Type 2 diabetes mellitus is associated with a significant increased morbidity and mortality resulting from microvascular and macrovascular complications, in particular diabetic nephropathy and cardiovascular disease. Treatment of these conditions has involved improving diabetic control, reducing blood pressure and addressing other cardiovascular risk factors. Dietary fibre has wide reaching health benefits, including improvement of diabetic control and blood pressure, potentially by alterations in colonic bacterial populations that result in changes in serum short chain fatty acids. An open labelled study with a washout period was undertaken to examine the potential effects of Gum arabic on blood pressure and renal function. A daily supplement of gum arabic (25g), a soluble dietary fibre, was administered for a period of 12 weeks. An initial pilot study was conducted in healthy subjects (n=10) and subjects with overt nephropathy (n=14). A follow on study investigated subjects with incipient nephropathy (n=23) in more detail. Measurements of renal function, including isotope GFR and ERPF, blood pressure and vascular stiffness (follow on only), and short chain fatty acids were measured. A significant drop in GFR was seen in the healthy individuals with no associated change in filtration fraction, which could convey some renal protective effect. No changes were seen in the diabetic subjects. Significant drops in blood pressure were seen each of the individual groups. Results of pulse wave analysis and central blood pressure measurements suggest this is not as a result of changes in vascular stiffness. Significant changes in short chain fatty acid production were seen, in particular an increase in acetate (p=0.033) in the incipient nephropaths and butyrate (p=0.03) in the healthy subjects. This study suggests that Gum arabic has beneficial effects on blood pressure but no immediate beneficial effects on renal function in either diabetic cohort.

All inflammatory events are mark by infiltration by leukocytes including neutrophils, which cross the endothelium before following migratory cues to the site of infection, and phagocytosing the infectious microorganisms. Before crossing the endothelial wall, the neutrophils spread on the endothelium. It has been proposed that the necessary additional membrane for cell spreading results from unfolding of wrinkled cell membrane held in place by molecule like membrane linker protein (such as talin or ezrin). Both talin and ezrin are potential substrates for cleavage by the Ca2+ activated proteolytic enzyme, calpain-1. It is possible that this mechanism underlies the membrane unwrinkling events but it is yet to be proved. The major aim of this thesis was to look for evidence that proteolysis and redistribution of these proteins occurred during neutrophil shape change. This thesis provide confirmatory evidence that ezrin is cleaved during extravasation of neutrophils and also provide evidence that the subcellular location of talin and ezrin protein can serve as an biological marker to identify extravasted neutrophils. The subcellular locations of talin and ezrin were identified using immunocytochemistry. Both ezrin (87%) and talin (92%) were detected at the cell membrane of neutrophils. This pattern was lost in polarized neutrophils, as well as after an elevation of cytosolic calcium level and also after transmigration through endothelial monolayers in vitro. Under these conditions, the detected ezrin and talin was mainly cytosolic. The same translocation was observed in extravasated oral neutrophils and also neutrophils which had extravasted under pathological conditions (gingivitis and osteoarthritis). GFP-tagged ezrin was expressed in RAW cells, in order to investigate the mechanism behind the relocation of ezrin. It was found to be triggered by an elevation of cytosolic calcium and was irreversible. It was also triggered locally during phagocytosis at the site where the membrane expanded. Western blotting showed that ezrin (72-69 kDa intact) was cleaved under similar conditions with fragments at 55kDa, 51kDa and 49kDa being generated by elavated calcium and extravasation. This cleavage was sensitive to calcium and calpain inhibition. It was concluded that ezrin is present in the plasma membrane wrinkles of resting neutrophils, but that changes when the cytosolic calcium level changes, as occur during extravsation and phagocytosis. Addition to this, any dynamic change in the surface area of the plasma membrane (phagocytosis), cause a relocation of ezrin away from the plasma membrane. Evidence was also provided that ezrin can be uses as a biological marker to identify extravasated neutrophils

In vitro models of infection were used to investigate the contribution of swarming to the blockage of urinary catheters

Mutants of Pr. mirabilis resistant to triclosan were generated in the laboratory, they had not gained resistance to antibiotics, but they were able to form crystalline biofilms in the bladder model on catheters exposed to triclosan.  Clinical trials of this strategy should therefore monitor the urinary flora for signs of triclosan resistance. If this strategy can be transferred successfully from the laboratory to the clinic, it could produce a major improvement in the care of the many elderly and disabled patients enduring long-term bladder catheterisation.

