Anaemia is a common health problem affecting women and children in the developing world. This condition is characterized by a reduction in erythrocyte density, primarily resulting from malnutrition and/or infectious diseases such as malaria. As red blood cells are the most important source of protein for haematophagous mosquitoes, any reduction could impede the ability of mosquito vectors to transmit malaria by influencing their fitness and/or that of the parasites they transmit. The aim of this study was to determine the impact of differences in the density of red blood cells in human blood on human malaria transmission (P. falciparum) by influencing: (i) mosquito vector (An. gambiae s.s.) fitness, (ii) the infectiousness of parasites to mosquitoes, (iii) parasite sporogonic success in mosquitoes, and/or (iv) the virulence of malaria parasites to mosquito vectors. The hypotheses tested are based on the premise that mosquito vector survival, fecundity and malaria parasite sporogony may be influenced by the nutritional value of infectious blood meals. Mosquitoes (An. gambiae) were offered blood meals at different Packed Cell Volume (PCV) of human blood consistent with those arising from severe anaemia (low PCV= 15%) and normal PCV (50%), and containing malaria parasite (P. falciparum) gametocytes infectious to mosquitoes. Mosquito fitness, the success of parasite development within them, and/or the virulence mosquitoes experience from infection were evaluated. The amount of protein that malaria vectors acquired from blood-feeding, and their resultant long term survival, was found to be positively associated with the PCV of human blood. Mosquitoes feeding on blood with low PCV had the same oviposition rates as those feeding on blood of normal PCV, but also showed an increase in fecundity of around 15%. Moreover, it was found that An. gambiae s.s. mosquitoes were substantially more likely to become infected after feeding on gametocyte-infected blood of low PCV rather than normal PCV. However, there was no evidence that blood PCV influenced either the oocyst intensity in infected mosquitoes, or the total number of transmission stage sporozoites they developed by 10 days after the infectious blood meal. Finally, there was no evidence that either consumption of P. falciparum infected blood or the subsequent development of oocysts reduced any measure of mosquito fitness (oviposition, fecundity, survival), in either mosquitoes infected from blood of low or normal PCV. The impact of blood PCV on the energetic reserves of both infected and uninfected mosquitoes was found to be relatively minor. These results demonstrate that variation in human erythrocyte of a magnitude likely to arise in malaria-endemic settings may have a significant impact of the outcome of vector–parasite interactions. Conditions such as severe anaemia, which reduce red blood density, could enhance malaria parasite transmission by increasing parasite infectivity to mosquitoes, while having no deleterious effects on mosquito survival.

Streptococcus pneumoniae or pneumococcus is included among major human pathogens and is responsible for a number of diseases including life-threatening conditions such as pneumonia, meningitis and sepsis. Though pneumococcal vaccines are available, they provide limited coverage against infections as pneumococcus shows extensive variation, which also allows escape from vaccines and antibiotic resistance. It is armed with several virulence factors including capsule, surface proteins, enzymes and toxins, which are variably expressed and altogether determine pneumococcal virulence. The aim of this project was to study pneumococcal genetic variation and its effect on virulence, with a focus on pneumococcal capsule, which is considered the major determinant of virulence and is involved in interaction with host immune system. It is the target for current vaccines and at least 93 pneumococcal serotypes are known, which differ in pathogenicity. To study the effect of capsule on the pneumococcal virulence, capsule-switch mutants were constructed in three genetic backgrounds; TIGR4 (serotype 4, virulent), 403 (serotype 4, avirulent) and D39 (serotype 2, virulent) and were studied for variation in their in vivo and in vitro characteristics. These mutants were compared with their parent strains and other mutants for effects of capsule switching on their growth, formation of capsular polysaccharide, capsular thickness, chain formation and virulence in murine models of infection using MF1 mice. Significant differences were observed in behaviour of parent and mutant strains. To develop a broader insight into pneumococcal virulence, avirulent derivative of strain TIGR4, 403 was genome sequenced and compared with TIGR4 for genetic mutations. To study differences in gene expression both the strains were also compared using microarrays. Genome analysis revealed only few mutations in strain 403 but microarray experiments showed 288 genes to be expressed differently in strain 403. Strain 403 was also tested as live attenuated vaccine to see if it could provide protection against the same and different serotypes, as it can be used as a vehicle for delivery of different polysaccharides to the host body along with the whole set of pneumococcal antigenome. Vaccine trials of 403 were not very fruitful as it failed to provide any protection through intranasal route though partial protection was observed in mice vaccinated intraperitoneally with significant differences in levels of bacteraemia, survival, weight and temperature losses on challenging with homologous strain.

