Obesity is known to be associated with obstructive sleep apnoea (OSA) and Type 2 diabetes mellitus (T2DM). The effect of OSA in very severely obese individuals is not well documented. In this thesis, I compared the effect of OSA in South Asians and white Europeans, examined the effect of OSA on glycaemic control among T2DM, and explored the relationship between OSA and diabetic retinal and kidney diseases in a severely obese population. I also systematically reviewed the effect of OSA on diabetic kidney and retinal diseases. Findings from this thesis were 1) severely obese South Asians had greater severity of OSA compared to white Europeans and the mechanisms mediating this require further investigation, 2) a high OSA prevalence in T2DM individuals with a positive relationship between nocturnal hypoxia and glycaemic control, 3) severity of hypoxaemia during sleep may be an important factor in the development of diabetic retinal complications, 4) duration of hypoxaemia during sleep were inversely associated with renal function in T2DM and 5) from the systematic review, there is a need for future large cohort studies with long term follow-up data to examine the long-term effects of OSA and other sleep parameters on diabetic retinal and kidney diseases.

The increased risk of cardiovascular disease (CVD) amongst South Asians (SAs) is unclear. This thesis examined potential biomarkers to address this. Cross-sectional data on SAs from community (n=1304) and hospital (n=148) populations was collected. Biomarkers were analysed by genotyping, mass spectrometry, automated-immuno-colourimetric-assays, ELISAs, and a new in-house assay for a novel marker, ferritin bound to apolipoprotein B. Diagnostic performance was assessed using receiver operating curves, logistic and linear regression models. C-reactive protein (CRP) was a comprehensive marker of CVD risk, where a range of 1.43-2.30 mg/L maximised sensitivity and specificity. CRP SNP (single nucleotide polymorphism) -390C>T/A contributed minimally to variation in CRP levels. Non-fasting triglycerides discriminated SAs at increased CVD risk, where APOA5 SNP -1131T>C was an independent predictor of triglycerides but APOC3 SNP -455T>C and -482C>T were not associated with triglycerides. The performance of IL-6, vWF, D-dimer and P-selectin were poor in comparison to CRP and triglycerides. BNP discriminated SAs with systolic heart failure with a cut off value of 36.4 pmol/l. Of the newly investigated biomarkers, a link between haemoglobin abnormalities and CVD was observed potentially through a mechanism involving iron transportation on lipoproteins. CRP and triglycerides should be considered in the routine CVD risk assessment of SAs.

Tuberculosis (TB) is an infectious bacterial disease mainly infecting the pulmonary system of the human body. It affects around 1.5 million people every year, most of whom live in developing countries. The incidence of TB has increased in line with the rise in incidences of Human Immunodeficiency Virus (HIV) infections and Acquired immune deficiency syndrome (AIDS). Due to the pressing concerns of TB, the World Health Organisation (WHO) came up with the Direct Observed Treatment (DOTS) programme. Unfortunately, the development of several resistant strains against first-line drugs and consequently second and third-line drugs have developed. As the current TB drug regimen is inadequate, a good screening strategy, discovery of newer drugs and identification of the mode of action would help in developing better treatment routines and determining bacterial pathways more clearly. Drug discovery follows two major routes, one leading from the drug to the target and the other from target to the drug. Both methods have been applied in this work in order to identify new drugs effective against mycobacteria. Screens performed against a drug library approved by the Food and Drug Administration (FDA) have resulted in some promising hits. Functional characterisation of a putative enoyl CoA hydratase EchA12, which was targeted by florfenicol, revealed a novel lipid chaperone functionality associated with cell wall lipid biosynthesis. Furthermore, a target based phenotypic drug screen of the GSK177 box set against Mtb-PrsA provided further evidence that this enzyme as a viable drug target (Ballell et. al., 2013).

