the funding bodies and UK Biobank Limited on the other. Academic scientists criticised the funding bodies for establishing UK Biobank in a way that departed from what I have termed ‘standard academic scientific practice’. Spokes felt they should receive some privileged access to data they would contribute to collecting, and felt that the set up did not recognise the performance indicators driving scientists and universities. Lack of clarity over who was in control of UK Biobank contributed to these tensions as both spokes and funders felt that the other exerted undue influence. Some mistrust developed between academic scientists and representatives of the funding bodies and UK Biobank Limited. Discussion: The configuration of UK Biobank was difficult for academic scientists and representatives of both the funding bodies and UK Biobank alike. Organisational issues, typical of those confronting Big Science initiatives, were largely responsible for this difficult legacy. Issues of leadership, the hub and spoke model, the sequencing of funding decisions, appointment of groups and committees and protocol development, uncertainties about who was in control, and ambiguities within the organisational structure as a whole were the most significant issues in the origins and development of UK Biobank, as the organisational changes in 2005 testify.
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Ferrari, Nicola. "Investigating RUNX transcription factors in mammary gland development and breast cancer." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4790/.
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Breast cancer is the third most common cause of cancer death in the UK, accountable for more than 11000 deaths in 2010 alone (www.cancerresearchuk. org). Developmental pathways commonly required for normal development are often hijacked during tumour progression, so a better understanding of mammary gland development is necessary to fully understand the roots of breast cancer. The Runx gene family are known to be important regulators of development in different lineages. In particular RUNX1 and RUNX2 have been widely studied in the context of haematopoiesis and osteogenesis respectively, but their role in epithelial tissue is much less well understood. In this thesis a role for RUNX1 and RUNX2 in mammary development and breast cancer has been identified. The first part of this study is focused on characterizing the expression and function of the Runx genes in the mammary epithelium. RUNX1 and RUNX2 protein levels fluctuate during embryonic and adult mammary development, and an in vivo conditional knockout strategy shows that both genes are important for maintenance of mammary epithelium homeostasis. Moreover, combined loss of RUNX1 and RUNX2 significantly perturbs the normal mammary architecture with an expansion of the basal population in vivo and the appearance of preneoplastic lesions in aged mammary glands. An exciting new role for RUNX2 in mammary stem cells has also been revealed showing that RUNX2 is important for the regenerative potential of mammary epithelial cells in vitro. Evidence is also presented to indicate that RUNX2 could be linked to regulation of quiescence and Wnt signalling in the stem cell compartment and during transformation. Finally, the role of these genes in breast cancer is discussed demonstrating involvement of RUNX1 and RUNX2 specifically in the triple negative (ER-PR-HER2-) subtype. In particular, for the first time, RUNX1 is revealed as an independent prognostic indicator correlating with poor prognosis in triple negative tumours. Meanwhile, evidence from various mouse models demonstrates that RUNX2 may be specifically involved in the squamous metaplastic form of this disease.
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Khosravi, Mardakheh Faraz. "Regulation of FGF Receptor signalling by SPROUTY and SPRED." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/730/.
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Signalling from Fibroblast Growth Factor Receptors (FGFRs) is under tight control by a wide variety of extrinsic and intrinsic regulatory mechanisms, many of which remain poorly defined. Sproutys and their related Spred proteins constitute two such families of signalling regulators with multiple developmental roles as well as potential tumour suppressive functions. However, the molecular mechanisms of these proteins have remained unclear and subject to many unresolved controversies. Using a mass spectrometry approach, several novel interacting partners of Sprouty with roles in endocytosis are identified here, suggestive of a potential endocytic-related function for Sproutys. In addition, comprehensive analysis of Sprouty phosphorylations and their dynamics by mass spectrometry reveal previously unknown but potentially crucial sites that might regulate Sproutys function. Next, a novel late-endosomal protein that directly binds to Spred is identified using a different approach. Neighbor of BRCA1-1 (NBR1) is shown to be necessary for attenuation of FGF signaling by Spred, and this is demonstrated to be via modulation of the trafficking itinerary of FGFRs. Finally, NBR1 is established as a novel regulator of RTK trafficking and signalling, and the interplay between its various regions for protein localisation and function is revealed.
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Machado, de Oliveira Gisela Sofia Mendes. "Modulation of calcium signalling in human sperm by nitric oxide." Thesis, University of Birmingham, 2008. http://etheses.bham.ac.uk//id/eprint/197/.
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Nitric oxide (NO) generation by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilization. In vitro studies were undertaken to assess the ability of human sperm and cumulus cells (surrounding the oocyte) to generate NO, investigate the mechanism of action of NO, the NO-mediated [Ca\(^{2+}\)]\(_i\) signalling pathways, the possible interaction of NO with progesterone (a product of cumulus) and its impact in the regulation of human sperm functions. Immunofluorescent staining revealed constitutive NOS in human cumulus. DAF-FM diacetate staining demonstrated NO production by cumulus cells. Human sperm exposure to NO donors caused mobilization of stored Ca\(^{2+}\) by a mechanism not requiring guanylate cyclase activation but mimicked by S-nitrosoglutathione (GSNO; an S-nitrosylating agent). Dithiothreitol application, to reduce protein –SNO groups, rapidly reversed the actions of NO and GSNO on [Ca\(^{2+}\)]\(_i\). The effects of NO, GSNO and dithiothreitol on protein S-nitrosylation, assessed using the biotin-switch assay, closely paralleled their actions on [Ca\(^{2+}\)]\(_i\). Progesterone mobilizes stored Ca\(^{2+}\) in human sperm, by a mechanism involving ryanodine receptor (RyR) activation. Pre-treatment with NO reduced the amplitude of the Ca\(^{2+}\) response to ryanodine (a RyR agonist), suggesting convergence of the actions of NO and ryanodine. Sperm pre-treatment with NO greatly enhanced the progesterone effect on [Ca\(^{2+}\)]\(_i\), causing a prolonged increase in flagellar excursion. We conclude that NO regulates mobilization of stored Ca\(^{2+}\) in human sperm by protein (possibly RyRs) S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilization.
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Zheng, Wei. "The LAR protein tyrosine phosphatase enables PDGF β-receptor activation and signal transduction." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/4364/.
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Many cellular activities including cell survival, proliferation, migration and differentiation are controlled by growth factors and their corresponding tyrosine kinases receptor (RTKs). Growth factor receptor activation is strictly regulated by protein tyrosine phosphatases (PTPs). Here I investigated whether the receptor protein tyrosine phosphatase (RPTP) LAR, which is known to modify the activity of several RTKs, also regulates platelet derived growth factor (PDGF) receptor activity and signalling. Mouse embryonic fibroblasts (MEFs) expressing mutant LAR lacking its phosphatase domains (LARΔP) showed reduced phosphorylation of PDGFβ receptor (PDGFβR) compared with wild type (WT) cells. This was rescued by re-expression of WT LAR. The decreased phosphorylation of the PDGFβR was independent of ligand concentration and occurred on all tyrosine residues, suggesting that LAR is required for full PDGFβR kinase activation. The decreased kinase activity reduced the amplitude or duration of the different signalling pathways activated downstream of the PDGFβR, and resulted in reduced proliferation in response to PDGF-BB. These findings demonstrate, for the first time, that LAR activity is required for PDGF-induced fibroblast proliferation. The inhibition of PDGFβR kinase activity in LARΔP cells was exerted via increased basal activity of the tyrosine kinase c-Abl and its substrate protein kinase Cδ (PKCδ). Ligand-induced PDGFβR dimerization is defective in LARΔP cells, possibly due to the observed increase in the Nherf2 protein associating with the PDGFβR. In summary, I have identified LAR as a new regulator of PDGFβR activity, and propose a novel mechanism where PDGF-induced activation of c-Abl serves as a negative feedback loop to terminate the PDGFβR kinase activity. This may occur via PKCδ activation promoting the association of Nherf2 with PDGFβR, thereby reducing ligand-induced receptor dimerization and kinase activation. In this model, LAR promotes PDGFβR activity by inactivating c-Abl.
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Chen, Jing. "Development of novel theory and methods for QTL analysis and inferring crossover interference in autotetraploids." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6703/.
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Polyploidization widely occurs in the evolutionary history of eukaryotes, especially for flowering plants. Exploring the evolutionary and agricultural important role by polyploidy is mystery and challenging job. In the first chapter of this thesis, I developed a quantitative genetics model based on orthogonal contrast scales, which would provide theoretical basis for any further bi-allelic QTL analysis in autotetraploid species. In the next chapter, I developed an interval mapping method for QTL mapping in outbred population for autotetraploid, considering both bivalent and quadrivalent pairing during meiosis. Extensive simulation work has been implemented to demonstrate the reliability of this method. This work would provide practical tools for breeding the world’s third most important crop, the cultivated potato. To give some insight into the evolutionary important role played by polyploidization, I developed a statistical method for inferring crossover interference based on three-locus analysis in autotetraploids in the third chapter and a method to predict genome-wide crossover rate for autotetraploid yeast in the final chapter.
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Williams, Susannah Jane. "Development and appraisal of MRI contrast agents for the in vivo analysis of stem cell grafts." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/43157/.
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The work reported in this thesis deals with the need for efficient in vivo tracking and monitoring of grafted cells and their subsequent survival. It is considered in the context of cell transplantation for neurodegenerative disorders, particularly Huntington’s and Parkinson’s disease, although the scope for a good contrast agent to monitor cells in vivo goes far beyond this. There is currently no routine method used to follow cells in vivo and it is crucial for the advancement of cell transplantation studies, both in terms of providing powerful longitudinal analysis and for decreasing the number of animals necessary per experiment. MRI allows good contrast resolution and provides details on soft tissue anatomy without being harmful to the subject. By labeling cells with an MRI contrast agent the labeled cells can be distinguished from the surroundings and information on location is attained. A good MRI contrast agent can provide more information than this though, but to date there hasn’t been an MRI contrast agent developed that can simultaneously provide good signal and be reflective of the graft changes, while not affecting the cell’s viability. The feasibility for in vivo MRI scanning of three MRI contrast agents were tested and detailed below. In Chapter 3 we looked to utilise SPIOs as contrast agents. Since SPIOs are the most widely used of contrast agents they were tested in mouse ES cells, expanded whole ganglionic eminence and rat ventral mesencephalon and successfully labeled all cell types. Problems were discovered in reference to the needle track leaving an MRI visible track that eclipsed the area of graft deposition, and while SPIOs did not hamper graft survival, only large grafts extending out from the needle track could be reliably measured. Chapter 4 examined the generation of ferritin constructs for use as contrast agents. Transgenes based on the ferritin subunits provide MRI contrast by increasing the iron content of a cell. Both subunits, heavy and light, were transfected into mouse ES cells and expressed to improve signal compared to overexpressing the ferritin heavy transgene alone, which has been done in the literature. The expected change in T2 relaxation compared to control cell lines was observed in vitro. In Chapter 5 the applicability for in vivo use of Chemical Exchange Saturation Transfer (CEST) was tested. CEST involves the selective saturation of protons of particular compounds that are then indirectly detected through the water signal. Current 2D RARE scans to pick up CEST are slow and not really transferable to animal studies, due to the large increase in scan time for each extra image slice required, here alternative 3D FLASH scans were developed that still allowed the CEST contrast change to be observed over a whole sample in a reasonable time frame. A transgene based on CEST was tested in vivo and expression was lost even under antibiotic selection. Work in this thesis contributes some understanding towards the promises and pitfalls of three MRI contrast agents, two of which may ultimately be used more routinely for cell tracking in the future.
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Gao, Xiao. "The role of oxidative stress and chitinase like proteins in Epstein-Barr virus latent membrane protein 1 induced chronic inflammation and carcinogenesis." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/7855/.
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Latent membrane protein 1 (LMP1) is regarded as the primary Epstein-Barr virus (EBV) oncogene, and is expressed in several EBV associated malignancies, including BL, HD and NPC. In order to understand the role of LMP1 in EBV associated epithelial malignancies, our lab generated transgenic mice expressing LMP1 in the epidermis. This mice display an age progressive inflammatory phenotype in the skin, progress to carcinogenesis. In order to investigate the rold of reactive oxygen species (ROS) in the phenotype, mice were treated with the antioxidant N-acetyl-L-cysteine (NAC). The treatment inhibited the inflammatory phenotype and this was quantified using an in vivo imaging system and flow cytometry. However NAC treatment did not appear to impact the activation of redox sensitive pathways including NF-κB, ERK and STAT3. In order to explore the role of ROS, inflammation and NAC treatment in carcinogenesis, mice underwent chemical carcinogen (CC) treatment. NAC treatment decreased the lesion load on transgenic mice, however, their concurrent decrease in body weight during CC treatment has complicated the interpretation of this finding. NAC treatment did not impact CC induced γH2AX detection or the growth of established carcinoma tumour cells in vivo. In the LMP1-expressing skin tissue, chitinase like proteins (Chil1, Chil3 and Chil4) are highly induced. Several assays were conducted to investigate the role and action of the mouse Chils, initiating with the purification of these three proteins. A glycan array experiment revealed strongest binding of mChil1 to maltohexaose, and Chil3 and Chil4 to acarbose. In addition mChil1/3/4 stimulated ERK1/2 signaling in serum starved 293 cells which suggest the Chils may have mitogenic activity. mChil1/3/4 induced several cytokines in vivo, including decorin, MIP-1α, E-cadherin and MIP-1γ which suggest that the Chils may be pro-inflammatory factors. In a preliminary experiment, mChil1/3/4 did not show any adjuvant effect, however this requires further study. Finally, optimal RNAi oligos were determined in culture. Using RNAi to silence mChil1/3/4 expression in vivo was attempted by testing different siRNA delivery routes, RNA modifications and carriers. It was found that using osmotic minipumps along with modified siRNA, achieved a partial inhibition of mChil1 expression, however no silencing effect was detected for Chil3 and Chil4. There data will allow a more informed approach to be designed to achieve silencing of Chil1, Chil3 and Chil4 in vivo.