The extracellular-Ca2+ sensing receptor (CaSR) is a G protein-coupled receptor which is essential for Ca2+ homeostasis in the body. The best studied function of the CaSR lies in the parathyroid gland, where hypercalcaemia activates the receptor which in consequence inhibits parathyroid hormone secretion. However, for other tissues like the vasculature the physiological and pathophysiological roles of the CaSR are a lot less well defined. The CaSR is expressed in all layers of blood vessels, the endothelium, the vascular smooth muscle cells (VSMC) and the tunica adventitia. Previous studies have suggested roles for the vascular CaSR in protection against vascular calcification and in blood pressure regulation. In the course of my thesis, I have investigated the specific roles of the vascular CaSR by characterising the phenotype of a transgenic mouse model of targeted CaSR deletion from VSMC (SM22α-Cre x LoxP-CaSR). In characterising this mouse model, I have made three principal discoveries: 1) The VSMC CaSR protects against vascular calcification induced by high Ca2+ and Pi in vitro. No calcification was discovered in mice lacking the VSMC CaSR in vivo, suggesting that this protective effect of the CaSR might only assert itself in pathological disease. 2) The VSMC CaSR contributes to blood pressure regulation. Mice lacking the VSMC CaSR exhibit hypotension which is due to impaired vascular contractility and therefore reduced total peripheral resistance. I propose a role for the VSMC CaSR as auto- / paracrine amplifier of VSMC contraction. Furthermore, knock-out mice exhibit bradycardia and sporadic cardiac remodelling. 3) Mice lacking the VSMC CaSR exhibit profound changes in mineral ion metabolism, together with severe hypercalcaemia, hypercalciuria, hyperphosphaturia and increased 1,25-(OH)2-Vitamin D3 and phosphaturic FGF23 levels, and decreased bone mineral density, consistent with a phenotype resembling primary hyperparathyroidism. The mechanisms behind this influence remain yet to be fully understood.

The Severn Estuary, UK is an important model estuarine environment due to its hyper-tidal range, leading to dynamic sediment environments. This work investigated the diversity and relationship with geochemistry of Severn Estuary sediment prokaryotic communities, which have not been previously described in detail. Focus was placed on the diversity and distribution of the largely uncultivated Chloroflexi, which were detected in high abundance in the deep subsurface but previous work has failed to address the diversity of subdivisions in dynamic surface sediments. Three geophysically different sampling sites were analysed from intertidal, shallow water and deep water areas of the Severn Estuary. Molecular profiling methods using the 16S ribosomal RNA (rRNA) gene, such as denaturing gradient gel electrophoresis (DGGE), ribosomal intergenic spacer analysis (RISA), quantitative PCR (qPCR) and 454 pyrosequencing were used. Novel qPCR and pyrosequencing methods were designed to target Chloroflexi subdivisions. Each of the sampling sites was characterised by differing prokaryotic communities depending on sediment turbidity and geochemistry, though the most abundant phyla, Proteobacteria, Firmicutes and Chloroflexi, were constant. The novel methods revealed surprising abundance and diversity of Chloroflexi subdivisions in Severn Estuary sediments, dominated by Anaerolineae, on a more detailed scale than previously reported in the literature. Further experiments described how sediment prokaryotic community structure and function changed over a wide temperature range and over 100 days. Slurries of Severn Estuary intertidal sediments were incubated between 1 - 80oC. A critical temperature window of 43oC indicated a shift in the bacterial community to thermophilic spore-forming Firmicutes and from heterotrophic to autotrophic sulphate reduction. In the Archaea community, methanogenesis shifted from chemoorganotrophic to chemolithotrophic dependent metabolism. These results extend our knowledge of the geochemical changes in temperature dependent sediment communities, which is important for the modelling of climate susceptible habitats, such as coastal sediments.