Recent studies have demonstrated a key role for platelets and microthrombi in the pathophysiology of intestinal ischaemia-reperfusion (IR) injury. The research described in this thesis investigates the role of the major platelet glycoprotein receptors in recruiting and activating platelets following intestinal IR injury, namely GPVI, GPIb-IX-V and the integrins IIb3 and 21. Intravital microscopy was utilised to monitor individual platelet adhesion and microthrombus formation in anaesthetised mice undergoing intestinal IR injury \(in\) \(vivo\) using a novel dual labelling methodology. This study focussed on the microcirculation of the mucosal villi as this luminal surface is most susceptible to IR injury. We demonstrate that it was necessary to inhibit both microthrombus formation as well as platelet-leukocyte-endothelial interactions in order to ensure longer lasting improvement in gut microcirculation and histology. This was achieved through a dual therapy that targeted both GPIb and P-selectin. The strongest anti-platelet effect was observed with blocking the IIb3 integrin, but this was also associated with a sustained bleeding from the mucosal surface. Overall, the research within this thesis suggests that therapeutic strategies targeting GPIb and P-selectin may prove beneficial in improving the clinical morbidity associated with gut IR injury.

The bacterium Vibrio cholerae is the causative agent of the acute diarrhoeal disease cholera. V. cholerae is naturally found in aquatic environments but can switch lifestyles to cause disease in humans. The lifestyle switch requires modulation of genetic systems. Much of the regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, I show that the global transcription regulator, cAMP receptor protein (CRP), plays an integral role in the regulatory network that controls lifestyle switching. I have identified two sites for CRP in the intergenic region between rtxHCA and rtxBDE, a locus which encodes the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin and toxin transport system respectively. Using a combination of genetics, biochemistry and in vivo animal studies, I have determined a CRP dependent regulation of gene expression for toxin transport in response to host infection. This work shows that rtxHCA is constitutively expressed and not subject to regulation by CRP whist CRP acts as a repressor of rtxBDE transcription. Examination of further CRP targeted genes reveals similar behaviour upon host colonisation. These findings suggest that toxin export occurs in nutritionally rich environments, where the MARTX toxin can exert cytopathic and cytotoxic effects on host cells.

Canonical WNT signalling plays a critical role in the regulation of ovarian development during embryogenesis; dysregulation of this pathway in adult ovary is associated with subfertility and tumourigenesis. The aim of the current study was to elucidate the previously unexplored roles of Adenomatous polyposis coli 2 (APC2), a WNT signalling pathway regulator, in the ovary using an Apc2 constitutive knockout mouse. For the first time, the current work demonstrated essential roles of APC2 in regulating ovarian WNT signalling and ovarian homeostasis. In early adulthood, APC2-deficiency resulted in WNT signalling activation and sub-fertility driven by intra-ovarian defects. Follicular growth was perturbed, resulting in a reduced rate of ovulation and corpora lutea formation, which was not rescued by administration of gonadotrophins. The current study provides fundamental new knowledge on the role of APC2 in ovarian tumourigenesis. APC2-deficiency (on the background of a hypomorph Apc- allele) resulted in a predisposition to granulosa cell tumour (GCT) formation, accompanied by acute tumour-associated WNT-signalling activation and expression of a histologic pattern and molecular signature seen in human adult GCTs. Hence, APC2 has an important tumour-suppressor activity within ovarian granulosa cells. However, APC2 is dispensable for ovarian surface epithelium (OSE) homeostasis. APC2 loss on its own, or combined with PTEN or APC loss in the OSE, failed to cause tumour development. Introducing APC2-deficiency to an ovarian endometrioid adenocarcinoma (OEA) mouse model, driven by loss of PTEN and APC in the OSE, resulted in early initiation of tumourigenesis, but attenuated tumour growth. This attenuation was accompanied by squamous metaplasia, decreased mitosis, decreased p-ERK1/2 expression and disrupted immune/inflammatory signalling. Thus, for the first time, an APC2 functional dualism in initiation and progression of WNT-driven OEA in mice is reported. RNA sequence analysis unraveled 2 transcripts (HAL and HUNK) associated with OEA progression and should be considered for future research.