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Cotton-Barratt, Rebecca. "Modelling biological form in evolution." Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/70973/.
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How are processes working at the individual level, the species level and the macro-ecological level connected? This thesis explores the theoretical and structural constraints on biological evolution. It does this by developing an evolutionary program to model biological form. This development was necessary as the existing models of evolution are poorly suited to modelling morphological constraint. The model of biological form developed in this thesis uses graphs to abstractly represent organisms and the relationships of their internal structure. We show that by increasing the number of degrees of freedom, or by increasing the ruggedness of the fitness landscape, higher levels of diversity are supported - particularly when there is strong directional selection. We explore whether meta-regulation is bounded in the model by using an analytical framework. We show that there is no analytical steady state, but that one can be induced in the model by selection effects. We find that a mixed strategy between increasing object complexity and increasing hierarchical complexity maximises the average degree of a vertex. This agrees with the evolutionary history of meta-regulation. We claim that the macro-ecological response to environmental perturbation is determined by both the characteristic time scale of mutation and the time scale of the environmental change. We show that for high amplitude changes the system can adapt provide the mutation time scale is smaller than the environmental change. We also show that low amplitude environmental changes cause rapid turnovers in species' diversity. Finally, we show that mass extinctions can be the result of species' interactions and background rates of extinction, and do not need large external perturbations to occur. This, combined with the results above, suggests that many of the trends seen over geologically long time periods can be explained as a result of the interacting processes at the individual and species level.
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Widdowson, Philip. "Biochemical and biophysical characterisation of Anopheles gambiae NADPH-cytochrome P450 reductase." Thesis, University of Liverpool, 2010. http://livrepository.liverpool.ac.uk/1496/.
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As the principal vector for the transmission of the Plasmodium falciparum parasite, and hence the spread of malaria in Sub-Saharan Africa, Anopheles gambiae is a globally significant species of mosquito. Over recent years, the efficacy of established insecticides has waned and there is a constant need for novel effective compounds. Cytochrome P450 reductase (CPR) is a diflavoprotein known to have a central role in phase I metabolism of xenobiotic compounds and, in mosquitoes, this involves the detoxification of insecticides. Due to an inherent lack of understanding regarding the mechanisms of action of A. gambiae CPR, the selective inhibition of this enzyme is a previously untried approach. This project aims to biochemically characterise A. gambiae CPR in direct comparison to the human enzyme. It was found that A. gambiae CPR was deficient in bound FMN, and to a lesser extent FAD, relative to human CPR with 20 % less FMN bound to the purified mosquito protein. Following the dissection of A. gambiae CPR into its constituent FMN- and FAD-binding domains, and using Isothermal Titration Calorimetry (ITC), a 4-fold decreased was observed for the binding affinity for FMN in the A. gambiae FMN-binding domain as compared to the equivalent human protein. The redox potential of the oxidised/semiquinone transition of the A. gambiae FMN-binding domain was -92 mV, much more negative than the published value for human FMN-binding domain. These data suggest a clear difference between these enzymes in the binding strength of FMN and its propensity to accept electrons. The binding characteristics of NADPH nucleotides were probed in some detail. Comparison of the binding of NAD+ and NADP+ revealed a strong bias for the phosphate containing NADP+. In addition, the position of the phosphate was important as 3’-AMP bound very poorly whilst 2’-AMP bound more strongly. 2’, 5’-ADP binding highlighted the importance of additional stabilising interactions involving the 5’-phosphate. Comparison of 2’, 5’-ADP and NADP+ binding confirmed that the 2’-phosphate interaction was the principal site for NADPH recognition and provided the majority of the binding energy for this interaction. A. gambiae CPR was shown to bind NADPH nucleotide analogues 2’-AMP, 2’, 5’-ADP and NADP+ much less strongly than the human enzyme highlighting a potentially significant difference in coenzyme binding. Binding affinities for the nucleotide ligand to intact CPR and the isolated FAD domain showed that the FAD-binding site is fully contained within the FAD-binding domain However, differences in the thermodynamic parameters between the intact enzyme and the isolated FAD-binding domain suggest that, although not directly involved in NADPH binding, the presence of the FMN binding domain had an effect on the overall binding energetics. Despite an apparent difference between A. gambiae and human CPR in flavin incorporation and NADPH binding affinity, it was interesting that the activity of cytochrome c reduction of both enzymes was similar. The measured Km with respect to NADPH corroborated the ITC data by suggesting a stronger interaction of the coenzyme with human CPR compared to A. gambiae CPR. There was an approximate 2-fold increase in potassium ferricyanide reduction with the isolated A. gambiae FAD-binding domain compared to the intact enzyme with the presence of the FMN-binding domain again seemingly imparting an effect of events involving the FAD-binding domain. In order to fully understand and rationalise all of the data, a comprehensive structural determination of A. gambiae CPR is required. With this in mind, isotopic labelling and subsequent biophysical analysis was carried out on the intact CPR and its FMN- and FAD-binding domains. Successful labelling was achieved for all samples, including the deuteration of the intact CPR and FAD-binding domain However, the greatest success involved the FMN-binding domain with sufficient triple resonance spectra collected for backbone assignments. Although this success could not be matched for the intact CPR and FAD-binding domain, the work has provided a solid base for more a comprehensive study in the future.
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Cesar, Christopher. "The roles of the cockle Cerastoderma edule L. on ecosystem functioning : cockle comings and goings." Thesis, University of Liverpool, 2009. http://livrepository.liverpool.ac.uk/1294/.
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There is increasing interest regarding the impacts of human activities on the functioning of marine systems. A primary driver of change to marine systems is through the impacts of fishing. Biomass-dominant target species have the potential to mediate a number of ecosystem functions, either directly or indirectly, through the influences that taxa have on ecological processes and/or other biotic or abiotic components of the system. This thesis investigates the roles of the cockle, Cerastoderma edule on ecosystem functioning within intertidal sedimentary systems. A series of investigations revealed that cockles have the potential to mediate benthic primary productivity through their roles in the recycling of nutrients and effects of sediment structure and have impacts upon assemblage biomass and functional diversity. However, the roles of cockles on other aspects of ecological functioning were less apparent. An investigation also assessed the suitability of the use of assigning taxa to functional groups when assessing functional diversity. Taxa were shown to have the potential to change their feeding activity following disturbance, with evidence suggesting a change to benthic-pelagic coupling. This change however, would not have been observed with investigations of functional traits alone and thus supports the use of direct measures of functions to support functional trait diversity measures. It is imperative for ecological investigations to consider long-term changes to population dynamics. However, particularly in marine systems, such data are generally lacking. This thesis presents a novel approach, using information from sea fisheries reports to gain a semi-quantitative 30-year data set on cockle landings within Morecambe Bay, north-west England. This technique revealed evidence of temporal changes to the functional processes at a large scale. This thesis provides evidence for cockles acting as key contributors to ecological functioning. The functional role of cockles is tempered by the high degree of redundancy within these assemblages, highlighting a number of issues relating to the use of field studies and encouraging a move towards increased use of traditional ecology in future studies.
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Miller, Angela. "Peptide based hydrogels in the study of mesenchymal stem cells for the purposes of regenerative medicine." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6880/.
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Regenerative medicine is a vastly expanding subject area, with a number of different strategies and substrates being studied to ultimately create a model for repairing diseased and injured tissue. Stem cells are a promising cell type in this field as they are known to differentiate into a number of cell types and contribute to normal cell repair. However, despite their potential as a useful cell choice in the field of regenerative medicine, stem cell based therapies have limited potential due to their ability to form tumours when implanted in the human body, problems arising with immunogenicity and the difficulty in obtaining adequate cell numbers for transplantation. To overcome these problems, many research groups are interested in using biomimetic substrates and scaffolds to mimic the architecture, chemical composition and stiffness properties of the in vivo cell niche and various human tissues, but in an in vitro setting. By doing so, the MSC behaviour can be studied and the differential lineages examined allowing the desired substrate to be tuned to obtain the desired cellular outcome. In this work, hydrogels composed of Fmoc diphenylalanine and Fmoc Serine have been characterised and used to study changes in Mesenchymal stem cell (MSC) responses in a two and three dimensional state. The results obtained from this work demonstrate that such hydrogels support MSC growth and produce a mixed phenotype population which differentiates over time. A further hydrogel paired with collagen has shown promise in promoting MSC differentiation down the osteogenic lineage and has potential for the future study in maxillofacial reconstruction models.
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Ganley, Robert. "Characterisation of distinct inhibitory interneuron populations in the spinal dorsal horn." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/7003/.
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The dorsal horn of the spinal cord is the first node in the somatosensory pathway, and is an area essential for controlling the flow of sensory information sent to the brain. Interneurons constitute the vast majority of neurons in this area, and between 25-40% of those in laminae I-III are inhibitory. These inhibitory interneurons are critical for normal somatosensation, for example, by suppressing pain in the absence of noxious stimuli. Interneurons of the dorsal horn are poorly understood due to their morphological and functional diversity, and this is a major factor limiting our understanding of the neuronal circuitry of the dorsal horn. In order to better understand sensory processing in the dorsal horn it is first necessary to characterise the neurons in this area, and to determine the neuronal circuits in which they are integrated. To address this issue, two separate and non-overlapping populations of inhibitory interneurons in the dorsal horn were thoroughly characterised in terms of their morphological and physiological properties. To achieve this, whole-cell recordings were taken from neurons labelled with green fluorescent protein (GFP) under the control of the Prion promoter (PrP) and the neuropeptide Y (NPY) promoter in spinal cord slices from mice. The recording electrodes contained Neurobiotin, which filled the cells during recording and was revealed with fluorescent molecules, enabling three-dimensional reconstruction of cell bodies and dendrites and axons of neurons. Slices containing these labelled neurons were then resectioned for immunocytochemical reactions to determine their neurochemical content and their synaptic inputs and outputs. This study demonstrated that both PrP- and NPY-GFP cells were morphologically heterogeneous although neither group contained islet cells, which are a distinct morphological class of interneuron. PrP- and NPY-GFP cells in lamina II could not be distinguished from each other by using hierarchical cluster analysis with measures of somatodendritic morphology. This suggests that morphological properties may not be useful in distinguishing these populations of interneurons. The vast majority of PrP- and NPY-GFP cells either displayed tonic or initial burst firing of action potentials. However, these groups of cells showed significant differences in some of their active and passive membrane properties, such as membrane resistance, spike frequency adaptation and mV drop in action potential height. When hierarchical cluster analysis was used to group these cells in lamina II based on physiological parameters, PrP- and NPY-GFP cells could be distinguished with some accuracy. This suggests that some physiological differences may exist between these two groups. Within the PrP-GFP group there was a subset that included lamina I among its synaptic outputs, and these cells could provide inhibition to the projection neurons located in this lamina, since GFP boutons from this mouse line can form synapses with giant cells and neurokinin-1 receptor (NK1r)-expressing lamina I neurons. Some PrP-GFP cells showed immunoreactivity for neuronal nitric oxide synthase (nNOS) or galanin, and these two groups had slight morphological differences, which included their laminar location and the spread of their processes. Several experimental approaches, such as electrophysiological, pharmacological and anatomical techniques, indicated that PrP-GFP cells received input from many different types of primary afferent fibre, including peptidergic and non-peptidergic C-afferents, as well as low-threshold mechanosensory fibres. Taken together these findings establish the PrP-GFP cells as a much more functionally heterogeneous group than previously reported. NPY-GFP cells were located in laminae II and III, but were preferentially found in lamina III. The lamina III cells had dendrites with a greater dorsoventral extent than the lamina II cells, and this extent was seen be more dorsal from the soma than ventral. Many NPY-GFP cells received synaptic input from C-fibres, and a subset of those tested lacked TRPV1. Since the TRPV1-lacking C-fibres mostly correspond to the non-peptidergic C-fibres, including non-peptidergic nociceptors and C-low threshold mechanoreceptors, this suggests that NPY-GFP cells could receive input from these fibres. Dorsal root stimulation experiments showed that labelled NPY-GFP cells with somata located in lamina III often received synaptic input from unmyelinated C-fibres, and NPY-expressing neurons in lamina III could respond to noxious mechanical stimuli. A select group of NPY-GFP cells were seen to innervate putative anterolateral tract (ALT) neurons located in lamina III, which could be identified by their dense innervation by bundles of axons containing either NPY or calcitonin gene related peptide (CGRP). Taken together these data suggest that the PrP- and NPY-GFP neurons are distinct populations based on their primary afferent input and post-synaptic targets, and that more than one functional population exists within each of these groups. Despite their many differences, morphological parameters do not appear to be useful in distinguishing the PrP- and NPY-GFP cells, or detecting different functional populations within these groups. The PrP-GFP cells are more morphologically heterogeneous than previous reports suggested, and due to similar features with cells that require the transcription factor Bhlhb5 to develop, they may include a population that are involved in suppressing itch-related signals. NPY-GFP cells could play a role in limiting the spread and intensity of noxious stimuli due to their input from C-fibres, and a small subset of these could inhibit ALT neurons in lamina III. These results further support the view that different neurochemical populations of inhibitory neurons have distinct functional roles, and also highlight the complexity of the neuronal circuitry in the superficial dorsal horn.