Gliomas are amongst the most malignant, invasive and recurrent forms of brain tumour with very short survival rate due to high chemoresistance. Recently, highly inducible molecular chaperones HSP70 and HSP90 are emerging as important anti-cancer targets. Previously, proteomic analysis had demonstrated that post-induction of HSP70 on HSP90 inhibition undermines the efficacy of treatment. The present study has quantified transcriptional levels and Akt/PKB activity of Hsp70 and Hsp90α in glioma cell lines. In order to evaluate the therapeutic value of both chaperones, HSP70 and HSP90 were targeted in glioma cells U87-MG using VER-155008 and 17-AAG, respectively. Improved efficacy of HSP70 and HSP90 inhibitors was evaluated using a chemosensitivity assay. MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules (21-24 nucleotides) that regulate simultaneously the expression of hundreds of mRNA targets, and are reported to be aberrantly expressed in glioma. Therefore, miRNA microarray technology was used to evaluate the efficacy of these inhibitory drugs compared with Temozolomide (TMZ) which is used as a standard treatment for glioma. Microarray data identified 154 miRNAs using either stringent or non-stringent parameters. 16 miRNAs were overlapped with treatments, 15 were upregulated, while 13 were overlapped between Temozolomide and VER-155008. In Temozolomide and VER-155008 treatment, Hsa-miR-194p was upregulated by 139 and 63 fold, respectively, Hsa-miR-215 was upregulated 165 and 61 fold, respectively, Hsa-miR-449a was upregulated by 62 and 77 fold, respectively and Hsa-miR-744-5p was upregulated by 63 and 43 fold, respectively. 17-AAG and VER-155008 treatment shown only one miRNA overlapping with 29 and 2 fold change, respectively. Hsa-miR-4636 was the only downregulated miRNA in TMZ and VER treatment with a 32 and 33 fold change, respectively. The miRNA target prediction software was used for the highly upregulated miRNAs: hsa-miR-194-5p, hsa-miR-215, hsa-miR-449a, hsa-miR-744-5p and hsa-miR-3161 correlating to Dnmt3a, Alcam, Cdk4, Dnajc16 (Hsp40) and R-Ras2 genes, respectively. Gene validation using qRT-PCR suggested no correlation between miRNA-mRNA levels, and thus, challenges the suitability of miRNAs technology as treatment predictors. In conclusion, the result for the protein data showed that HSP70 was inhibited on treatment with Temozolomide, 17-AAG and VER-155008 to 13, 0 and 20 %, respectively, while HSP90 inhibition was 84, 43 and 65 %, respectively, reflecting the affinities of these three compounds towards HSP90 compared to HSP70, and therefore infers that HSP70 could be a stronger therapeutic approach. In conclusion the result of the study has clearly demonstrated that HSP70 can be better therapeutic biomarker for treatment of glioma.

Background. Numerous studies into the effect of probiotic supplementation in the infirm have been carried out. However, research into the effect of long-term probiotic supplementation in healthy individuals is lacking. With this in mind the PROHEMI study, a randomized, double-blinded, multi-centre and long-term (6 months) probiotic feeding study in healthy males was designed and carried out. Through the use of varied culture dependent and independent techniques, the effects of long-term probiotic consumption were researched. In addition, a study into the effect of freezing faecal material on its bacterial composition was also carried out. Results. Through a community fingerprinting technique and next generation sequencing it was shown that the distal gut bacterial community is unaffected by probiotic supplementation. Functional screening of faecal material showed a reduction in bacteria expressing protease activity when probiotic supplementation began. In addition, bacteria expressing β-galactosidase and β-glucuronidase activity increased during probiotic supplementation. Metabonomic analysis showed no difference in metabolite profiles attributable to probiotic supplementation. However, differences between the gut bacterial community, metabonomic profiles, and bacteria expressing functions were observed between the two study centres. Freezing of faecal material at -20°C detrimentally affected its bacterial composition between 2 weeks and 3 month storage time-points. Significant reductions in the abundance of the Bacteroidetes phylum observed following 6 months of storage at -20°C. Conclusions. Long-term probiotic administration in healthy individuals did not seem to affect the distal gut bacterial community in these individuals and did not affect metabonomic profiles. However, some functions expressed by the resident distal gut bacterial community were significantly affected during probiotic supplementation. DNA extraction from faecal material should ideally be carried out from fresh samples. Failing this it is not recommended to store samples at -20°C for longer than 2 weeks prior to DNA extraction.