Human African trypanosomiasis and leishmaniasis are caused by parasites belonging to the genera Trypanosoma and Leishmania, respectively. Significant numbers of people are affected by these diseases worldwide, which are fatal if untreated. Animals can also be infected, posing agricultural and economic hindrances, especially in poor countries. Although chemotherapy can be used for treatment, many problems are associated with it, including drug toxicity, resistance, lack of guaranteed supply, and high treatment cost. Therefore, there is an urgent need for new treatment approaches. Here, we aim to examine the in vitro efficacy of curcumin and phosphonium compounds against these parasites, assay their toxicity to human kidney cells in vitro, and investigate the mechanism of antiparasite activity of curcumin. The Alamar blue assay was used to test 158 curcumin analogues against Leishmania major promastigotes and Leishmania mexicana promastigotes and axenic amastigotes to obtain in vitro EC50 values. Many curcumin compounds such as AS-HK122 and AS-HK126 exhibited anti-leishmanial activities similar to or better than the current clinical drug pentamidine. Similarly, EC50 values of 83 phosphonium compounds against Trypanosoma brucei brucei bloodstream forms were determined. More than 20% of the tested compounds were found to be more active than the standard veterinary drug diminazene aceturate. Multi-drug resistant strains were used to determine that there is no cross-resistance between the tested compounds and the diamidine or melaminophenyl arsenical classes of trypanocides. Structure activity relationship (SAR) analysis revealed that mono-O-demethylated curcumin compounds showed 10-fold higher activity against the parasites than curcumin. The addition of one or two pentyl pyridinium (C10H15N) groups on specific positions of the aromatic ring also increased the activity of these compounds. Furthermore, curcumin compounds with an isoxazole ring instead of the diketo motif showed higher activity and the lowest EC50 values. Similarly, pentyl bromide (OC5H10Br) substitutions on the phenyl rings improved the antiparasitic activity. Curcuminoids with trienone linkers showed increased antiparasitic activity against all parasites tested. Eighty-three phosphonium analogues were tested against T. brucei brucei. SAR analysis indicated that the bulky substituents surrounding the bisphosphonium cations led to strong antiparasitic activity while the nature of the linker had less effect on the activity. Some monophosphonium analogues registered the lowest EC50 values of all the phosphonium compounds. The toxicity of the curcumin and phosphonium analogues to HEK cells was analysed in vitro. All curcumin and phosphonium compounds demonstrated lower toxicity to HEK cells than to the parasites. Of the 83 phosphonium compounds, 60 displayed >200-fold in vitro selectivity index (SI). We also investigated the mode of antiparasitic activity of curcumin compounds. Preliminary toxicity tests had revealed that AS-HK014 caused rapid depletion of glutathione content in rat hepatocytes. Therefore, we tested AS-HK014 activity in the presence of different concentrations of L-glutathione, and AS-HK014 activity was found to decrease with increased L-glutathione concentrations, strongly suggesting that glutathione reacted with the active compound. Indeed, a chemical adduct was observed between the two compounds and identified through mass spectrometry. A trypanosome cell line (TA014) adapted to AS-HK014 was produced. TA014 and wild-type T. brucei brucei were treated with AS-HK014 and compared with each other and with untreated controls. The glutathione and trypanothione levels were lower in the treated WT cells than in the untreated cells. However, there was no change in the glutamate, ornithine, or spermidine levels, providing no evidence for the inhibition of trypanothione synthesis, suggesting that the effect is probably not metabolic but chemical. AS-HK014 did not significantly affect thiol levels in TA014; this might reflect a higher level of trypanothione synthesis through increased glutathione synthetase (GS) and/or γ-glutamylcysteine synthetase (γ-GCS) expression. Therefore, we analysed the protein levels using western blotting, and sequenced the encoding genes in both WT and TA014 to identify any mutations in the open reading frames (ORFs). However, we found no changes in the GS and γ-GCS protein levels in resistant trypanosomes and no mutations were found in the GS and γ-GCS ORFs. It is clear that the resistance is to the reactive enone motif of AS-HK014 rather than to curcumin and curcuminoids in general, since TA014 only displayed resistance to AS-HK014 analogues bearing the enone motif while sensitivity to curcumin remained unchanged, confirming that this motif is responsible for the higher activity of AS-HK014 compared to curcumin. The effects of bisphosphonium analogues on T. brucei brucei bloodstream forms were investigated to identify the target. All tested analogues rapidly reduced the T. brucei brucei mitochondrial membrane potential Ψm and decreased the intracellular ATP level after one hour of incubation, suggesting that the compounds may be targeting the mitochondria. The intracellular Ca2+ levels increased gradually after eight hours, suggesting that the damaged mitochondria are unable to retain the stored Ca2+ as their membrane potential dissipates. We also studied the trypanosome cell cycle after incubating the parasites with bisphosphonium compounds. The cell cycle defects became apparent after eight hours of incubation: DNA synthesis could not be initiated, leading to a dramatic reduction of cells in the S phase. This result was also confirmed by fluorescence microscopic assessment of DNA configuration. After eight hours of incubation with the bisphosphonium compound CD38, the number of 2K1N cells significantly decreased as compared with the control. There may be a causal relationship between mitochondrial damage and cell cycle defects. Transmission electron microscopy images of the cells obtained after 12 h of exposure to CD38 also revealed the presence of mitochondrial damage. We tested whether bisphosphonium compounds can induce programmed cell death in trypanosomes. A TUNEL assay was used to detecting DNA fragmentation; the results showed increased DNA fragmentation after 24-h treatment with two different bisphosphonium compounds, CD38 and EFpI7. This result indicates is consistent with apoptosis occurring in treated cells but there was no evidence suggesting that bisphosophonium-induced cell death in trypanosomes is dependent on new protein synthesis. In conclusion, curcumin and phosphonium analogues exhibit promising antiparasitic activity, and some analogues could be optimised for in vivo evaluation. Further investigations on the site of action of phosphonium compounds in the mitochondrion are in progress.