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Critchlow, Rob. "Ecological patterns and predictors of parasite sharing among domestic and wild mammals." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5099/.
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Multi-host pathogens of domestic and wild mammals have significant socio-economic, animal health and conservation consequences. However, despite the interest in diseases at this interface, most research has focused on only a few key pathogens. Studies of focal systems provide limited information on the broad scale ecological patterns of pathogen occurrence and the factors that might drive these distributions, yet large scale studies may have important consequences for disease control and pathogen surveillance. This thesis aims to quantify the abundance and distribution of pathogen sharing among domestic and wild mammals, and develop and understanding of the processes that determine this distribution. Using comparative methods and comprehensive databases this thesis provides the first systematic assessment, on a global scale, of which domestic mammal pathogens have been reported in wild mammals in natural settings. Assessing the extent of pathogen sharing and the characteristics of the pathogens involved showed that the occurrence of domestic mammal pathogens in wild mammals was greater than previously recorded, with an additional 28.5% of domestic pathogens reported to infect wild hosts. Pathogens with the broadest host range had the greatest sharing probability and, in general, pathogen transmission strategies did not limit the degree of sharing. Importantly, from analysing reporting trends, the majority of shared pathogens are already likely to be known, but these are still being reported from novel host-parasite combinations suggesting that the opportunities for pathogen transmission continue to occur, especially since the majority of pathogens have been reported in wildlife multiple times. Most wild ungulates (artiodactyla and perissodactyla) have evidence of infection with domestic livestock parasites, and these hosts are also dominated by those more closely related to livestock. However, phylogenetic relatedness did not appear to be a barrier of infection. The diversity of sympatric wild species was associated with a greater proportion of shared viruses and bacteria, but a lower proportion of shared helminths. These differences among parasite groups are potentially due to variation in parasite transmission strategies. Hosts of conservation concern were not more likely to be infected with domestic mammal pathogens than un-threatened species, suggesting that domestic hosts do not directly contribute to parasite driven declines of wild mammals. Assessing the spatial reporting of wild mammal parasites and what global environmental drivers determines the occurrence of shared parasites may have important implications for disease control and surveillance. Although there are bias in reporting, the majority of wild mammal sampling locations reported pathogens also found in domestic mammals. Livestock densities did not predict the occurrence of pathogens in wild ungulates, but human densities (a proxy for domestic carnivores) did predict the occurrence of pathogens in wild carnivores. For both host groups economic variables were also informative. The probability of parasite sharing was lower in protected area systems, suggesting that these areas may have an valuable role in wildlife disease management. Pathogen sharing among domestic and wild mammals is ubiquitous. Therefore, systematic surveillance for shared pathogens or those shared pathogens that are likely to cross the species barrier in the future is arguably not beneficial. Instead, the informative drivers determined from this macro-ecological analysis, and the identification of areas and hosts that have an increase risk of pathogen sharing may help inform disease management and surveillance strategies.
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Lee, Louisa Chi Yin. "Investigation of mesenchymal stem cell response to bioactive nanotopography." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7180/.
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Mesenchymal stem cells (MSC) are multipotent stem cells that have much potential for application in regenerative medicine, with their ability to self-renew and to undergo differentiation into several lineages, including those that comprise fat, bone, and cartilage. Studies on MSCs have mainly focused on exploiting their capacity to differentiate, rather than self-renewal, yet understanding of the latter process is pivotal for the expansion of these cells to sufficient numbers for use in future clinical treatments. Aspects of MSC behaviour can be induced by culture on nanopatterned substrates, known as nanotopography. Use of established nanopit features, in arrangements known to maintain MSC multipotency over long periods in culture (SQ), in addition to an osteogenic promoting arrangement (NSQ), were used as a tool to study self-renewal in MSCs, and begin to elucidate some of the potential mechanisms underlying the effects of the nanotopographies on stem cell fate. This study utilised patient-specific primary MSCs derived from bone marrow, which were optimised in terms of initial seeding density, to make efficient use of our nanotopographies. Once the fundamental details pertaining to optimal conditions for use of the substrates and cells were established, exploration of changes in metabolism, cell cycle and gene expression were carried out. Results indicated that MSCs on SQ contained more unsaturated metabolites, and that cell cycle may be altered, which warranted further investigation. Further study identified some differences in cell cycle regulatory proteins when compared to NSQ and flat controls. Further inferences were achieved by analysis of transcript abundances and differential expression, supporting the hypothesis of a heterogeneous population existing on NSQ, and activation of pathways linked to differentiation, consistent with previous work. A greater percentage of MSCs on SQ were shown to be in the early G0/G1 stages of the cell cycle in comparison to those on flat, suggesting that cell cycle is altered and further establishes a link between self-renewal and cell cycle regulation. Nanotopography was assessed for a novel application, namely the potential of SQ to reprogram differentiating MSCs. Nanotopography was used to induce and reverse the onset of osteogenic differentiation, and evaluated in conjunction with the addition of an epigenetic modifier, valproic acid. Results did not indicate that nanotopographical cues were able to reprogram MSCs. However, promising indications that stem cell marker STRO-1 levels increased, is consistent with SQ being a surface that maintains ‘stemness’ and multipotency of MSCs with culture in vitro.
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Hopkins, Charlotte Rachael. "Considering climate change in the development of Marine Protected Areas." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7265/.
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Marine ecosystems are facing a diverse range of threats, including climate change, prompting international efforts to safeguard marine biodiversity through the use of spatial management measures. Marine Protected Areas (MPAs) have been implemented as a conservation tool throughout the world, but their usefulness and effectiveness is strongly related to climate change. However, few MPA programmes have directly considered climate change in the design, management or monitoring of an MPA network. Under international obligations, EU, UK and national targets, Scotland has developed an MPA network that aims to protect marine biodiversity and contribute to the vision of a clean, healthy and productive marine environment. This is the first study to critically analyse the Scottish MPA process and highlight areas which may be improved upon in further iterations of the network in the context of climate change. Initially, a critical review of the Scottish MPA process considered how ecological principles for MPA network design were incorporated into the process, how stakeholder perceptions were considered and crucially what consideration was given to the influence of climate change on the eventual effectiveness of the network. The results indicated that to make a meaningful contribution to marine biodiversity protection for Europe the Scottish MPA network should: i) fully adopt best practice ecological principles ii) ensure effective protection and iii) explicitly consider climate change in the management, monitoring and future iterations of the network. However, this review also highlighted the difficulties of incorporating considerations of climate change into an already complex process. A series of international case studies from British Columbia, Canada; central California, USA; the Great Barrier Reef, Australia and the Hauraki Gulf, New Zealand, were then conducted to investigate perceptions of how climate change has been considered in the design, implementation, management and monitoring of MPAs. The key lessons from this study included: i) strictly protected marine reserves are considered essential for climate change resilience and will be necessary as scientific reference sites to understand climate change effects ii) adaptive management of MPA networks is important but hard to implement iii) strictly protected reserves managed as ecosystems are the best option for an uncertain future. This work provides new insights into the policy and practical challenges MPA managers face under climate change scenarios. Based on the Scottish and international studies, the need to facilitate clear communication between academics, policy makers and stakeholders was recognised in order to progress MPA policy delivery and to ensure decisions were jointly formed and acceptable. A Delphi technique was used to develop a series of recommendations for considering climate change in Scotland’s MPA process. The Delphi participant panel was selected for their knowledge of the Scottish MPA process and included stakeholders, policy makers and academics with expertise in MPA research. The results from the first round of the Delphi technique suggested that differing views of success would likely influence opinions regarding required management of MPAs, and in turn, the data requirements to support management action decisions. The second round of the Delphi technique explored this further and indicated that there was a fundamental dichotomy in panellists’ views of a successful MPA network depending upon whether they believed the MPAs should be strictly protected or allow for sustainable use. A third, focus group round of the Delphi Technique developed a feature-based management scenario matrix to aid in deciding upon management actions in light of changes occurring in the MPA network. This thesis highlights that if the Scottish MPA network is to fulfil objectives of conservation and restoration, the implications of climate change for the design, management and monitoring of the network must be considered. In particular, there needs to be a greater focus on: i) incorporating ecological principles that directly address climate change ii) effective protection that builds resilience of the marine and linked social environment iii) developing a focused, strong and adaptable monitoring framework iv) ensuring mechanisms for adaptive management.
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Alba, Katerina. "Isolation, characterization and functional properties of okra pectin." Thesis, University of Huddersfield, 2015. http://eprints.hud.ac.uk/id/eprint/26440/.
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Pectin was isolated by aqueous extraction at pH 6.0 or 2.0 from okra (Abelmoschus esculentus L.) pods. An isolation protocol was designed to extract pectin and study the influence of the extraction pH on its chemical composition, macromolecular and functional properties. The extraction protocols resulted in the isolation of pectin of high purity as evidenced by their high total carbohydrate (70.0 – 82%) and low protein (4.3 – 6.3%) contents. Samples contained between 47-57% galacturonic acid, had broad molecular weight distributions, a low degree of methylation (40 and 25 %) and high degree of acetylation (52 and 38 %). Neutral sugar analysis showed that pectin extracted at pH 6.0 contained more neutral sugars, particularly, galactose, rhamnose and arabinose than that extracted at pH 2.0 indicating variations in fine structure. In addition, molecular parameters of the isolated pectins, such as intrinsic viscosity (2.8 – 4.4 dL g-1), critical concentration (0.15 – 0.45 dL g-1) and coil overlap parameter (0.66 –1.51), showed that extraction conditions resulted in pectin with different chain macromolecular characteristics. Following extraction, the functional properties of okra pectin were investigated in high and low moisture systems and also in colloidal dispersions. It has been shown that okra polysaccharides are non-gelling pectins and their inability to form ordered structures was attributed to the high degree of acetylation and branching of the side-chains. The pH sensitivity of okra pectins has been further demonstrated in high solid systems, where the mechanical relaxation of LM-pectin in the presence of co-solute has been altered by pH. It has been shown that high pH values result in extended chain conformation and early vitrification events. In contrast, viscoelastic functions of polyelectrolyte decreased and resulted in delayed vitrification events at low pH. The next step of present work was focused on potential utilization of okra polysaccharides in fabrication of oil-in-water emulsions for food and pharmaceutical applications. The emulsifying properties of crude okra extracts and okra isolates (rich in pectin) have been investigated under different conditions (e.g., oil volume fraction, biopolymer concentration, pH values, energy input methods) in order to produce fine emulsions with long-term stability. It has been shown that pH of extraction has a pronounced effect on the interfacial activity of both crude extract and pectin isolates. Extracts or isolates obtained at high pH demonstrated higher emulsifying capacity than those extracted at low pH. In general, okra pectin isolates were more efficient in emulsion stabilisation than crude extracts by producing emulsions of smaller droplet sizes. Moreover, emulsifying capacity of okra pectin was affected by the pH and stable emulsions were produced only at low pH values (pH 2.0 or 3.0). It has been shown that okra pectin-stabilized emulsions evolve under the effects of Ostwald ripening and coalescence during the long-term storage. The present work shows the potential of okra pectins as emulsifiers under acidic conditions and serves as the basis for the development of such systems in encapsulation technology of bioactive components.
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Nikukar, Habib. "Nanomechanotransduction of human mesenchymal stem cells : an application of medical nanobiotechnology." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4752/.
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In this project the influences on human adult mesenchymal stem cells using nanomechanical stimulation techniques has been explored. It is expected that human mesenchymal stem cells will find use in many autologous regenerative therapies and in tissue engineering. However, the ability to control stem cell growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, unpredictable number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, etc.) culture substrates. According to known cellular reactions to environmental stimuli, it was expected that human cells show some reactions to nanoscale vibration that in the case of stem cells it could be a differentiation response. This thesis gives a first demonstration of using nanoscale mechanotransductive protocols (10-14 nm vertical displacements at 1 kHz frequency), “nanokicking”, to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, laser interferometry was used to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of biological techniques has then been used to demonstrate osteoblastogenesis. Furthermore, RhoA has been implicated as being central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol.
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Hodges, Holly R. "An investigation of social structure in housed dairy cows." Thesis, University of Essex, 2018. http://repository.essex.ac.uk/23324/.
