The family of mammalian 2-oxoacid dehydrogenase complexes (PDC, BCOADC and OGDC) are stable, high Mr assemblies composed of multiple copies of 3 separate enzymes (E1, E2 and E3) that catalyse key stages in carbohydrate and amino acid metabolism. Their respective E1 and E2 enzymes are complex-specific while E3 is the identical gene product in all 3 complexes. In general terms, the oligomeric E2 ‘cores’ provide the structural and mechanistic framework to which their partner E1 and E3 enzymes are tethered tightly but non-covalently. However, the mode of E1 and E3 binding differs significantly from complex to complex. In the BCOADC, its cubic E2 core is composed of 24 identical subunits to which E1b and E3 bind stably in a mutually-exclusive fashion via multiple subunit binding domains (SBDs). In a variation of this theme, the icosahedral (60-meric) E2-PDC core comprises 2 types of subunit, E2 and an E2-related polypeptide, E3 binding protein (E3BP). In this case, E1p and E3 bind independently to specific SBDs located on E2 and E3BP, respectively. In contrast, OGDC differs significantly from its counterparts as its 24-meric E2 core does not contain any apparent SBDs. In addition, there is no equivalent to E3BP in this complex. Hence, how stable complex formation is achieved for the OGDC is still an area of active research, particularly in view of increasing evidence implicating OGDC deficiency as a major causative factor in a variety of neurodegenerative and oxidative stress disorders. Previous subunit-specific proteolysis, enzymatic and immunological studies on native bovine OGDC in our laboratory have suggested that an intact E1o N-terminal region is vital for maintaining the structural integrity of the complex. In particular, a single cleavage of E1o at Arg77 results in complete loss of OGDC function stemming from dissociation of both E3 and a large, active E1o species (E1') from the native E2 core assembly. The principal aim of this thesis was to establish the location and precise nature of the domains responsible for protein-protein interactions between the constituent E1o, E2o and E3 enzymes of OGDC and their roles in assembly, taking into account our previous data and the unique domain organisation of E2. It was also a goal to produce a recombinant version of the human OGDC for future biomedical studies including genetic analysis of naturally-occurring mutant forms. Initially, the cloning, expression and purification of a series of E1o N-terminal constructs (His-tag, GST or MBP fusion proteins: 60, 90 and 153 a.a.s in length) is described extending from Ser1 to Phe153 of mature human E1o. Access to 10-30 mg of highly-purified E1o N-terminal peptides was required to enable testing of the ability of this region to interact with E3 (and also E2) employing a range of biochemical and biophysical techniques. High-level expression of full-length E1o was also achieved; however, attempts to produce active E1o in soluble form proved unsuccessful. Recombinant human E2o and E3 were both produced as soluble active enzymes in high yield. A preliminary structural characterisation of the E1o N-terminal region was also undertaken employing synthetic peptides, circular dichroism and a basic bio-informatics approach. These studies demonstrated that the N-terminal region had the potential to form 2 short α-helical segments linked by regions of unstructured and flexible polypeptide chain. Moreover, a 3D-structural prediction for mature, full-length human E1o confirmed that its N-termini were highly accessible, extending above the enzyme surface and situated in close proximity at one end of the homodimer. Although there was no apparent sequence homology, our data also suggested that this region had several of the main structural features of the E3-SBD located on E3BP. Direct evidence that the N-terminal region of E1 bound to E3 post-translationally was obtained using peptide array technology, alanine scanning, isothermal titration calorimetry (ITC), affinity chromatography and gel filtration (GFC). Interaction of E1o N-terminal peptides with E3 was salt-sensitive and reduced over the range 0-0.5M NaCl, i.e. similar to that required to promote E3 