Leishmania spp. are protozoan parasites that infect humans and other vertebrates to cause a spectrum of disease, ranging from cutaneous ulceration to visceral dissemination dependent on the species. Leishmaniasis is prevalent across the developing world and is a major global health issue, yet difficulties in the efficacy and administration route of current anti-leishmanial treatments means the existing drug repertoire is inadequate. To address this, further research and development measures are necessary to identify Leishmania proteins representing useful targets for drug inhibition. Essential genes encode proteins that are necessary for parasite survival and therefore represent suitable drug targets, but the study of such genes is limited by the absence of a conditional deletion system. A family of proteins which has previously been shown to regulate crucial aspects of Leishmania biology are the protein kinases. Protein kinases have been validated in mammalian systems as drug targets in cancer therapy, therefore they represent a promising avenue for research into anti-leishmanial drugs. The cdc-related kinases CRK3 has been studied in particular depth in Leishmania, and current reverse genetic techniques have implicated expression of CRK3 as essential to promastigote survival. CRK3 regulates the cell cycle as demonstrated by treatment of cdc2 inhibitors, but a lack of a system to regulate expression prevents more specific phenotypic dissection of the role of CRK3. In addition the validation of CRK3 as a drug target has been limited by an absence of a conditional genetic system to ablate the gene in mammalian infective amastigotes. To regulate CRK3 expression in a conditional manner to assess its function in the cell cycle of promastigotes and validate it as essential for amastigotes, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) for use in L. mexicana. Cre recombinase mediates the excision of DNA sequences flanked by 34bp loxP sites (‘floxed’). diCre is encoded as two separate subunits each linked to rapamycin binding domains (FRB and FKBP12); therefore recombinase activity is induced by rapamycin treatment which causes dimerisation of the subunits. Our method involves replacing both CRK3 alleles with a ‘floxed’ CRK3 open reading frame and the diCre coding sequence through promastigote transfection and homologous recombination. Induction of diCre through rapamycin treatment of promastigotes results in highly efficient deletion of CRK3 and a distinct growth arrest phenotype corresponding to a block in G2/M. Induced loss of CRK3 can be complemented by expression of a CRK3 transgene but not by expression of an inactive site (T178E) CRK3 mutant, showing that protein kinase activity is crucial for CRK3 function. Significantly, inducible deletion of CRK3 in stationary phase promastigotes prevents the establishment of murine infection, thereby demonstrating an essential role in the amastigote cell cycle to further validate CRK3 as a drug target. Promisingly, inducible deletion is functional in lesion-derived amastigotes and will enable direct phenotypic assessment following essential gene loss in this life cycle stage. To establish a basis for future in vivo application of diCre in Leishmania, a murine infection model was developed with which to track bioluminescent parasite burden by in vivo imaging and assess innate immune cell recruitment to the site of infection by flow cytometry analysis. The combination of functional gene regulation in amastigotes and measures of parasite burden and immune response will yield a powerful tool for the further study of Leishmania genes encoding suitable drug targets. The application of the diCre technique to Leishmania would be greatly benefitted by targeting genes where there is evidence of a regulatory role of orthologous genes in model organisms. The utilisation of genome or protein family-wide RNAi screens in Trypanosoma brucei has identified a number of protein kinases which regulate the differentiation of the parasite between life cycle stages. The repressor of differentiation (RDK1) protein regulates bloodstream form to procyclic form differentiation in T. brucei, and the identification of a protein in L. mexicana with high sequence identity suggested a potentially analogous role in preventing Leishmania from undergoing amastigote to promastigote differentiation in vivo. To assess this, a cell line was generated deficient in RDK1 but no effect on differentiation was identified, as parasites were able to maintain murine infection and differentiate between life cycle stages. This study represents an important addition to the reverse genetic toolkit to study aspects of cell cycle regulation in vitro, and further assess essential genes as drug targets by deletion in amastigotes. The application of the diCre conditional deletion method will enhance the discovery and evaluation of suitable drug targets in Leishmania by phenotypic analysis.