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The changing landscape of the UK’s dairy farms poses increasing challenges to farm staff in terms of monitoring individuals behaviour within increasing herds, and more intensive conditions. Failure to detect behavioural changes may be costly, both from a welfare and financial perspective, as such alterations may indicate underlying disease or other challenges with corresponding impacts on yield and animal well-being. Social behaviour may provide a useful indicator of normal animal activity, and subsequent changes with health status, particularly if automatically monitored to reduce labour. This thesis applies a local positioning system (LPS) to collect social proximities of dairy cows, to investigate the social structure of a housed herd via social network analysis, and any relationship with traits or health. The LPS was validated by comparing sensor reported, with human observed proximities, and accurately detected proximities at lying, feeding and in direct interactions. Use of this data to construct social networks indicated a highly connected structure, with some substructure becoming evident after filters were applied. An approaching significant effect of parity on sociality was found, but stage of lactation had no effect. Temporally, the network showed some stability but a much greater amount of variation. When divided into ‘functional area’ (feeding, non-feeding and milking), the non-feeding area of the shed yielded the most loosely connected network with likely most interest for further analysis due to its potential basis in choice, as opposed to forced proximity. In these functional area networks, some evidence exists for homophily (association with similar cows – based on parity and days in milk). Finally, sociality was investigated alongside health status, with evidence for a tendency for greater betweenness in lame cows than non-lame. The results suggest that sociality is a highly variable trait, and that further investigation is required to assess its suitability as a disease indicator.
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Howson, Emma Lucy Anna. "The development and application of molecular tools for the diagnosis of foot-and-mouth disease in field and low-resource laboratory settings." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8607/.
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The requirements for prompt diagnosis of foot-and-mouth disease (FMD) during outbreaks, and the need to establish robust laboratory testing capacity within FMD-endemic countries, have motivated the development of point-of-care tests (POCTs) to support current diagnostic strategies. Despite numerous publications detailing the design of platforms and assays for this purpose, the majority have only been evaluated in laboratory settings, using protocols incompatible for use in challenging environments. To address this gap, this thesis describes the development of an end-to-end molecular toolbox for the detection and characterisation of FMD virus (FMDV) RNA in decentralised settings. A critical review and multiway comparison of seven assay formats and 11 sample preparation methods revealed that reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time reverse transcription PCR (rRT-PCR) POCT-formats exhibited comparable analytical and diagnostic sensitivity to their laboratory-based equivalents. Additionally, reagent lyophilisation provided a solution for cold chain and storage considerations, whilst not compromising assay performance. Both assays were compatible with simple sample preparation methods, removing the requirement for nucleic acid extraction. For example, dilution of samples in nuclease-free water enabled FMDV RNA to be detected in multiple sample types (epithelial tissue suspensions, serum, oesophageal-pharyngeal fluid and lesion swabs), from as early as one day post infection. Notably, when the robust field-ready protocols were deployed into challenging low-resource laboratory and field-settings within East Africa, POCT results (rRT-PCR = 144; RT-LAMP = 145) were consistent with clinical observations and a reference rRT-PCR, with FMDV detected from acutely infected as well as convalescent cattle. Furthermore, transitioning of East Africa-specific FMDV-typing rRT-PCR assays (for serotypes O, A, Southern African Territories [SAT] 1 and SAT 2) into a multiplex POCT-format enabled rapid identification of FMDV serotype in situ, confirming active outbreaks of both O and A. This thesis also describes the development of GoPrime, a novel real-time PCR (rPCR) primer/probe validation tool. By parameterising GoPrime with experimental data, collected to investigate the effects of primer/probe-template mismatches on cycle threshold and limit of detection, it was possible to quantitatively predict the performance of rPCR assays in silico. The work of this thesis supports the deployment of molecular POCTs into non-specialised, resource-limited and challenging settings for simple, highly sensitive and rapid detection and/or characterisation of FMDV.
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Batas, Anastasios. "Protein trafficking and autophagy in the moulting cycle of C. elegans." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8856/.
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Endosomal trafficking and autophagy are two fundamental processes of eukaryotic cell biology, from unicellular organisms such as yeast to multicellular metazoans such as C.elegans and Humans. Both processes are involved in a diverse number of physiological processes and implicated in a number of pathologies. A recent study has exhibited a mutation on the SM protein Vps45 as a cause of severe congenital neutropenia in humans. The same mutation in yeast causes defects in endosome to vacuole trafficking in S.cerevisiae as well as a temperature sensitive lethality at the non-permissive temperature. A null allele of vps-45 in C.elegans results in developmental arrest during the highly secretory phase of moulting in a similar temperature conditional manner to yeast and defects in yolk protein trafficking. The work presented in this thesis aims to provide basic understanding in an animal model of the impact of loss of Vps45 function that might be informative of the reason for the death of the highly secretory neutrophil cells under the absence of a functional Vps45 protein. The vps-45 and unc-51 mutants as well as a novel unc-51 vps-45 double mutant where possible, were characterised for lifespan, duration of post- embryonic development as well as moulting duration. Reduced embryonic viability, reduced lifespan as well as delays in the moulting process were identified. Data suggested that both autophagy and protein trafficking play a role in C.elegans development through unc-51 and vps-45 respectively. In addition to this, the seam cells of both the vps-45 and unc-51 defective C.elegans were observed during the moult using an autophagy marker. An increase in autophagic activity during the moult was observed, which was more pronounced in the case of the vps-45 mutant. As such the obtained data suggest autophagy and endosomal trafficking play an important role in the moulting process. Following up to previous work conducted in our lab in yeast defective for Vps45 trafficking which exhibited increased sensitivity to oxidative stress, the redox state of the vps-45 and unc-51 animals as well as their sensitivity to oxidative stress was assessed using a set of ER and cytosolic GFP markers and killing assays. Both the vps-45 and unc-51 mutants showed a higher sensitivity to oxidative stress, with the unc-51 exhibiting the more pronounced phenotype overall. These results came in agreement with the shorter lifespan phenotypes exhibited by both mutants in the previous experiments, possibly as a result of accumulation of ROS, as well as the severe defects of the double mutant. Finally, a suppressor identified for the moulting death of the vps-45 mutant was characterized for a set of phenotypes, in order to exclude suppression of any of the other phenotypes identified for the vps-45 mutant. Furthermore, the suppressor was identified as being autosomal and recessive and as thus an SNP full genome sequencing technique was employed, which gave rise to two suppression loci in two different chromosomes, along with two different subpopulations corresponding to these loci which exhibited different growing patterns.
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Bennett, Mark. "Dependence of cellular behaviour on viscosity defined ligand mobility in supported lipid bilayers." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30587/.
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This thesis has explored the nature of cellular behaviour in response to the mobility of ligands presented on supported lipid bilayers of varying viscosity (diffusive characteristics). This was inspired by the various characteristics of the in vivo microenvironment, controlling the cell response. For example, the viscoelastic, topographical, or chemical nature of the extracellular matrix can control cellular behaviours, such as adhesion, proliferation, migration and differentiation. Numerous biomaterials have alternately sought to understand the nature of the cell response and also to take advantage of it; the current work predominantly falls into the former of these categories. Whereas elastic stiffness is one side of the coin of viscoelasticity, viscosity is the other. Further, while much work has sought to understand the nature of both the elastic and viscoelastic nature of the cell response, as of yet few have sought to understand the role of viscosity in isolation. This is despite some work seeking to take advantage of this viscosity, by observing cellular behaviour on surfaces with known viscous components. This work has noted that cellular adhesion and spreading, focal adhesions, and differentiation are all affected by the viscous component of the surface without addressing why. Supported lipid bilayers (SLBs) present an excellent opportunity to understand these mechanisms. Commonly used as biosensing platforms, non-fouling coatings and model systems, they have also found use in both cell culture systems and in understanding mechanobiology. Individual lipids may exhibit a phase transition, Tm, at a temperature defined by the chemistry of the SLB component lipids; as such, they can exhibit significantly different, viscosity-defining, diffusive characteristics. This work describes the use of SLBs of differing Tm¬ that exhibit fluid-like or gel-like properties in cell culture conditions. These non-fouling SLBs were functionalised with the cell adhesive ligand RGD, derived from the matrix protein fibronectin, with the response of the cell on both the cell-wide and molecular scale determined. The cell response was then understood via pathways related to the mechanical sensing of the environment. Further, initial forays into the nature of the response of human mesenchymal stem cells (hMSCs) was determined, to test the applicability of the system to the overall field of biomedical engineering.
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O'Leary, Jade. "Multi-dimensional mycelia interactions." Thesis, Cardiff University, 2018. http://orca.cf.ac.uk/111801/.
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The activities of competing wood decay fungi bring about the decomposition of deadwood. The rate of decomposition is determined by the community composition, which is shaped by competitive interactions. Fungi compete with one another for territory and resources within 3 dimensional space via the production of an arsenal of inhibitory chemical compounds, yet interspecific interactions and chemical warfare within communities have never before been studied in 3 dimensional resources. This thesis, therefore, aims to determine how interactions cause metabolic processes to change, and, by the use of novel 3 dimensional systems, how those processes are affected by increased species diversity and spatial heterogeneity. Overarching hypotheses which have driven investigations are: (1) secondary metabolism and interaction outcome are inherently linked, both of which are affected by environmental conditions, (2) changes to spatial distributions and increased species diversity cause interaction outcomes and community dynamics to alter, and (3) changes to metabolic strategies for antagonism and resource utilisation will reflect alterations to community dynamics. Transitive interactions in pair wise 2 dimensional systems often became intransitive in species richer 3 dimensional systems, and the extent of patch fragmentation determined the length of coexistence of individuals within communities. Production of secondary metabolites comprising the fungal chemical weaponry, namely extracellular lignocellulolytic enzymes and volatile organic compounds (VOCs), was significantly different between 2 and 3 dimensional systems, between systems of varied species diversity, and between different spatial distributions of fungi, reflecting the observed changes to community dynamics. Furthermore, the production of intracellular metabolites which function in the biosynthesis pathways of antimicrobial compounds and agents of decay, was affected by species diversity, spatial distributions and territory fragmentation. Additionally, secondary metabolism and interaction outcome were found to be inherently linked, and affected by abiotic conditions. Specifically, interaction outcomes changed under different temperatures, and when wood was pre colonised for different lengths of time. Changes to the production of both enzymes and VOCs reflected the different outcomes, as either the cause or effect of outcome changes. These conclusions not only confirm the thesis hypotheses, but also demonstrate the importance of environmental conditions, species diversity and spatial dynamics to fungal community dynamics and forest ecosystem functioning.
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Gu, Xu. "Systems biology approaches to the computational modelling of trypanothione metabolism in Trypanosoma brucei." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/1618/.
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This work presents an advanced modelling procedure, which applies both structural modelling and kinetic modelling approaches to the trypanothione metabolic network in the bloodstream form of Trypanosoma brucei, the parasite responsible for African Sleeping sickness. Trypanothione has previously been identified as an essential compound for parasitic protozoa, however the underlying metabolic processes are poorly understood. Structural modelling allows the study of the network metabolism in the absence of sufficient quantitative information of target enzymes. Using this approach we examine the essential features associated with the control and regulation of intracellular trypanothione level. The first detailed kinetic model of the trypanothione metabolic network is developed, based on a critical review of the relevant scientific papers. Kinetic modelling of the network focuses on understanding the effect of anti-trypanosomal drug DFMO and examining other enzymes as potential targets for anti-trypanosomal chemotherapy. We also consider the inverse problem of parameter estimation when the system is defined with non-linear differential equations. The performance of a recently developed population-based PSwarm algorithm that has not yet been widely applied to biological problems is investigated and the problem of parameter estimation under conditions such as experimental noise and lack of information content is illustrated using the ERK signalling pathway. We propose a novel multi-objective optimization algorithm (MoPSwarm) for the validation of perturbation-based models of biological systems, and perform a comparative study to determine the factors crucial to the performance of the algorithm. By simultaneously taking several, possibly conflicting aspects into account, the problem of parameter estimation arising from non-informative experimental measurements can be successfully overcome. The reliability and efficiency of MoPSwarm is also tested using the ERK signalling pathway and demonstrated in model validation of the polyamine biosynthetic pathway of the trypanothione network. It is frequently a problem that models of biological systems are based on a relatively small amount of experimental information and that extensive in vivo observations are rarely available. To address this problem, we propose a new and generic methodological framework guided by the principles of Systems Biology. The proposed methodology integrates concepts from mathematical modelling and system identification to enable physical insights about the system to be accounted for in the modelling procedure. The framework takes advantage of module-based representation and employs PSwarm and our proposed multi-objective optimization algorithm as the core of this framework. The methodological framework is employed in the study of the trypanothione metabolic network, specifically, the validation of the model of the polyamine biosynthetic pathway. Good agreements with several existing data sets are obtained and new predictions about enzyme kinetics and regulatory mechanisms are generated, which could be tested by in vivo approaches.
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Morrison, Daniel Scott Simpson. "Controlled surface nanotopography and oxygen plasma treatment of PEEK to improve cellular response." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7804/.