The surfactants did not inhibit IL-8 induced by IL-1[Special character omitted.] in A549 cells suggesting that they act at the level of the LPS receptor complex. Disruption of membrane micro-domains ('rafts') with methyl-[Special character omitted.]-cyclodextrin significantly inhibited bacteria and LPS induced IL-8 in A549 cells. Isolation of membrane raft containing fractions by sucrose density gradient ultra-centrifugation showed that TLR4 is recruited into membrane lipid rafts on cell stimulation with the bacteria or LPS. The surfactants inhibited the bacterial and LPS mediated translocation of TLR4 into raft domains suggesting that their mechanism of action involves inhibition of LPS receptor complex formation in lipid raft domains.

The respiratory tract is exposed to a wide range of environmental constituents including potentially infective agents. This thesis presents two series of papers concerning two atypical organisms and their sequelae or potential sequelae – Coxiella burnetii, which causes Q fever, and flagellated protozoa, which is associated with respiratory symptoms and asthma. The first series of papers examine a Q fever outbreak and its sequelae. They describe one of the largest Q fever outbreaks in the UK and demonstrate the benefit of facsimile cascade in supporting case searching in such an outbreak. Chronic fatigue (Chalder Fatigue scores) and depression (PHQ-9 scores) were raised in post Q fever patients six years later (p<0.05 in both cases). Concordance regarding serological status across three international reference laboratories was as low as 35%, indicating a major problem with international standardisation in this area. Unpublished supplementary clinical and serological findings are also presented to inform future outbreak investigation. The second series of papers examine the role of flagellated protozoa in respiratory disease. Flagellated protozoa were shown to be present in a case series of inpatients. In a subsequent community-based case control study, protozoa were present in 67% (20/30) of induced sputum samples taken from asthmatics and 31% (4/13) of samples from non-atopic controls (p=0.046). In another study of inpatients, 67% of those who were on oral steroids had protozoa in their sputum, compared to 35% of those not on oral steroids. In this study, 45% of smokers/ex-smokers had protozoa in their sputum compared to 30% of non-smokers. Unpublished data using molecular techniques to identify eukaryotes in sputum is also presented. The findings provide a basis for a RCT of antibiotics in patients with post Q fever chronic fatigue and a trial of anti-protozoal agents to eradicate flagellated protozoa in patients with asthma.

Controversy still exists as to the incidence, role and impact of lactose sensitivity in inflammatory bowel disease. The thesis shows that there is a higher than previously reported incidence of lactose sensitivity determined by a combination of genotype, breath test and symptoms after a lactose challenge. Lactose sensitivity in patients with inflammatory bowel disease who are in remission is 70%. There was no difference compared to healthy volunteers in terms of lactase genotyping; however there was a significantly greater prevalence of positive breath test and symptoms after lactose challenge. This suggests that lactose sensitivity in inflammatory bowel disease is related to the disease itself or a consequence of it and not due to a genetic predisposition. A significant proportion of inflammatory bowel disease patients [16%] are methane producers which warrants further investigation. A pilot study of reduced lactose intake in patients with Crohn’s disease and lactose sensitivity, who were in remission, showed a promising improvement in symptoms reported and quality of life scores. The Real-Time Polymerase Chain Reaction is simple and quick compared to Restrictive Fragment Length Polymorphism for assessing the lactase genotype. The Quintron MicroLyzer to assess breath samples after lactose challenge is preferred to the hand held Micro H2 meter. This detects methane in addition to hydrogen and without this a number of cases of lactose sensitivity would be II missed. It may be possible to predict a negative breath test with the absence of any GI symptoms after a breath test and vice-versa a positive breath test is very likely if multiple GI symptoms are reported. The ‘hidden’ lactose in drugs used to treat inflammatory bowel disease and co-existing conditions should be considered as it is present in many drugs and can make a significant contribution to the amount of lactose ingested; lactose free alternatives are widely available.