dissociation from intact OGDC. Only the longer E1o-90 and E1o-153 constructs complexed with E3 and evidence suggested that steric hindrance by the fusion partner blocked E3 binding to the short E1o-60 GST fragment. As expected, in the absence of any obvious SBD on E2o, no direct interaction could be detected between E2o and E3 using ITC or GFC. In addition, no post-translation association occurred between our purified E1o constructs and fully-assembled, oligomeric E2o. In contrast, co-expression of E1o-90 and E1o-153 constructs with E2o (but not E1o-60) resulted in the formation of an E1o-90/E2o GST or E1o-153/E2o GST sub-complex. Moreover, these N-terminal E1o/E2o sub-complexes supported stable E3 binding as judged by affinity chromatography and GFC. Taken together, the data presented in this thesis have established that the E1o N-terminal region is pivotal for mediating formation of a stable multi-enzyme assembly by directing self-integration with the E2o core during or immediately after synthesis and subsequently promoting high-affinity E3 binding in a manner reminiscent of E3BP integration with the oligomeric E2-PDC core. To test the functional importance of the putative E3-binding domain on E1o, the E1o constructs were employed as inhibitors of OGDC and PDC activity since it was anticipated that they should displace E3 from its normal binding site in the intact complexes. Incubation of OGDC or PDC with the E1o-90 and E1o-153 (but not E1o-60) constructs caused preferential inhibition of OGDC activity. Conversely, an E3BP-SBD construct was a more effective inhibitor of PDC suggesting that the mode of E3 interaction differs significantly in these 2 complexes. In summary, our data have provided (a) new insights into the structure, organisation and mode of assembly of the mammalian OGDC; (b) suggested new approaches to producing a recombinant model OGDC as an important biological tool for future biomedical and genetic studies and (c) raised new questions concerning the subunit composition, architecture, and stoichiometry of its core assembly.

Bcl-2 proteins are very well known regulators of the programmed cell death. Accumulating evidence suggests that they are also involved in regulation of calcium signalling events. Bcl-2 has been reported to affect calcium release from the intracellular calcium stores through regulation of inositol trisphosphate receptors and endoplasmic reticulum calcium pumps. Physiological and pathological processes in pancreatic acinar cells are controlled by calcium. Intracellular Ca2+ signals regulate not only gene expression and trigger enzyme secretion but also might contribute to premature trypsinogen activation and development of pancreatitis, which is characterised by extensive necrosis of the pancreatic tissue. Detailed investigation of Bcl-2 family-dependent mechanisms of intracellular Ca2+ regulation and its association with cell death induction is required for understanding of the basic physiological signalling pathways as well as pathophysiological processes leading to development of severe diseases of pancreas. This study investigates the effects of Bcl-2 family proteins on intracellular calcium homeostasis, with particular focus on their involvement in Ca2+ fluxes and CICR phenomenon. Also, the Ca2+-related actions of different doses of ethanol in pancreatic acinar cells and their contribution to pancreatitis are presented and assessed. The results indicate that pharmacological inhibition of anti-apoptotic Bcl-2 and Bcl-xL proteins with BH3 mimetics BH3I’-2' or HA14-1 sensitizes pancreatic acinar cells to CICR; and overexpression of Bcl-2 has the opposite effect significantly decreasing rising phase of CICR-types of responses in pancreatic cell line AR42J. Responses to BH3 mimetics are at least partially dependent on both IP3Rs and RyRs, 5 since inhibition of either of them results in a substantial decrease in Ca2+ release from the intracellular stores. However, simultaneous blockade of IP3Rs and RyRs did not completely abolish BH3 mimetic-elicited Ca2+ release, which indicates engagement of other factors in the development of the response. Importantly, the effects of BH3 mimetics on intracellular