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Poly(aryl-ether-ether-ketone) (PEEK) is a semi crystalline polymer which exhibits properties that make it an attractive choice for use as an implant material. It displays natural radiolucency, and MRI compatibility, as well as good chemical and sterilization resistance, both of which make it of particular interest in orthopaedic implants. However, PEEK has demonstrated poor cellular adhesion both in vitro and in vivo. This is problematic as implant surfaces that do not develop a layer of adhesive cells are at risk of undergoing fibrous encapsulation, which in turn leads to lack of a strong interface between the implant device and the patient tissue, which can in turn lead to failure of the implant and revision surgery . As incorporating nanotopography into a polymer surface has been demonstrated to be able to direct the differentiation behaviour of stem cells, a possible solution to PEEKs underlying issues with poor cellular response would be to incorporate specific nanoscale topography into the material surface through injection moulding, and then analysing if this is a viable method for addressing PEEKs issues with cellular response. In addition to nanoscale topography, the experimental PEEK surfaces were treated with oxygen plasma to address the underlying cytophobicity of the material. As this type of treatment has been documented to be capable of etching the PEEK surface, experiments were carried out to quantify the effect of this treatment, both on the ability of cells to adhere to the PEEK surface, as well as the effect it has upon the nanotopography present at the PEEK surface. The results demonstrated that there were a range of plasma treatments which would significantly improve the ability of cells to adhere to the PEEK surface without causing unacceptable damage to the nanotopography. Three different types of cells with osteogenic capacity were tested with the PEEK surfaces to gauge the ability of the topography to alter their behaviour: SAOS-2, osteoprogenitors and 271+ MSCs. Due to PEEKs material properties (it is non transparent, exhibits birefringence and is strongly autofluorescent) a number of histological techniques were used to investigate a number of different stages that take place in osteogenesis. The different cell types did display slightly different responses to the topographies. The SAOS-2 cells cultured on surfaces that had been plasma treated for 2 minutes at 200W had statistically significantly higher levels of von Kossa staining on the NSQ surface compared to the planar surface, and the same experiment employing alizarin red staining, showed a statistically significantly lower level of staining on the SQ surface compared to the planar surface. Using primary osteoprogenitor cells designed to look into if whether or not the presence of nanotopography effected the osteogenic response of these cells, we saw a lack of statistically significant difference produced by the surfaces investigated. By utilising HRP based immunostaining, we were able to investigate, in a quantitative fashion, the production of the two osteogenic markers osteopontin and osteocalcin by cells. When stained for osteocalcin, the SQ nanotopography had total percentage of the surface with stained material, average area and average perimeter all statistically significantly lower than the planar surface. For the cells that were stained for osteopontin, the SQ nanotopgraphy had a total percentage of the surface with stained material, average area and average perimeter all highly statistically significantly lower than those of the planar surface. Additionally, for this marker the NSQ nanotopography had average areas and average perimeters that were highly significantly higher than those of the planar surface. There were no significant differences for any of the values investigated for the 271+ MSC’s When plasma treatment was varied, the SAOS-2 cells demonstrated an overall trend i.e. increasing the energy of plasma treatment in turn leads to an increase in the overall percentage of staining. A similar experiment employing stem cells isolated from human bone marrow instead of SAOS-2 cells showed that for polycarbonate surfaces , used as a control, mineralization is statistically significantly higher on the NSQ nanopattern compared to the planar surface, whereas on the PEEK surfaces we observe the opposite trend i.e. the NSQ nanotopography having a statistically significantly lower amount of mineralization compared to the planar surface at the 200W 2min and 30W 1min plasma treatments. The standout trend from the PEEK results in this experiment was that the statistically significant differences on the PEEK substrates were clustered around the lower energy plasma treatments, which could suggest that the plasma treatment disrupted a function of the nanotopograhy which is why, as the energy increases, there are less statistically significant differences between the NSQ nanotopography and the Planar surface This thesis documents the response of a number of different types of cells to specific nanoscale topographies incorporated into the PEEK surface which had been treated with oxygen plasma. It outlines the development of a number of histological methods which measure different aspects of osteogenesis, and were selected to both work with PEEK, and produce quantitative results through the use of Cell Profiler. The methods that have been employed in this body of work would be of interest to other researchers working with this material, as well as those working with similarly autofluorescent materials.
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Gehl, Bernadette. "Functional and molecular characterisation of two stomatin-like proteins from Arabidopsis thaliana." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/640/.
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Stomatins belong to the band-7 (or SPFH domain) family (short for Stomatin, Prohibitin, Flotillin HflC/K) of diverse membrane proteins. This protein family is evolutionary conserved with members found in all sequenced eukaryotes and in most prokaryotes. Band-7 family proteins have the ability to oligomerise and generally aid in the assembly and regulation of large membrane-bound protein complexes. In animals, stomatins have been demonstrated to regulate ion channels by direct protein interactions. Additionally, they localise to membrane microdomains where they actively contribute to their assembly by binding sterols, and they also associate with the actin cytoskeleton. The Arabidopsis genome encodes for two structurally similar stomatin-like proteins that are functionally completely unknown yet. They will be referred to as AtSlp1 (for Arabidopsis thaliana stomatin-like protein) and AtSlp2. The aim of this thesis was to provide a detailed characterisation of these two genes on a molecular and functional level. Both proteins are expressed ubiquitously throughout plant development, but they accumulate at particularly high levels in pollen and other metabolically active cells. Phylogenetic analysis reveals that AtSlps are homologous to stomatin-like proteins of type 2. Amongst these, the human stomatin-like protein 2 (HsSlp2) is localised to mitochondria where it participates in large membrane-bound protein complexes and is also involved in the proliferation of cancer cells. Evidence is provided here that demonstrates mitochondrial localisation of both Arabidopsis Slp proteins in vitro and in vivo. On a functional level, mitochondria from an slp1 knockout mutant plant have a decreased mitochondrial membrane potential and increased oxygen consumption rates. This is interpreted as a defect in coupling efficiency and an impairment of the mitochondrial inner membrane integrity. This defect results in a variety of other growth phenotypes that are related to metabolically active tissues and cell types. Knockout plants are delayed in overall growth of shoots and roots and have decreased seed germination rates. Additionally, these plants are less resistant to conditions of high salinity and are less fertile. Overexpression of a protein acting as a putative dominant-negative Slp fragment results in plants with a dwarf phenotype and early onset of leaf senescence. This phenotype correlates with increased levels of reactive oxygen species and altered organelle ultrastructure. Guard cells from these plants in particular have enlarged chloroplasts and are impaired in transpirational control. It is concluded that also in plants, stomatins act together with other band-7 family proteins as parts of large protein complexes that have regulatory roles important for development and stress responses. Their main role is probably to provide membrane scaffolds that affect mitochondrial function and morphology during cell division and in situations of mitochondrial stress.
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Miles, William Thomas Stead. "Ecology, behaviour and predator-prey interactions of Great Skuas and Leach's Storm-petrels at St Kilda." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/2297/.
Full textAbstract:
At the St Kilda archipelago, Outer Hebrides, declines have been recorded in the Leach's Storm-petrel breeding population, the largest in Britain and Ireland, and rapid increases in the population of Great Skuas. Leach's Storm-petrels have frequently been found in the diet of Great Skuas at St Kilda, where storm-petrels are active on land only at night and, unusually, skuas often hunt after dark. Apparent severe skua predation of Leach's Storm-petrels has raised conservation concerns regarding the sustainability of the St Kilda Leach's Storm-petrel population. However, it was recognised that this particular predator-prey relationship is a globally rare phenomenon, had not previously been studied for long at St Kilda (and never elsewhere), and warranted further research before conservation management interventions could be considered. Additionally, research on Leach's Storm-petrels was desirable in its own right, because the species had rarely been studied in the UK, due to its highly pelagic lifestyle and very remote breeding locations. The aim of this study was to increase our understanding of the ecology, behaviour and predator-prey interactions of Great Skuas and Leach's Storm-petrels at St Kilda. Results showed that Great Skua predation of Leach's Storm-petrels was considerable and sustained. Estimated numbers of Leach's Storm-petrels consumed annually by skuas were variable but averaged approximately 21,000 individuals per year. There was strong evidence from storm-petrel ringing and behavioural observations conducted at night that skuas fed predominantly on non-breeding Leach's Storm-petrels, which likely visit the archipelago in very large numbers each year, from huge colonies elsewhere, and probably play and important role in reducing impacts on the breeding population at St Kilda. It was found that Leach's Storm-petrels did not exhibit any specialised counter-predator adaptations to Great Skuas, and were very easily captured at night on the surface of the breeding colonies by skuas on foot. However, prey specialisation by skuas on nocturnally active seabirds (predominantly storm-petrels) did not create fitness advantages over prey specialisation on diurnally active seabirds or fish. Leach's Storm-petrel specialist skua pairs were very few and all pairs exhibited a tendency to feed on a diversity of prey and to switch prey-types between years. Adult and juvenile Leach's Storm-petrels were highly sensitive to light, and artificial light reduction measures in autumn helped prevent storm-petrel attractions and mortality in the village on Hirta. The St Kilda Great Skua population was found to be declining slightly, in contrast to the exponential growth recorded between 1990 and 2000, and Leach's Storm-petrel conservation issues now appear less severe than had been expected.
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de, Wert Leoni. "Anti-predator adaptations and strategies in the Lepidoptera." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3541/.
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This thesis examines visual anti-predator strategies employed by the Lepidoptera. I examine key aspects of pattern and behaviour and how they relate to the reduction of an individual’s predation risk. Symmetrical patterns have been found to be easier to remember and pick out, suggesting that symmetry is beneficial to aposematic displays. This suggests that symmetry may be maladaptive in cryptic patterning and asymmetry beneficial. In Chapter one, I report the results of a field experiment using artificial prey and wild birds to investigate how asymmetry and symmetry affect the efficiency of cryptic patterning to reduce predation. I found that asymmetry does not affect predation rate, in agreement with previous work. Yet, there is still the problem of how to mesh this with the potentially conflicting conclusions of symmetry studies. Chapter two examines aspects of the intimidation hypotheses of Lepidopteran eyespots. These address the generally larger and more centrally placed spots found on Lepidopteran wings and state that they startle or intimidate predators, providing time for escape. While it is agreed that eyespots intimidate or startle predators, the mechanism has not been agreed. There are two competing lines of thought 1) that ‘eyespots’ intimidate because they resemble the eyes of the predators’ own predators and 2) that it is the conspicuous colouration of the pattern that induces the startle or avoidance behaviour. The first experiment utilised artificial prey with differing ‘directions of gaze’ in a field setting. If purely conspicuous patterns direction of gaze should have no influence on prey survival. The results indicate that patterns imitating staring or upward gazes provide the greatest protection, suggesting that in some cases eyespots may be being perceived as eyes and not simply as conspicuous patterns. I wanted to see if it would be possible to find a way in which to measure or quantify the reaction of an animal to ‘real’ eyes, in order to compare it to the reaction to eyespots. Recent trials investigating human reactions to eye contact suggested a computer based method may be possible. In this second experiment we examined whether the direct gaze of a predator might produce a measurable effect in human subjects. I was not able find any effect, but it is unclear as to whether this is due to problems with the experimental set up. In Chapter three I investigate a factor often over looked in the study of crypsis, that of the behavioural adaptations that can enhance its efficiency. The larvae of the early thorn moth (Selenia dentaria) masquerade as twigs, using both colouration and behaviour adaptations. I compared the angle at which the larvae rested, to the angle at which real twigs deviate from the main stem. The results found that the larvae showed variation in their angle of rest and do not appear to match the angle of real twigs on the host tree. This result suggests that perfectly matching the angles of real twigs is not necessary to twig mimicry. While carrying out this experiment it was noticed that a breeze appeared to increase larval activity and induced a ‘swaying’ behaviour. This led me to examine whether mimic species may utilise the visual ‘noise’ produced by windy conditions to camouflage movement. Firstly, a small ‘proof of concept’ pilot was carried out, followed by a larger study using 2 different twig mimic species. The study involved measuring movement and swaying behaviour in 3 conditions (still air, wind setting 1 and 2). The results suggest that cryptic and mimetic lepidopteran species may use windy conditions to camouflage their movements and that some species may employ specialised ‘swaying’ behaviours. Cryptic species are limited in opportunities to move between foraging sites without increasing detection by predators, therefore, any adaptation that might reduce detection is extremely advantageous. In Chapter four I examine how conspicuousness and colouration are affected by living in a group, particularly in relation to other group members. A field experiment using groups of artificial prey, with differing densities and group sizes was used to explore the effect of group size and density on the predation risk and detectibility of cryptic prey. My results show that, as expected, larger groups are more likely to be detected, but that the increase is much slower than a linear increase. This suggests that groups must increase considerably in size before any individual group member will suffer increased predation risk. The second experiment examines the ‘oddity effect’ and how it affects predation. This hypothesises that when confronted by grouped prey, predators can increase their kill rate by concentrating their efforts on capturing unusual or ‘odd’ prey, a strategy that reduces the ‘confusion effect’. A field experiment was conducted with groups composed of differing proportions of two artificial cryptic prey types. Groups with odd individuals did not suffer an increase in conspicuousness and were not attacked more often. However, once located and attacked the groups did suffer a greater predation rate. Odd individuals were predated at a greater rate than normal individuals and the rate did not change as more or less odd individuals were added to the group. A computer based ‘game’ was used to further investigate the oddity effect. The results from the initial run of the game appeared to show strong evidence for the oddity effect, with a further significant increase in this effect when attention is split between foraging for prey and scanning for predators. To be confident of this result the experiment was repeated with the ‘odd’ and ‘normal’ seed patterns reversed. The new data set strongly suggested that much of the effect seen in the previous experiment was due to a difference in pattern visibility between the two seed patterns. Nevertheless, the results indicated that selecting odd seeds is quicker than selecting normal seeds. The results from both the field and computer trials suggest that preference for odd prey may improve predator foraging speed and efficiency. Chapter five investigates whether cryptic and non-defended prey could reduce their predation risk by grouping with aposematic and defended prey. This was tested using artificial prey in a field setting. My results show that undefended non-aposematic prey can benefit by grouping with aposematic prey with no evidence that predation rates for aposematic prey were adversely affected by this association. If confirmed this might illuminate the origins of Batesian mimicry. I have investigated a range of anti-predator adaptations and strategies in the Lepidoptera and in particular pattern elements and use of crypsis and aposematic displays. These anti-predator strategies are important in that they modify predation rate and so directly influence the evolution of species. While I have been able to provide evidence for some current hypotheses, in many respects my results demonstrate that there is still a lot to learn about visual anti-predatory strategies.