The platelet receptor CLEC-2 is the only known endogenous ligand for the transmembrane receptor podoplanin, which is expressed on lymphatic endothelial cells (LEC) as well as a number of other cell types. Both CLEC-2 and podoplanin are required for normal lymphangiogenesis as mouse embryos lacking either protein develop a phenotype in which blood is detected in the lymphatic vessels. This thesis examines the role of the podoplanin-CLEC-2 interaction in the migratory and tube-forming capabilities of LEC. Addition of platelets or antibody-mediated podoplanin crosslinking both inhibited migration of LEC in transfilter migration assays in the presence, but not absence, of vascular endothelial growth factor (VEGF)-C. Similarly, platelets and podoplanin crosslinking reduced stability of LEC networks formed in co-cultures with fibroblasts. We also found that siRNA-mediated knockdown of podoplanin negated the pro-migratory effects of VEGF-C and VEGF-A. Furthermore, we obtained evidence that podoplanin signalling may involve RhoA and Rho-kinase, and that the effect of podoplanin might be linked to its phosphorylation by protein kinase A downstream of VEGF receptor signalling. These data suggest that the interaction of podoplanin and CLEC-2 prevents connection between blood and lymphatic vessels through reductions in LEC migration and stability of cell-cell interactions.

Coronary artery disease (CAD) is a disease of inflammatory aetiology, and remains the commonest cause of death globally despite therapeutic advances. Monocytes are implicated in the pathogenesis of CAD, but also in reparative mechanisms after myocardial infarction (MI) due to subset heterogeneity. The aim of this thesis was to provide a detailed phenotypic comparison of differences between the three human monocyte subsets in CAD and after MI, with particular emphasis on CD16+ monocytes which have previously been analysed as a single population rather than two distinct subsets. Longitudinal changes were analysed following MI and relationships explored with plasma cytokines. Multiple significant novel changes in monocyte phenotype attributable to specific subsets were identified, particularly related to the CD14++CD16+CCR2+ ‘Mon2’/‘Intermediate’ subset which increased in number on day 1 after MI and appeared highly functionally active. There were significant changes in expression of a range of receptors associated with inflammation, migration and reparative processes. Significant relations to plasma cytokines and the degree of myocardial damage were observed. Most monocyte parameters predictive of left ventricular ejection fraction six weeks after MI were related to the Mon2 subset. This suggests an important role for this subset in the acute phase of MI.