Ca2+ were effectively inhibited by loss of Bax protein, suggesting Bax involvement in the regulation of Ca2+ release from the ER. Further, the results presented here demonstrate that moderate concentrations of ethanol (10 - 100 mM), although having only a minor effect on intact cells, induce substantial Ca2+ release from both the ER and the acidic store in permeabilized cells, and trigger intracellular trypsinogen activation - the hallmark of acute pancreatitis. The data suggest that calmodulin, which is present in intact cells but is lost in permeabilized cells, constitutes a part of natural defence mechanism responsible for the differences in the severity of the responses to ethanol. What is more, the evidence indicates that specific pre-activation of calmodulin by Ca2+-like peptides boosts this defence and reduces the pathological calcium responses to ethanol as well as to BH3 mimetics in pancreatic acinar cells decreasing necrosis. Finally, the effects of Bcl-2 protein on calcium fluxes in pancreatic acinar cells were investigated. Cells lacking functional Bcl-2 showed substantially more rapid clearance of thapsigargin / high Ca2+-induced cytosolic calcium plateau as compared to wild type cells. This effect has been explained by increased activity of the PMCA that results in a marked increase of apoptosis / necrosis ratio in oxidative stress induced cell death. Overexpression of Bcl-2 in AR42J cells reduces ER calcium content, while silencing of Bcl-2 expression by siRNA results in substantially increased releasable calcium 6 pool in the ER. The data indicate that at least a fraction of Bcl-2 in both AR42J cells and pancreatic acinar cells locates in the ER and is present in close proximity to the plasma membrane, making possible the direct regulation of the PMCA. In conclusion, this study provides new insights into roles of Bcl-2 family proteins and calmodulin in intracellular calcium homeostasis. This thesis presents evidence for involvement of anti-apoptotic Bcl-2 members in Ca2+-induced Ca2+ release; demonstrates previously unknown regulation of the PMCA by Bcl-2; and suggests involvement of Bax protein in regulation of Ca2+ release from the intracellular stores. The study also proposes that Ca2+-like peptides can boost natural protective mechanisms and suggests their potential applications as therapeutic agents.

The feto-maternal interface is vital to promote growth and development of the placenta while maintaining tolerance and surveillance through the immune system. Pathogens are detected through pattern recognition receptors, which have a key role in the innate recognition by transducing signals from pathogen-associated molecular patterns. Lipopolysaccharide, a PAMP of Gram-negative bacterium, is recognized by the Toll-like receptor. Cluster of differentiation (CD) 14 and macrophage receptor with collagenous structure (MARCO) are key players in innate recognition. The hypothesis derived from the above is that MARCO might be expressed on trophoblast cells and plays a valuable role in association with CD14. The interaction of CD14 and MARCO was explored with the use of confocal microscopy which showed physical associations, most likely contributing to their function at the feto-maternal interface. Trophoblast responses to LPS indicated a significant role in regulating the expression of CD14 and MARCO and NF-κB translocation and activation. It was also hypothesized a correlation between the down-regulation of Myoferlin and vascular endothelial growth factor (VEGF), and the decreased cell proliferation of trophoblast cells upon treatment with LPS. Myoferlin and VEGF quantification estimated by flow cytometry and western blotting showed significant decrease in LPS treated JEG-3 cells in time and dose dependent manner. The cell proliferation assay revealed a significant decrease in trophoblast cell growth of LPS treatment suggesting it is associated with the decrease of Myoferlin of which the expression is reported to be correlated with VEGF. These data suggest that CD14 and MARCO can be modulated and thus might contribute to feto-maternal tolerance and surveillance preventing pregnancy complications such as preeclampsia.