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Muinonen-Martin, Andrew James. "Melanoma cells induce LPA gradients that drive chemotactic dispersal and invasion." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4836/.
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Melanoma is notoriously resistant to immuno- and even targeted chemotherapeutic strategies despite recent advances in drug development. The overall mortality of melanoma correlates with its ability to metastasise. Breslow thickness or the depth of invasion remains the most useful prognostic indicator, thereby linking the ability of the cells to invade with their propensity to metastasise. Invasion occurs early during tumour development, but the factors driving this process remain poorly understood. There is a growing appreciation that chemotaxis plays an important role in driving the migration and invasion of melanoma cells, but the key stimuli are not known. Through the generation and validation of an improved chamber for cancer cell chemotaxis, melanoma cells are shown to create chemotactic gradients that drive or disperse themselves outwards with remarkable efficiency. This process is driven by strikingly positive chemotaxis and depends on the melanoma reaching a critical density to generate the gradient. The principal attractant is the inflammatory signal lysophosphatidic acid (LPA). Unexpectedly, it is active across all stages of melanoma evolution and LPA is both necessary and sufficient for chemotaxis in 2D & 3D assays. Growth Factors were previously considered to play essential roles in driving directed migration, but instead facilitate LPA chemotaxis. Sampling across the margins of melanomas in vivo, gradients of LPA are reliably identified, which are capable of driving accurate chemotaxis. This not only confirms the physiological importance of the results, but also is the first time a chemoattractant gradient has been measured in vivo. The corollary of these findings is that, provided with an external homogenous source of LPA, a large enough melanoma will degrade the local LPA to generate an outward gradient. Therefore it is the ability to degrade the gradient that acts as the signal to drive chemotaxis and invasion rather than the presence of LPA per se. In the chambers, cells are observed dispersing in a wave and in addition to driving efficient melanoma invasion, this may be responsible for the patterning of melanocytes across the skin during embryogenesis. Ultimately, identifying key aspects of and targeting the LPA-axis may prove a novel prognostic indicator and therapeutic target for invasion and metastasis.
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Surman, Susanne Barbara. "The integration of an avirulent Legionella pneumophila into aquatic biofilms." Thesis, University of Central Lancashire, 1994. http://clok.uclan.ac.uk/1773/.
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A continuous culture model system was set up in the laboratory and inoculated with a diverse range of microorganisms, including several bacteria and protozoa, obtained from the local mains water tap supply. This inoculum and was added to the system without any prior culture or other selection process. Biofilms readily developed on glass tiles suspended in the planktonic phase of the system. An avirulent Legionella pneumophila was inoculated into the system and was subsequently isolated from both biofilms and also from the aqueous(planktonic) phase of the chemostat. The attenuation of this strain, determined by its inability to cause disease and death in guinea pigs, remained unaltered despite the long term survival of this strain within the system. Investigations to determine whether the avirulent L. pneumophila was able to infect and proliferate within protozoa were carried out. The results of the present study show that this avirulent L. pneumophila did not proliferate intracellularly and suggest that the association of L. pneumophila with protozoa although probably important in the long term survival of this bacterium especially during periods where adverse conditions prevail, is not essential but opportunistic. In chapter 3 the importance of the presence of these non-legionellae bacteria, which included Flavobacterium sp., Acinetobacter spp. and several species of Pseudomonas, was investigated. The results suggest that the presence of the non-legionellae are relevant to the survival of Legionella especially in environments which favour it's growth, for example water distribution systems. In order that we may gain a further insight into the ecology of microorganisms in their natural environment, it is necessary to visualise them in conditions which allow them to interact in a way which mimics as closely as possible the natural environment. Biofilms were developed on surfaces which could be removed from the model system in their entirety. Direct visualisation techniques, including atomic force microscopy and Hoffman modulation microscopy could then be used which allowed the in vivo examination of biofilms in situ on the surface upon which they had developed. More traditional microscopy methods were also used. Atomic force microscope images of biofilms and individual biofilm bacteria including Legionella were obtained, which clearly showed the presence of exopolymeric substances (EPS). Hoffman modulation contrast microscopy and scanning electron microscopy showed the diverse nature of the biofilms being studied. The results of these investigations suggest that a more complete understanding of the complex nature of biofiims is achieved by the use of a combination of several microscopy techniques. The response of a L. pneumophila serogroup 6 and the avirulent L. pneumophila serogroup 1 to a commercially available biocide, Vantocil IB, was investigated. Both the serogroup 6 and the avirulent serogroup 1 could not be detected following biocide teatment in either the planktonic phase or biofilms. These results suggest that this avirulent L. pneumophila is a suitable model substitute for the virulent L. pneumophila.
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Manganas, Phanee. "Oxidative regulation mechanisms in the mitochondrial intermembrane space." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8568/.
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Oxidative stress occurs when cells are unable to cope with the levels of various reactive oxygen species (ROS) that arise as part of regular cellular metabolism or in response to ionising radiation (H2O2, O2-, OH-). The most well studied ROS is H2O2, due to its dual role as a mediator of oxidative stress and a signalling molecule for many cellular pathways. Cells possess a number of different mechanisms to combat ROS, in order to prevent their levels from becoming toxic. In this thesis, we studied three different aspects of the antioxidant defence in Saccharomyces cerevisiae. In the first part, we explored the role of erythroascorbic acid – the yeast analogue of ascorbic acid (vitamin C) – and attempted to determine its role as an antioxidant in yeast. Our results were inconclusive, though there were indications that the presence of erythroascorbic acid may have a protective effect on the mitochondrial inner membrane potential (ΔΨ), protecting it from depolarisation. The second part focused on elucidating the mitochondrial targeting of the main H2O2 sensor Gpx3 and, more specifically, whether the Yap1-binding proteins, Ybp1 and Ybp2, have an effect on the import of Gpx3 in yeast mitochondria. Our results show a slight effect of Ybp1 (but not Ybp2) on the import of Gpx3, indicating that Ybp1 may act as a chaperone for the more efficient targeting of Gpx3 from the cytosol to the outer mitochondrial membrane and, as a result, its eventual translocation into the IMS. The final part of this thesis focused on elucidating the import of Trx1 and Trr1 in the mitochondrial IMS, as well as their function in this particular subcompartment. The discovery of two members of the thioredoxin system in the IMS is important, due to the absence of a known reducing mechanism in this oxidising compartment. Our results determined that several well-known import factors are dispensable for the import of either Trx1 or Trr1, indicating that they follow a yet unknown pathway for their translocation into the IMS. Importantly, we showed that Trx1 is reduced (and thus, active) in the IMS and that it can interact in vitro with both components of the MIA machinery (Mia40 and Erv1), while in organello experiments showed that Trx1 most probably interacts with a large number of Mia40 substrates.
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Sarhan, Adil Rashid. "The regulation of cell signalling by LAR protein tyrosine phosphatase." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7256/.
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Signal transduction pathways are mainly depending on phosphorylation events, which are controlled by the activity of phosphatases and kinases. Although kinases have been widely studied, however, much less is known about the contribution of phosphatases to the regulation of cell signalling pathways. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is involved the regulation of a number of receptor tyrosine kinases (RTKs) including platelet-derived growth factor receptor (PDGFβR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. The differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP) was analysed. A significant change in abundance of phosphorylation on 270 phosphorylation sites from 205 proteins was associated with the lack of LAR phosphatase activity. Gene ontology analysis revealed an enrichment of LAR-mediated phosphorylation events on proteins involved in many signalling transduction pathways including those regulating the actin cytoskeleton, cell adhesion, endocytosis and cell metabolism. Analysis of putative kinases upstream of LAR-dependent phosphorylation events revealed a role for LAR in regulating signalling through mTOR and JNK. In summary, this thesis identifies an important role for LAR phosphatase in the regulation of signal transduction, cell adhesion and cell metabolism.
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Wen, Jikai. "Characterization of nonsense mediated mRNA decay in Schizosaccharomyces pombe." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/490/.
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Nonsense mediated mRNA decay (NMD) is a translation-coupled process that preferentially destroys mRNAs harboring premature translation termination codons (PTCs). In mammalian cells, NMD is linked to pre-mRNA splicing. Typically, PTCs elicit strong NMD only if positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD. However, recent studies have questioned the importance of splicing and the EJC in NMD and instead they have proposed that, from yeast to mammalian cells, NMD is mostly determined by the distance of the PTC from the 3’ end. In this study, to investigate the link between pre-mRNA splicing and NMD, I used the fission yeast Schizosaccharomyces pombe. Many genes carry introns in fission yeast and unlike in Saccharomyces cerevisiae, the genome encodes for proteins homologous to EJC components. S. pombe is a powerful model organism which is easily genetically manipulated and we envisaged that studying NMD in this organism should facilitate the understanding of the molecular mechanism and in particular the link between NMD and splicing. During my PhD research, I have developed a versatile gene reporter system to study NMD; with it I discovered that splicing strongly enhances NMD in fission yeast and, surprisingly, that the EJC does not appear to be required. Unexpectedly, I found that splicing enhances NMD when the intron is either before or after the PTC, and furthermore, it does so only when the intron is close to the PTC. These observations suggest that the effect of splicing on NMD is direct and not a secondary consequence of splicing enhancing translation. Splicing is not an absolute requirement for NMD; I found that PTCs located early in the coding region could induce NMD even in the absence of a nearby intron. However, against the prediction of current models, I found no strong correlation with the distance of the PTC from the 3’ end. In summary, during my PhD a versatile system has been developed to study NMD in fission yeast and what I have observed challenges current NMD models and provides new mechanistic insights into NMD.
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Thomas, Huw. "A systems based approach to neutrophil gene expression." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/2008185/.
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Neutrophils are the major cellular constituent of blood leukocytes and play a central role in the inflammatory response, expressing an array of destructive molecules and antimicrobial processes that characterise the cells as front-line defenders of the innate immune system, thus neutrophils are crucial to host defence. It is now appreciated that neutrophils produce and respond to a variety of inflammatory signals and are able to regulate both the innate and adaptive immune response. The molecular changes that underlie this regulation are poorly defined, yet represent an attractive area of research to fully elucidate the role and regulatory capacity of neutrophils within the immune response. RNA-Seq provides an accurate and robust mechanism for global characterisation of cellular transcripts. Neutrophils were isolated from healthy donors and incubated with or without inflammatory cytokines for 1 h. RNA was extracted and analysed by RNA-Seq using the SOLiD or Illumina platforms. Raw data was quantified using a number of software packages which formed a bioinformatic pipeline for data analysis which was developed during the course of the research. Results were validated by a selection of traditional laboratory functional assays. Priming of neutrophils by GM-CSF and TNFα was found to induce differential gene expression and activation of transcription factors, which led to differential regulation of apoptotic pathways. Stimulation of neutrophils with inflammatory cytokines/chemokines (IL-1β, IL-8, G-CSF, IFNγ) resulted in expression of discrete gene sets and differential activation of signalling pathways. Stimulation of neutrophils with IL-6 did not induce any significant expression of genes but result in activation of STAT signalling. Comparison of gene expression of neutrophils isolated by density gradient and magnetic bead preparation revealed significant differences in gene expression and function, in part attributable to levels of contamination associated with each isolation method. Bead isolation was found to enrich a more heterogeneous neutrophil population including a subpopulation of neutrophils expressing transcripts previously associated with low density granulocytes. Thus, RNA-Seq and bioinformatic analysis has provided a full characterisation of neutrophil gene expression under inflammatory conditions and identified several new areas of research that could lead to targeted drug design for the treatment of inflammatory disease.
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Said, Amira. "Urinary pharmacokinetic methodology to determine the relative lung bioavailability of inhaled beclometasone." Thesis, University of Huddersfield, 2011. http://eprints.hud.ac.uk/id/eprint/12297/.