Single Nucleotide Polymorphisms (SNPs) are used as markers in association studies and may contribute directly to inherited disease. Here, we investigated high throughput in silico and in vitro methods for identifying SNPs and applied these to large scale genomic projects. We evaluated the utility of the Transgenomic NavigatorTM software to facilitate automated detection of aberrant denaturing high performance liquid chromatography (dHPLC) elution profiles. 3,747 dHPLC profiles were analysed with this software and 98.3% of products with profiles distinct from wild type harboured novel variants (Chapter 3). We applied this software to investigate whether rare inherited variants in genes that play a role in oxidative DNA damage repair (OxDR) predispose to colorectal adenomas (CRA) and found that unlike MUTYH, inherited variants in other OxDR genes are unlikely to be a frequent cause of CRA (Chapter 4). We evaluated the sequence analysis packages Sequencher, InSNP, Mutation Surveyor and Staden to identify variants in patients with CRAs (using >4,000 chromatograms). Staden and Mutation Surveyor correctly identified 76/77 (98.7%) SNPs and 96.7% and 99.3% of the genotypes respectively (Chapter 5). We compared an optimised version of Staden against Sequencher for variant detection over a 2.5kb region of the adenomatous polyposis coli (APC) gene in 969 healthy controls. We found 100% concordance between these packages and found that rare nonsynonymous variants in APC were significantly over-represented in patients with CRAs as compared to healthy controls (32/480 vs. 37/969, P=0.0166) (Chapter 5). We evaluated data held in dbSNP (build 129) for common variants in the Caucasian population by examining ten DNA repair genes and subsequently developed software for automatically extracting selected information from dbSNP (Chapter 6). This program was used to rapidly identify 221 common nonsynonymous SNPs in every DNA repair gene in the human genome; these were subsequently typed in 480 publically-available human lymphoblastoid cell lines generating a resource for functional analyses (Chapter 6). We also assessed the role of these SNPs in susceptibility to CRC and response to therapy by exploiting data and samples for the randomised controlled trial COIN (Chapter 7). Finally, we used Next Generation Sequencing (NGS) to discover SNPs in the exomes of 10 patients that exhibited peripheral neuropathy in response to chemotherapy and discovered that ERCC4 nonsense mutations may contribute to this toxicity (Chapter 8). NGS is likely to become the key SNP discovery technique over the next decade due to its potential to comprehensively search an entire genome at a comparatively low cost.

Ca²⁺ release via the cardiac ryanodine receptor (RyR2) is a fundamental event in excitation-contraction coupling. Point mutations in the gene encoding RyR2 are associated with arrhythmogenic right ventricular dysplasia type 2 (ARVD2), a disease likely characterised by abnormal release of Ca²⁺ that may result in sudden death. GFP-tagged RyR2 mutants (R¹⁷⁶Q/T²⁵⁰⁴M, L⁴³³P and N²³⁸⁶I) were generated and expressed in a human embryonic kidney (HEK) cell model, enabling profiling of the amplitude and temporal characteristics of caffeine-evoked Ca²⁺release through homotetrameric channels using confocal microscopy. Mutants were functionally heterogeneous and demonstrated profound differences in Ca²⁺ release when compared with WT channels, including the novel observation that one of the mutants (L⁴³³P) exhibited reduced sensitivity to caffeine activation. The molecular basis of this heterogeneity was investigated by determining the sensitivity of the mutant channels to cytoplasmic Ca²⁺. This was achieved by evaluation of caffeine-induced Ca²⁺ release from WT or mutants channels in streptolysin-O permeabilised HEK cells, where the cytoplasmic Ca²⁺ concentration was clamped. Although resting ER Ca²⁺store and cytoplasmic Ca²⁺ levels were comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca²⁺-dependent inactivation. We also investigated whether these mutations disrupted the interaction between RyR2 and accessory proteins involved in normal channel function. cDNA encoding mutation susceptible regions were constructed and screened against a human cardiac cDNA library using a yeast two hybrid system. The N²³⁸⁶I mutation abolished association of the RyR2 domain with two cardiac proteins, which robustly occurred with the corresponding WT domain. These findings demonstrate that ARVD2-linked RyR2 mutations critically affect channel activation and suggest that differential sensitivity to cytoplasmic Ca²⁺ may be a causative mechanism in the pathogenesis of this disease.

This project developed a solution for verifying external photon beam radiotherapy. The solution is based on a calibration chain for deriving portal dose maps from acquired portal images, and a calculation framework for predicting portal dose maps. Quantitative comparison between acquired and predicted portal dose maps accomplishes both geometric (patient positioning with respect to the beam) and dosimetric (2D fluence distribution of the beam) verifications. A disagreement would indicate that beam delivery had not been according to plan. The solution addresses the clinical need for verifying radiotherapy both pre-treatment (without patient in the beam) and on-treatment (with patient in the beam). Medical linear accelerators mounted with electronic portal imaging devices (EPIDs) were used to acquire portal images. Two types of EPIDs were investigated: the amorphous silicon (a-Si) and the scanning liquid ion chamber (SLIC). The EGSnrc family of Monte Carlo codes were used to predict portal dose maps by computer simulation of radiation transport in the beam-phantom-EPID configuration. Monte Carlo simulations have been implemented on several levels of High Throughput Computing (HTC), including the Grid, to reduce computation time. The solution has been tested across the entire clinical range of gantry angle, beam size (5 cm x 5 cm to 20 cm x 20 cm), beam-patient and patient-EPID separations (4 cm to 38 cm). In these tests of known beam-phantom-EPID configurations, agreement between acquired and predicted portal dose profiles was consistently within 2% of the central axis value. This Monte Carlo portal dosimetry solution therefore achieved combined versatility, accuracy and speed not readily achievable by other techniques.