The mammary gland is a complex organ, relying upon multiple cell types and signalling pathways to orchestrate its predominantly postnatal development. To obtain a detailed understanding of such development, and also the mechanisms whose deregulation lead to mammary tumourigenesis, a physiologically relevant, quantifiable and qualitative model system is required. In vitro, currently available mammary culture systems have allowed studies into prospective stem cell populations, cell-cell interactions, differentiation, proliferation, paracrine networks and hormonal responses, but are limited in the degree to which they can truly recapitulate in vivo mammary biology. Three dimensional organoid culture systems from many tissues have recently been shown to concurrently allow sustained stem-cell maintenance, proliferation and functional differentiation; however, no such recapitulative in vitro model currently exists for the mammary gland. This thesis therefore details the development of a novel murine mammary organoid culture system. Culture conditions described, including R-Spondin1 and Neuregulin1, enable the development and expansion of mammary epithelial organoids for up to 2.5 months in culture. Organoids possess distinct basal and luminal compartments - functional steroid receptors in the latter allowing hormonal responses - and regenerative cell populations enabling mammary gland reconstitution in vivo. Additional conditions are also described promoting the growth of abnormal, tumour-like organoids. Utilisation of the organoid model allowed the study of key signalling pathways associated with mammary development; most notably revealing the critical importance of the Wnt signalling pathway in regulating normal development, its role in mammary tumourigenesis and demonstrating its potential as a target in anti-tumour therapy. Cell-cell signalling, individual cell populations and behaviours, and tumour organoid growth were studied, the latter offering a platform in which to study potential therapeutic compounds. Evidence provided furthers the understanding of mammary biology and supports that the organoid model comprises a suitable physiologically relevant tool for use in mammary research and drug discovery.

No description available.

Introduction: The risk of chronic kidney disease (CKD) aith impaired glucose regulation (IGR) is not well characterised. It is not clear whether individuals with IGR are at risk of developing CKD or whether the risk is confined to individuals who progressed to overt diabetes. Methods: This study consists of three main parts: 1) systematic review, 2) analysis to determine incidence and prevalence of IGR, and 3) analysis to determine risk of CKD in IGR. A systematic review was undertaken using a large dataset of patient records to describe the incidence and prevalence of IGR and to investigate the relationship between IGR and CKD. Results: The systematic review found no evidence associating risk of CKD in young adults aged 18 to 40 years with IGR. The THIN database analysis shows that incidence and prevalence increased consistently throughout the study period. Incidence of CKD was 4 times higher in IGR than normoglycaemia. The incidence of CKD stage (3-5) was approximately 4 times higher than the incidence of CKD stage (1-2) in the IGR cohort. Conclusion: The systematic review demonstrates that the risk of CKD in young adults with IGR remains to be elucidated. The THIN database analyses provides evidence of an increased risk of CKD amongst young adults with IGR. It also showed that IGR patients are at higher risk of CKD stage (3-5) compared to CKD stage (1-2).

Synovial fibroblasts and macrophages are incriminated in rheumatoid arthritis (RA) but the interactions between these cells and the roles of cellular subsets are only partially understood. To address this, fibroblasts isolated from normal, resolving arthritis, very early RA, established RA, and longer duration RA patients were co-cultured in vitro with myeloid cells; synovial fibroblast and macrophage subsets were identified, and the transcriptomes of synovial cells were analysed. Co-culture of fibroblasts and monocytes elicited an increase in IL-6 release and a reduction in CCL2/CCL4 levels. No difference in response was elicited by fibroblasts from different stages of RA. Fibroblast and macrophage markers were defined in frozen tissue sections. A synovial tissue digestion protocol was then developed and used to isolate synovial cells. Fibroblast and macrophage populations were identified and the proportions of fibroblast subsets correlated with clinical variables. Transcriptional analysis identified differentially expressed genes between fibroblast subsets and one distinct macrophage population. Analysis of previously generated transcriptome data for fibroblasts from RA stages identified cassettes of genes differentially expressed at each disease stage. This work expands previous findings on fibroblast function by beginning to assign functions to subsets and demonstrating that fibroblasts from stages of RA have distinct gene expression.