P2X receptors and pannexins (Panx) are eukaryotic ion channels that are implicated in a range of diseases and conditions including cancer, inflammation and pain sensation and as a result, are important therapeutic targets. Deducing their 3D-structures would enable the use of structure-based drug design to identify novel agonists or antagonists. However, solving eukaryotic membrane protein structures is a significant challenge due to the requirement for high yields of purified folded, functional protein, which are not readily obtainable with conventional over-expression systems. By using P2X receptors and pannexins as model ion channel targets, this thesis aims to test Drosophila melanogaster as a system for the over-expression and functional analysis of eukaryotic ion channels. A number of epitope-tagged P2X and Panx protein constructs were generated and first expressed in HEK-293 cells (rat P2X2-GFP, human P2X4-GFP, rat Panx1-GFP and human P2X4-int-CBD (chitin binding domain)) to allow their expression, glycosylation and oligomeric states to be investigated as markers of protein folding and quality. Subsequently, rat P2X2-GFP and rat Panx1-GFP constructs were successfully expressed in the photoreceptor cells of Drosophila melanogaster, where the photoreceptive membrane in the visual system is organised into a densely packed brush of microvilli, the rhabdomere. This system provides a large surface area of membrane for protein expression. Although the yields of purified protein were lower than expected, rat Panx1-GFP was successfully purified and used for low resolution structural studies with transmission electron microscopy. Rat P2X2-GFP was also expressed in the nervous system of Drosophila under control of a pan-neural, C155-Gal4 driver and was shown to be functional by measuring ATP-evoked action potentials using electrophysiological recordings of the Drosophila taste sensilla. This system was also used to test the activity of an adenosine nucleotide library of 80 compounds. Three nucleotides were identified that elicited responses similar to ATP; these were 2F-ATP, ATPαS and ATPγS.

In the last 10 years biocides have been used increasingly and questions have been raised about their contribution to the reported increase in biocide and antibiotic resistance in pathogenic bacteria. The EU Biocidal Product Regulation (BPR) now requires information on the risk of resistance development in organisms targeted by the biocidal product. There is no current protocol available to predict the likelihood of bacteria becoming resistant to a biocidal product or biocides contained therein. This study aimed to identify useful markers of biocide resistance and develop a step-by- step protocol predictive of bacterial biocide resistance and antibiotic cross-resistance following biocide exposure. A range of experimental techniques with the potential to generate markers of biocide resistance were explored. These included minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC)/antibiotic susceptibility determination, flow cytometry, efflux activity measurements, outer membrane protein changes, real-time PCR and microarrays. Salmonella enterica serovar Typhimurium strains SL1344 and 14028S, and Burkholderia lata strain 383 were exposed to low concentrations of chlorhexidine gluconate and benzalkonium chloride as test biocides. Baseline and post-exposure data were then compared. Techniques used to understand any change in antimicrobial susceptibility were assessed in terms of practicality, cost and ease of use, and a step-by-step protocol was put together accounting for each of these factors. Increases in biocide MIC and MBC of up to 100 fold were observed in SL1344 and 14028S after exposure to both biocides. However these changes were not stable after subculture of surviving organisms in the absence of either biocide. No such dramatic changes were observed within B. lata. Up-regulation of efflux activity was observed as a result of CHG/BZC exposure and the efflux regulatory gene acrR underwent a >100 fold down-regulation in both Salmonella strains after CHG exposure. Flow cytometry experiments performed using SL1344 and 14028S indicated that at low CHG/BZC concentrations (0.0001 – 0.0004 %) greater than 50 % of the population were not killed and that these organisms could be sorted and further investigated to determine the mechanisms behind their survival. Reduction in the expression of two outer membrane proteins was observed in strain SL1344 after exposure to 0.0004 % CHG but further protein sequencing would be required to identify these. Changes in phenotype and genotype of biocide-exposed bacteria were identified using different experimental techniques. Some of these changes e.g. increased MIC/MBC values, altered antibiotic susceptibility, up-regulated efflux activity, alterations in the expression of specific genes and surviving organisms identified by flow cytometry represent useful markers of biocide resistance. A preliminary step by step protocol incorporating these techniques was successfully developed and allows for the rapid identification of biocide resistance and antibiotic cross-resistance as a result of biocide exposure, and will prove particularly useful in light of the recent changes to the BPR.