Full textAbstract:
Urinary pharmacokinetic methods have been introduced to identify the relative lung and systemic availability of inhaled drugs but have not been extended to corticosteroids. The main aims were to validate the urinary pharmacokinetic methodology when applied to inhaled beclometasone dipropionate (BDP), demonstrate the usefulness of the method and compare its indices to the in-vitro characteristics of the emitted dose. A simple and sensitive LC-MS method for quantifying BDP and its metabolites in methanol (for in-vitro studies) and urine samples was identified and validated in accordance with the FDA and ICH guidelines. The accuracy, precision, and recovery of the method were within acceptable limits (±15%). Twelve healthy volunteers completed the in-vivo urinary pharmacokinetic validation of the methodology to determine the relative lung bioavailability of inhaled beclometasone following inhalation. Twelve healthy volunteers received randomised doses, separated by >7 days, of 2000μg BDP solution with (OralC) and without (Oral) 5g oral charcoal, ten 100μg inhalations from a Qvar® Easi breathe metered dose inhaler (pMDI) with (QvarC) and without (Qvar) oral charcoal and eight 250μg inhalations from a Clenil® pMDI (Clenil). Subjects provided urine samples at 0, 0.5, 1, 2, 3, 5, 8, 12, and 24 hours post study dose. Urinary concentrations of BDP and its metabolites, 17-beclometasone monopropionate (BMP) and beclometasone (BOH) were measured. No BDP, BMP, or BOH was detected in any samples post OralC dosing. Post oral dosing, no BDP was detected in any of the urine samples and no BMP or BOH was excreted in the first 30 minutes. Significantly more (p<0.001) BDP, BMP and BOH was excreted in the first 30 minutes and cumulative 24 urinary excretions post Qvar and Clenil compared to Oral. Using 30 minute urinary excretion the mean ratio (90% confidence interval) for Qvar compared to Clenil was 231.4 (209.6, 255.7). The results confirm that the relative lung and systemic bioavailability can be identified from urinary excretion of BDP and its metabolites over the first 30 minutes and 24 hours respectively. The 2-fold difference between Qvar and Clenil is consistent with related clinical and pharmacokinetic studies. The low inter and intra-subject variability of the study confirms the reproducibility of this method. When compared to the in-vitro aerodynamics characteristics of the emitted dose, using standard compendial methods, the in-vivo indices showed a relationship to the fine particle dose (FPD) and the emitted dose (ED), respectively. The application of this urinary pharmacokinetic method was demonstrated in further studies to compare the effect of different spacers and different washing methods on the in-vivo drug delivery post inhalation from Clenil and Qvar inhalers in healthy volunteers. In addition, the in-vitro aerodynamic particle size distribution of the same inhalation methods has been investigated using the Andersen Cascade Impactor according to the standard compendial methodology. Urinary excretion, using 24 hour excretion, revealed that relative bioavailability to the body was reduced with spacers for both inhalers. There was no increase in the relative lung bioavailability when Qvar was used with spacers. When Clenil was attached to a spacer (either AeroChamber or Volumatic) the relative lung bioavailability was significantly greater only if the spacers were not rinsed after washing with detergents. Consistent with the above study there were correlations between the in-vivo urinary indices and the in-vitro characteristics of the emitted dose. The thesis highlights the extension of the urinary pharmacokinetic method to inhaled beclometasone dipropionate and provides further evidence of in-vitro in-vivo correlations between the urinary methodology and the aerodynamic characteristics of the emitted dose.
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Ghori, Muhammad U. "Release kinetics, compaction and electrostatic properties of hydrophilic matrices." Thesis, University of Huddersfield, 2014. http://eprints.hud.ac.uk/id/eprint/24269/.
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This thesis illustrates the behaviour of cellulose ethers during powder processing, compaction and drug release, as these are frequently employed in the fabrication of compressed hydrophilic matrices. The handling operations can give rise to the electrification of powder particles, which can affect the end product‘s quality. Controlling the parameters which can dictate the quality of compressed matrices is an ambition inherent in the development of pharmaceutical formulations. Thus, the aims and objectives of this thesis were to firstly study the electrostatic, surface adhesion, dissolution and compaction properties of plain polymers and model drugs. Secondly, binary mixtures of fixed drug to polymer ratios were made in order to investigate the effect of polymer concentration and physico-chemical attributes (particle size, chemistry and viscosity) on the tribo-electric charging, surface adhesion (SA), swelling, erosion, drug release kinetics and compaction properties of model drugs. It can be discerned that the both drugs charged negatively, whereas the methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) particles charged positively. The physico-chemical properties associated with MC and HPMC, such as particle size, chemical heterogeneity and molecular size of cellulose ethers all have a significant effect on charging and adhesion behaviour of plain MC and HPMC particles. Moreover, the concentration, particle size, chemical heterogeneity and molecular size of MC/HPMC all significantly affect the charging and SA propensity of the model drugs studied. The swelling and dissolution results confirm that the extent and rate of swelling, swelling exponent, dissolution rate and drug release kinetic parameters were affected by physico-chemical attributes (concentration, particle size, substitution and viscosity) of MC/HPMC and drug solubility. The mechanism of swelling and drug release was found to be anomalous. However, it inclined towards more diffusion-oriented swelling/drug release with higher MC/HPMC levels, viscosity, Hpo/Meo substitution ratios, drug solubility but smaller MC/MC particle size. The matrix erosion results obtained from newly developed phenol-sulphuric acid assay (PSA) method confirmed that the solubility of the drug, and levels of HPMC in a particular matrix tablet, significantly affect the matrix erosion rate and the results were similar to those determined using the much more labour-intensive gravimetric method. Moreover, the combination of conventional UV drug analysis technique and PSA assay can be used to simultaneously quantify the matrix erosion, polymer dissolution and drug release kinetics in a single set of experiments avoiding the need for separate studies. The compaction results confirmed that the FBP has poor compaction as compare to THP. The particle size, substitution ratios and molecular size of MC/HPMC affect the compaction and consolidation behaviour of plain MC/HPMC compacts. Furthermore, it can be noticed that the concentration and physico-chemical attributes (particle size, chemistry and molecular size) of MC/HPMC have a significant influence on the densification and consolidation process of hydrophilic matrices. In summary, the information obtained can be used in the future to develop and adopt strategies for development and further optimization of compressed hydrophilic matrices.
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Velanis, Christos N. "Regulation of transcription by Ultraviolet-B radiation in Arabidopsis thaliana." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6204/.
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Plants are sessile photo-autotrophic organisms and need to adapt constantly to a dynamic environment. Light is of utmost importance for plants to be able to monitor their surroundings. Ultraviolet-B radiation (UV-B; 280-315 nm) is an intrinsic part of sunlight and, depending on the wavelength and the fluence rate, it may be a stressful signal or an “informational” one. The so called photomorphogenic responses of plants to UV-B are largely mediated by the UV-B specific photoreceptor UV RESISTANCE LOCUS 8 (UVR8), which “senses” UV-B via a tryptophan based mechanism. UVR8 is localised in the cytoplasm and the nucleus mainly as a homodimer. Upon UV-B irradiation it splits to its monomers and accumulates in the nucleus where it has been found to interact with the E3 Ubiquitin ligase COP1. In the nucleus UVR8 has been shown to associate with chromatin on loci of UV-B responsive genes, including that encoding for the bZIP transcription factor (TF) ELONGATED HYPOCOTYL 5 (HY5), a key effector of UVR8-dependent signalling pathways. The binding of UVR8 to chromatin appears to take place via interaction with histones (H2B in particular) rather than DNA itself. However, this association with chromatin seems not to be UV-B specific. The above data suggest a mechanistic basis for an assumed function of UVR8 in the regulation transcription. It seems likely that UVR8 interacts with other proteins associated with chromatin to promote remodelling and/or recruits/activates TFs which in turn stimulate transcription of its target genes. The main objective of this study was to address the above working hypothesis.
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Guerrero, Flores Jimena Jazibel. "Conservation genetics of neotropical otters (Lontra longicaudis) in México." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/6247/.
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In this thesis I aimed to provide base-line data to inform conservation of neotropical otters (Lontra longicaudis ) at both the range-wide and local (Mexico) scale. In Chapter 2, I compared three commonly used preservation methods for faecal DNA in order to identify the best method for neotropical otter faeces under challenging field conditions and long-term storage: 1) ambient-temperature drying, 2) a two-step protocol involving incubation in 95% ethanol and posterior silica desiccation, and 3) RNAlater. The results of this experiment showed that that RNAlater provides the highest mtDNA amplification success. In Chapter 3, I looked into the demographic history, genetic diversity and genetic structure of L. Longicaudis in Mexico using mtDNA. I found high genetic structure among North and South regions of the country, potentially due to geographic formations. Analyses of demographic history in Mexico indicated a recent expansion coinciding with the end of the Pleistocene. Given that recent evidence supports the existence of three subspecies of L. longicaudis across its range, I combined mtDNA haplotypes identified in this study with available Central and South American haplotypes in order to examine phylogeographic patterns; as a result, a distinct lineage distributed in North and Central America (NCAM) was identified. Due to the monophyly of this lineage, I propose to consider it a distinctive Evolutionary Significant Unit (ESU). In Chapter 4, I used landscape genetics to identify landscape features that affect otter geneflow in Mexico by means of microsatellites. I looked into the effect of elevation, slope, river networks and land cover on geneflow at a country-wide scale and two regional scales (North and South Pacific). I used Bayesian clustering to examine country-wide genetic structure. In terms of landscape genetics, elevation and slope were the only variables that explained genetic distance among individuals at the range-wide and North Pacific scale, respectively. The results of Bayesian clustering indicated two population clusters roughly distributed in the North and South of Mexico. The results of this thesis suggest that non-invasive methods can be applied to inform conservation efforts for Neotropical otters. I suggest that the NCAM lineage should be considered a distinct Evolutionary Significant Unit (ESU) throughout the range of L.longicaudis. Within Mexico, it is recommended to plan conservation corridors for the species where naturally low elevations and slopes allow genetic connectivity.
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Jones, Nathaniel Gadsby. "Validating protein kinases of Trypanosoma brucei as drug targets." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4007/.
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Trypanosoma brucei spp. are protozoan parasites that cause Human African Sleeping Sickness and Nagana, a disease of cattle. The diseases have a very high mortality rate if untreated and the current drug treatments are inadequate due to toxicity and resistance. In order to develop new treatments, potential drug targets can be investigated in a candidate approach by genetic validation. In trypanosomes this can be performed by using RNA interference (RNAi) technology, which is well developed for this organism. One category of potential targets are protein kinases; members of this enzyme family have been shown to be suitable targets for relatively specific and selective, therapeutic inhibition- especially in the field of human oncology. T. brucei possesses 183 eukaryotic protein kinases, atypical protein kinase and pseudokinases that may be suitable for chemical inhibition. This study intended to genetically validated these targets and pursue interesting leads as potential drug targets. In order to perform RNAi studies on large numbers of candidate genes an existing stem-loop RNAi system was adapted to contain Gateway recombination sites. This created a highly efficient system, allowing the rapid generation of RNAi plasmids to target the entire complement of the predicted kinome of this organism. This collection of plasmids was used to generate a library of RNAi cell lines in bloodstream form parasites (BSF) which were screened for growth defects using an alamar blue cell viability assay. This identified 53 genes which were essential or important for proliferation of the BSF parasites. During this screen, a cell line was identified that contained an RNAi construct targeting two NEK kinases (NEK12.1: Tb927.8.7110, NEK12.2: Tb927.4.5310) which displayed a severe growth defect. This was replicated in a mouse model of infection. NEK 12.1 possesses an alanine gatekeeper residue and molecular modelling and virtual screening predicted this would allow the accommodation of bulky protein kinase inhibitors and therefore was investigated in more detail. Expression and purification of NEK12.1 in kinase active and dead forms allowed activity assays to be performed; these could be inhibited with the bulky inhibitor 3-MB-PP1, which also possessed activity against BSF parasites. Experiments to confirm the individual essentiality of NEK 12.1 (and its activity) remain to be performed, therefore NEK 12.1 has been partially genetically and chemically validated in this study. A unique “Orphan” kinase, containing a putative zinc-finger domain and a potential homologue of PDK1 were also investigated after the pTL RNAi screen. In vivo RNAi studies (using a mouse model) correlated well with the in culture RNAi data, demonstrating that these targets are essential in mammalian infections. During the pTL RNAi screen it was noted that the knockdown of one protein kinase, (DFK) led to a change in the morphology of the cells, yet no reduction in the alamar blue ratio was observed. Further investigation showed that after 72 h of RNAi induction 17% of cells expressed EP procyclin. This was associated with detectable changes in the transcriptome (ascertained by RNA -Seq), that were consistent with a BSF-PCF differentiation event. DFK was predicted to contain multiple transmembrane domains and features suggestive of a receptor kinase. Epitope tagging of DFK followed by cell fractionation and immunofluorescence microscopy demonstrated that the protein localised to the cell membrane. Transmission immuno-electron microscopy confirmed the cell membrane localisation and suggested that the protein kinase domain of DFK was intracellular. This study reveals several protein kinases as new drug targets, in turn identifying one as a key regulator of BSF-PCF differentiation. Due to the tractability of T. brucei it also provides a collection of plasmids and cell lines that should further the investigation of other key biological processes in this important pathogen.
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Pieszko, Marta. "Molecular regulation of the macroschizont to merozoite differentiation in Theileria annulata." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6079/.