Increasing lines of evidence place platelets as having a central role in liver disease. Platelets are recruited to the liver and, depending upon stage and type of liver injury play varying roles ranging from driving liver fibrosis to aiding regeneration. However the molecular basis and consequences of platelet activation in the liver are less clear. The work presented in this thesis demonstrates for the first time that platelet activation via CLEC-2 is important in the pathogenesis of liver disease. In chronic human diseases (CLD) such as Primary Biliary Cirrhosis, and Alcoholic Liver disease I have demonstrated that the ligand for CLEC-2, podoplanin is upregulated on portal venules and increases proportionately to disease activity. I also note podoplanin staining on macrophage populations in CLD. Furthermore I show that this enhanced podoplanin expression may be a useful predictor of portal venous thrombosis, and correlates with MELD score for some categories of disease. In acute liver injury, CLEC-2-depended platelet activation has a profound effect on disease development. Here podoplanin expression occurs upon Kupffer cells in both humans and mice. Using carbon tetrachloride and paracetamol to induce acute liver injury in mice, I show that macrophage-expressed podoplanin activates platelets via CLEC-2. This interaction worsens liver injury, I next show that by blocking this interaction (using either CLEC-2 or podoplanin-deficient mice, or by using a function- blocking podoplanin antibody) liver recovery from toxic liver injury was remarkably enhanced. This was dependent upon enhanced hepatic neutrophil recruitment in a TNF dependent fashion.

Antibiotic resistance is one of the greatest scientific and medical challenges of the 21st century. However, our arsenal to combat this surge is limited to a small number of antimicrobial drugs. Plasmids are major contributors to the problem, yet specifically targeting them to reduce the burden of resistant infections has not been fully exploited. It is possible to repress plasmid’s replication and eliminate them from a population by harnessing their innate replication and maintenance functions. This concept has been applied in this study to construct conjugative broad-host range plasmids aimed at curing either IncF or IncK plasmids based on IncPα RK2 and pUB307 backbones. pUB307- based pCURE gave a stronger curing phenotype as tested by a novel simple curing experiment and long-term invasion assays. The curing potentiation was investigated and found to be due to the absence of iteron 10 in oriV and a resulting higher copy number. The clinical potential of the technology was demonstrated by successfully curing a novel resistance plasmid, pEK499 and by in vivo mice experiments where pCURE conjugated into and cured target plasmids from E. coli established within the gastrointestinal tract. With further work to ensure long-term curing and biosafety, pCURE could improve the treatment options for multi-drug resistant infections.

Over the last decade, the transmission of zoonotic malaria from non-human primates to humans has emerged as a public health problem and possible threat to malaria elimination in Southeast Asia. A major outbreak of the macaque malaria parasite Plasmodium knowlesi in humans began in Malaysian Borneo in 2004 and is now the primary cause of malaria in this region. This simian parasite is transmitted by mosquitoes in the Anopheles leucophyrus species complex. The emergence of P. knowlesi has been tightly linked to land-use change, particularly the widespread deforestation occurring in the state of Sabah where the largest focus of human infection is found. Efforts to combat this disease and understand its emergence and future spread in humans are hindered by limited knowledge of mosquito vector ecology and behaviour; and the risks of exposure to vectors in changing landscapes. This PhD aimed to address these knowledge gaps by carrying out a series of field studies near the epicentre of human P. knowlesi cases in Sabah, Malaysian Borneo, to elucidate P. knowlesi vector ecology, behaviour and transmission potential, verify associations between land-use type and human exposure risk, and characterize the dynamics of transmission within reservoir macaque populations. In combination this information will deepen understanding of P. knowlesi transmission and emergence, and provide insights for the control of this and other emerging zoonotic malarias. My initial study evaluated new sampling methods for collecting resting P. knowlesi vectors. Resting collections are valuable for characterization of mosquito habitat and host species choice, however no standard methodology is currently available for P. knowlesi vectors. I evaluated two simple traps, resting buckets and sticky resting buckets, for sampling resting P. knowlesi vectors within two villages in Kudat District, Sabah. The performance of traps was evaluated, and the relative abundance and host choice of resting mosquito vectors was compared across