Introduction: The human gut microbiota outnumbers our own human cells by 100-1 and is often considered an extension of our genome harboring fundamental functions of which we are genetically incapable. However, it can also be a significant liability and has been implicated in numerous diseases, particularly Inflammatory Bowel Disease (IBD). The efforts of this research entailed evaluating a specific molecular mechanism of the gut bacterial metagenome; proteolytic activity, collectively known as the Degradome due to their putative role as significant virulence factors in IBD. In order to access this degradome of the gut microbiota, firstly, novel functional metagenomic (FM) tools were developed with an aim of facilitating the isolation of proteases. Secondly, a comprehensive cohort study was conducted comparing faecal protease activity, 16S rRNA microbial community structure and the potential of the faecal degradome to act as virulence factors between a group of IBD patients and a group of healthy volunteers to begin to determine the role of microbial proteases in disease aetiology. Results:-The IBD cohort exhibited significantly higher protease activity than the healthy cohort. Inhibitor assays also showed that the IBD cohort contained different types of proteases to the healthy cohort with significantly higher levels of metalloprotease activity. 16S rRNA gene analysis of the microbial community also revealed a dysbiosis of the gut microbiota between the IBD cohort and the healthy cohort. Dysbiosis was also observed between the high protease producers and the low protease producers and protease activity in the IBD cohort was able to decrease trans epithelial resistance in an HT-29 cell line and increase cellular permeability. Functional metagenomics tools were also assessed for isolation of proteases from the gut microbiota. The ability of a protease deficient strain; Bacillus subtilis WB800N to express proteases was compared with E. coli to determine its usefulness as a host for FM screening for proteases. B. subtilis WB800N was able to express gelatinase E and neutral protease while E. coli was not suggesting B. subtilis WB800N was more suitable as a host. However, when the gut microbiota was screened for proteases using this host, none were isolated suggesting improvements still needed to be made. Conclusions: - Compositional alterations of the gut microbiota appear to be associated with high and low levels of protease activity. The IBD cohort had elevated activity and an expanded repertoire of faecal proteases which also appear to have the potential to act as a virulence factor by disrupting epithelial cell barrier integrity. Proteases remain elusive via FM screening; however this study has highlighted the areas that need improvement to optimize future screens for accessing the degradome of the gut microbiota.

WNT signalling is a major driving force in cartilage development, chondrocyte differentiation, and homeostasis. Due to its major role in cartilage homeostasis, deregulation of this pathway was suggested to be involved in the pathophysiology of osteoarthritis, which causes cartilage degeneration. Studies have identified mutations in inhibitors of WNT signalling in OA tissue in addition to up-regulation of related WNT components. However, these studies have focused on the end stage of the disease and have not factored in mechanical loading which is one of the main regulators of cartilage homeostasis. Results: Canonical WNT signalling was activated in response to both tensile and compressive loading. Furthermore, WNT signalling was observed to be differentially regulated in bovine cartilage explants in response to physiological (2.5MPa, 1Hz, 15 minutes) and degradative (7MPa, 1Hz, 15 minutes) compressive loading in a zone dependent manner as indicated by β-catenin nuclear translocation. Based on periods post-cessation of load, physiological and degradative loads have induced differential matrix gene expression. In addition, both loading regimes have shown differential regulation of WNT signalling components displaying more WNT signalling activation in degradative loading regime. DKK-1 and NFATC, WNT signalling components which have shown to have a role in cartilage homeostasis, have been chosen for functional analysis studies. Treatment of recombinant DKK-1 in combination with degradative load have shown that DKK-1 primarily inhibited canonical WNT signalling pathway, whereas treatment with NFAT inhibitor induced inhibition of both WNT signalling pathways. In addition, both treatments were observed to inhibit some of the load-induced matrix gene changes. Conclusions: WNT signalling is mechano-regulated in articular cartilage. The differential expression of WNT signalling components in response to different mechanical load regimes indicates a role for these components in maintaining cartilage homeostasis.