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Theileria annulata is an intracellular, tick-transmitted apicomplexan parasite, which causes tropical theileriosis in cattle. It undergoes a complex life cycle with several distinct stages occurring within the bovine host and tick vector. ApiAP2 proteins are key candidate transcription factors for regulation of stage specific gene expression across apicomplexans. They are differentially expressed in specific developmental stages and certain ApiAP2s bind specifically to unique DNA sequence motifs. Identification of stage-specific expression of putative transcriptional regulators, the motifs they bind to and potential target genes provided the rationale for this study to understand the molecular mechanisms that control stage differentiation to the merozoite in T. annulata. The results demonstrated that T. annulata ApiAP2s show marked differences in expression levels during the parasite life cycle. ApiAP2 target DNA motifs orthologous to those in Plasmodium and Cryptosporidium were also discovered in Theileria intergenic regions, indicating that the genes downstream are potential targets of Theileria ApiAP2s. These motifs were also found in upstream regions of up-regulated TaApiAP2 genes, suggesting possible auto-regulation and an interaction network of ApiAP2 transcription factors. Importantly ApiAP2 fusion proteins up-regulated during differentiation to the merozoite stage bound to their predicted specific DNA motifs validating that ApiAP2 DNA-binding domain structure is conserved across Apicomplexa genera. Evidence was also produced that AP2 proteins play important roles in steps that commit a cell to differentiate: TA13515D is the orthologue of the AP2G factor in Plasmodium that is a major regulator of gametocytogenesis: TA16485 may be involved in down-regulation of genes during merogony and expression of TA11145 at a higher level in a cell line competent for merogony relative to a line severelly attenuated indicated involvement in regulation of this differentiation step. Discovery of multiple nuclear factors binding to a 2x(A)CACAC(A) motif implicated in autoregulation of TA11145, together with phylogenetic evidence for a clade of related domains that bind this motif suggest that multiple competing ApiAP2s may operate to regulate stochastic commitment to merozoiteproduction. Based on this data an updated stage differentiation model has been generated, with up regulation of the TA11145 gene a key event. A C-box motif association with genes implicated in establishment of the transformed host cell (TashAT, SVSP) suggests it could be important for deregulation of this event as the parasite undergoes stage differentiation. In contrast the inverse G-box was found associated with genes up-regulated from merozoite to piroplasm. EMSA analysis of parasite nuclear extract with a G/Cbox motif probe showed that the motif is an active binding site for a stage regulated nuclear factor. Specific binding of candidate TA12015 protein to the G/C-box motif was unable to be confirmed. Taken together, these results provided evidence that ApiAP2 proteins are regulators of stage-specific gene expression in T.annulata. They also provide insight into probable ApiAP2 interaction networks and support the postulation of a differentiation mechanism conserved across the Apicomplexa. Finally, the data suggests that this mechanism is stochastic and is likely to occur via a positive feedback loop generating a threshold that commits the cell to differentiate to the next stage of the life cycle.
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Hernandez-Martinez, Juan-Manuel. "Role of kynurenines and oxidative stress in the differentiation of SH-SY5Y cells." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6133/.
Full textAbstract:
Neuroblastoma is the most common solid extracranial tumour in children. The neuroblastoma SH-SY5Y cell line is a third successive subclone established from a metastatic bone tumour biopsy. It can be induced to differentiate (regress) into a neuronal phenotype when treated with any of several molecules including retinoic acid (RA). This characteristic has been exploited in several studies that use the SH-SY5Y cell line as a neuronal model. These studies have had far- reaching implications in shaping our understanding of certain key aspects of neurotoxicity and neurodevelopment yet their genuine relevance becomes evident when approached from an oncological point of view, as they provide information about the process underlying tumour regression which in turn can lead to the development of better therapies for the clinical management of this malignancy. It has been shown both in vitro and in vivo that several tumours constitutively catabolize the essential amino acid tryptophan (Trp) promoting cancer- associated inflammation, immune response suppression, immune escape and tumour outgrowth. The main degradation pathway of Trp is the kynurenine pathway: it involves its transformation into several bioactive compounds such as kynurenic acid (KA) and quinolinic acid (QA). QA has been implicated in several neurodegenerative diseases where it is believed to induce excitotoxic neuronal death through the activation of the N-methyl-D-aspartate (NMDA) receptor, a type of ionotropic glutamate receptor, as well as by causing oxidative stress and energy metabolism disruption. Conversely, KA acts as an NMDA receptor antagonist and exerts neuroprotection. Similarly, glutamate signaling and its dysregulation has been implicated in the development and progression of cancer. Furthermore, several glutamate receptor antagonists, including kynurenic acid, have been shown to inhibit the proliferation and migration of neoplastic cells. Conversely, it has recently been reported that QA increases the proliferation of SK-N-SH cells and protects gliomas against oxidative stress by acting as a precursor of NAD+. In view of all that has been mentioned thus far, the SH-SY5Y cell line was used as a model to investigate the effect of certain Trp metabolites such as QA, KAand 3-hydroxyanthranilic acid (3-HAA) on cellular morphology, viability and neurite extension. An important part of this study was to determine whether the available methods could reliably be employed to investigate these parameters in the SH-SY5Y cell line. It was confirmed that the acquired SH-SY5Y cell line retains its ability to differentiate and to die, and that both processes can be accurately quantified. Additionally, the optimal culturing conditions for the SH- SY5Y cell line were determined. Treatment with RA (10 μM) was used as a positive control of differentiated SH- SY5Y cells. Overall, the morphology adopted by cells after QA (50 μM) treatment was similar to the one that follows RA-induced differentiation. It was demonstrated that QA caused an increase in the neurite/soma ratio in SH-SY5Y cells, which was confirmed by Western blot analysis as evidenced by an increase in the total cellular content of β3-tubulin. These results were also confirmed by a neurite outgrowth assay that selectively quantified the neuritic mass present in cultures. However, unlike RA, QA did not decrease the levels of the neuronal proliferation marker doublecortin; the term neuritogenesis is therefore more appropriately used to refer to the series of morphological and molecular changes induced by QA in SH-SY5Y cells. The morphological changes induced by QA were not reproduced by application of NMDA, nor were they inhibited by blockade of the NMDA receptor with MK-801. Furthermore, SH-SY5Y cells were not susceptible to NMDA excitotoxic death. In view of this, the expression of GluN1 protein was determined by Western blot. GluN1 could not be detected in either undifferentiated or differentiated SH-SY5Y cells, confirming that QA-induced neuritogenesis occurs through a mechanism independent of NMDAR activation. The results herein contained suggest that the SH-SY5Y cell line does not have functional NMDARs, nonetheless it is recognized that a more exhaustive study would be necessary to fully establish which glutamate receptor subtypes are found in the SH-SY5Y cell line. The effect of QA on the production of reactive oxygen species (ROS) was also investigated. QA caused an increase in the intracellular levels of ROS as evidenced by an increase in the fluorescence of oxidised ethidium. Additionally QA-treatment caused an increase in the expression of NRF2, a transcriptionfactor that responds to oxidative stress and which has been implicated in ROS- induced differentiation in SH-SY5Y cells. In contrast, superoxide dismutase (SOD; 300 U/ml) significantly reduced the levels of ROS induced by QA treatment, which in turn caused an increase in cell proliferation and a reduction in the number of neurites. Similarly, diphenylene iodonium (DPI; an inhibitor of NADPH oxidase) also inhibited QA-induced neuritogenesis. These results suggest that the action mechanism of QA is mainly via the production of ROS, most likely superoxide (O2•-) through NADPH-oxidase. Interestingly, nicotinamide (1 nM-1mM; another precursor of NAD+) caused a dose dependent increase in the number of neurites and in the expression of β3- tubulin, which suggests that the action mechanism of QA may be mediated by metabolites of the nicotinate and Nam pathway, including NAD+ either before or after the induction of ROS. Cells were treated with 3-hydroxyanthranilic acid (3-HAA) in order to ascertain whether other pro-oxidant molecules could induce neuritogenesis as well. Single and repeated application of 3-HAA (100 μM) induced cell death in SH-SY5Y cells. Furthermore, when 3-HAA was delivered in combination with SOD, there was a shift in the IC50 values indicating that toxicity was potentiated by SOD. Catalase (CAT; 100 U/ml) afforded complete protection from the exacerbated damage induced by the single co-application of 3-HAA + SOD. However, when repeated treatments were performed, CAT no longer afforded any protection. Interestingly, the serum concentration in the medium did not affect the IC50 of 3-HAA but it did modulate the response to CAT, indicating that the specific ROS produced after 3-HAA treatment depend on the medium in which 3-HAA is delivered. At sublethal doses, 3-HAA interfered with the expression of NeuN (neuronal marker) through a mechanism that involves high production of ROS. The ability of some kynurenines to induce differentiation and cell death in SH- SY5Y cells may open new and exciting avenues of research. If these results can be confirmed in vivo they could impact the way in which certain neuroblastomas are treated.
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Khojah, Sohair Mohammed. "Ageing in the mammalian brain." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6244/.
Full textAbstract:
With a globally ageing population diseases associated with this natural process are becoming major issues worldwide. Research into the process of ageing and its concomitant issues is rapidly expanding; the need for new tools and models to investigate this rapidly expanding arena of research is paramount. The discovery of a spontaneous mutation in the AS rat strain which introduces a premature stop mutation into the gene encoding protein kinase C γ (PKCγ) lead to the development of a model for one such age related disorder, Parkinson's disease. Consequently, this model has been selected to investigate age related changes in specific areas of the brain (the cerebellum, basal ganglia, cerebral cortex and brainstem). These regions were selected because they have previously been shown to demonstrate changes with age (cerebellum, cerebral cortex and basal Ganglia), they show differences between the AS and AS/AGU strains (basal ganglia) or they show differences in PKCγ knockout models (cerebellum). The Brainstem was selected as it shows little change due to age and shows no differences in PKCγ knockouts or AS/AGU rats. This study used established qPCR methods to measure a validated biomarker of ageing, CDKN2A (the transcript for p16INK4a) in the brains of these rats to determine whether this model is in fact a genuine model for accelerated ageing. This thesis demonstrates that CDKN2A expression, in combination with senescence-associated β-galactosidase staining, provides clear evidence of accelerated ageing in the brains of AS/AGU rats when compared with the parent AS strain. These investigations were furthered by an investigation of members of the Sirtuin family of proteins. The changes in expression of these Sirtuins indicates that there may be increased levels of cellular stress, disruption of metabolism and DNA damage in the AS/AGU rats, this would be congruent with the accelerated ageing phenotype present in this strain. Furthermore, the levels of these Sirtuins were in line with the predictions from the MTR trinity in regards to the accelerated ageing phenotype. Whilst some of the changes in senescence and metabolic disruption may be attributable to the PKCγ mutation in the AS/AGU rats, it would appear that there is some element of accelerated ageing that is independent of this mutation.
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Welden, Natalie Ann Cooper. "Microplastic pollution in the Clyde sea area : a study using the indicator species Nephrops norvegicus." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6377/.
Full textAbstract:
Microplastic pollution has been identified as an ever increasing proportion of marine litter. Despite an increase in microplastic awareness over the last decade, it represents an as yet unquantified threat to the marine environment. The relatively few studies that monitor its distribution and impact have illustrated a range of worrying effects on marine habitats and communities. The Clyde Sea Area (CSA) is subject to many sources of terrestrial and maritime plastic input. The use of plastics in recreational and commercial vessels throughout the CSA is believed to result in large levels of microplastic fibres, which have previously been seen to be ingested by a range of marine organisms. In a study of the breakdown of commonly used polymers in benthic environments, it was found that ropes of 10 mm diameter in sub-tidal conditions release between 0.086 and 0.422g of microfibers per meter per month in the early stages of degradation. This rate would be expected to increase over subsequent months, releasing substantial amounts of fibres into the CSA environment. In addition to the presence of numerous sources of microplastics, the CSA is relatively enclosed, and may accumulate high levels of debris as a result. Monthly sampling of the water and sediment in the CSA revealed contamination similar to that observed in other near-shore environments. Thus, it is expected that the potential threat to organisms in other areas will be similar to that observed in the CSA. One organism known to take up microplastics is the Norway lobster, Nephrops norvegicus, the target of the main fishery in the CSA. In this work we examined the levels of microplastic in the gut of N. norvegicus from the Scottish waters. Examination of individuals from the CSA revealed both a high occurrence and high accumulation of microplastic. This was found to be much greater than in N. norvegicus sampled from more remote Scottish waters. As a result, N. norvegicus from the CSA are most likely to suffer from the negative impacts associated with microplastic ingestion than those in offshore or in areas of low anthropogenic activity. In order to determine the potential impacts of microplastic ingestion on N. norvegicus, we first examined the mechanism by which N. norvegicus retain and egest microplastic. The position of microplastic aggregations in the foregut indicates that the gastric mill is the main obstacle to microplastic egestion. Inducing moult in microplastic-fed individuals demonstrated that expulsion of the gut lining during ecdysis enables N. norvegicus to reduce their plastic load, limiting plastic aggregation to the length of a single moult-cycle. In an 8 month controlled-feeding experiment retained plastic was seen to have a range of impacts on N. norvegicus. Feeding rate and body mass was seen to decrease in plastic loaded N. norvegicus, and a reduction was observed in a number of indicators of nutritional state. The results presented in this thesis have a number of implications to the CSA and wider marine environment. The similarity in the level of microplastic observed in the CSA to that of other studies of inshore waters indicates the potential for high microplastic uptake by crustaceans in those areas. The high variability in observed microplastic abundance suggests that small-scale monitoring is unsuitable for monitoring marine microplastic debris, and that use of an indicator species may provide a more reliable method of monitoring that is not subject to small-scale heterogeneity in distribution. The seasonal retention of microplastic by N. norvegicus indicates that crustaceans may provide a suitable indicator of local contamination. However, in the CSA, the high level of fibre aggregation and observed impacts of prolonged retention indicate that microplastic may be causing further pressure on an already exploited resource, reducing the stability of the valuable N. norvegicus population.