eight different habitat types representing a gradient of deforestation. In 5748 trap days, a total of 2212 mosquitoes were collected in resting collections, but none were malaria vector species. Culex and Aedes genera dominated collections; with the former being most abundant in resting bucket traps and CDC aspirator catches, and the latter in sticky resting bucket traps. Several other vector species were collected including the sylvatic dengue vector Aedes albopictus, and Culex vectors of filarasis and Japanese encephalitis. Consequently these simple resting traps could be effective for studying the ecology of a range of other important mosquito vectors in Sabah even if not those responsible for malaria. In a following study I investigated associations between habitat and human exposure to P. knowlesi vectors, and tested for associations between vector abundance and human infection risk across a broad geographic range in Sabah. Previous studies indicated that the primary P. knowlesi vector was An. balabacensis. This vector was more abundant in a village than forest site, conflicting with the original hypothesis that humans are at greatest risk of infection in forests and suggested the possibility of peri-domestic transmission. However this inference was drawn from a limited number of sampling sites in only one district, Kudat, within Sabah. To test this hypothesis over a broader geographical scale, I conducted extensive entomological sampling across four districts in Sabah. Human landing catches were performed to measure human biting rates in forest, farm (plantation) and peri-domestic habitats in 11 villages. Prior to entomological sampling survey of human sero-positivity to P. knowlesi was conducted in all villages, carried out as part of a larger research programme. Making use of this data, I tested for associations between vector abundance and human infection risk at the village level. The primary vector An. balabacensis was found in all four districts, but at much lower relative abundance than in pilot work from Kudat. Additionally this vector was more abundant in forest and farm habitats than in peri-domestic settings. Only 1 of the 32 An. balabacensis collected in this study tested positive for P. knowlesi; an individual caught in a forest site. No significant association between the mean abundance of An. balabacensis and human P. knowlesi sero-positivity was detected in this study. However the relatively small sample size of mosquitoes and sites used here meant there was relatively low power to detect such an effect. This study highlights the importance of incorporating geographical heterogeneity and replication when assessing mosquito-habitat associations, and the need for more intensive longer-term sampling to establish potential entomological indicators of P. knowlesi infection in humans. A final study was conducted to investigate the transmission dynamics of P. knowlesi in macaque reservoir populations. Most studies of P. knowlesi vectors have been conducted in or near disturbed forest, where both humans and macaques are in contact. It is unknown whether the same vector species involved in human-macaque infection also mediate transmission between macaques. To investigate this and other aspects of macaque-mosquito interactions, I conducted a field study within the Danau Girang Field Centre in Sabah where there is a large population of long-tailed macaques. First I evaluated the use of Mosquito Magnet Independence Traps (MMIT) as a non-invasive means to sample mosquitoes host seeking near macaque sleeping sites. The MMIT performed well relative to the human landing catch, with both methods collecting An. balabacensis and other malaria vector species. Second, MMITs were used to sample mosquitoes host seeking near trees where macaques were sleeping and at unoccupied control trees. Additionally, macaque faecal samples were tested for malaria as an estimate of infection rate in the reservoir population. Anopheles balabacensis was more abundant at macaque sleeping sites than control trees indicating this vector has a specific propensity for feeding on macaques. Approximately 37% (n = 17/46) of macaque stool samples tested positive for Plasmodium infection but none of these were identified as being P. knowlesi. Two Anopheles vectors tested positive for Plasmodium which was subsequently confirmed as the primate parasite P. inui. Thus P. inui is likely the major source of malaria infection in this primate population. This study indicates that not all macaque populations pose a P. knowlesi risk, but other malaria parasites are common and should be monitored to assess for future spillover. In combination, this research expands knowledge of P. knowlesi transmission in Malaysian Borneo, and has implications for planning surveillance and control. Notably it emphasizes the value of larger-scale surveillance of vector and macaque populations to assess human exposure risk, as and requirement of an integrated One Health approach to tackle zoonotic malaria.