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Quorum sensing (QS) acts as an important communication wiring in Pseudomonas aeruginosa. The bacteria use the system to regulate production of autoinducers for several adaptation and survival advantages. The system controls the production of key factors like proteases and rhamnolipid. Due to the substantial genome size and plasticity of P. aeruginosa, other factors are increasingly being linked with QS calling for the need of a better understanding of this complex system. The initial part of the thesis acknowledges the QS adaptation in P. aeruginosa clinical isolates from Cystic Fibrosis (CF) patients. Much of the factors that are deemed important for early infection establishment become redundant, as observed through loss of QS behaviour in most of the late isolates investigated in this study. This was particularly validated through complete loss of QS gene expressions and virulence factors like elastase, pyocyanin and rhamnolipid in a late clinical isolate, PA80. However, this cannot be regarded as an "universal adage" as isolates with and without QS activity were detected from the same patient in late or chronic stage of infection. The observations recorded in the study helps us to sketch a natural system where isolates having both QS activity and inactivity can co-exist in harmony in a chronic CF lung. In a stressful lung environment, it would be beneficial to have only certain isolates producing the expensive QS metabolites, in concentrations sufficient for the entire community, allowing the cell energy to be reserved for other functions. P. aeruginosa is a notorious nosocomial pathogen which is intractable with most of the common antibiotics. The resistance towards most antibiotics emphasises the need for alternative strategies to reduce virulence without eliciting a selection pressure on the bacterial population. Targeting QS with natural compounds is a promising strategy. This work, using a reliable and accurate RT-qPCR method developed in­house, demonstrated that trans-cinnamaldehyde and salicylic acid could attenuate expressions of QS genes at sub-inhibitory concentrations. Trans-cinnamaldehyde was effective in downregulating the Iasi and lasR genes by 13- and 7-fold respectively while salicylic acid caused smaller reductions of 3-fold in Iasi and 2-fold in lasR compared to untreated samples. The ability to interfere with the master QS genes had a consequent inhibitory effect on the production levels of QS controlled virulence factors (elastase, protease, pyocyanin). Rhamnolipid, an important QS metabolite, was extensively studied and showed to be significantly downregulated following treatment with the inhibitors. The use of QS inhibitors alone may not completely abrade the infection but can reduce the pathogenic load by decreasing the levels of QS regulated virulence factors. This could allow for effective use of antibiotics at much lower concentrations than used now. Rhamnolipid is not only regarded as an important clinical factor but also holds promise as "green surfactant" due to its amphipathic structures allowing for characteristics like chemical surfactants. Therefore, an ability to regulate the rhamnolipid production would be useful in both industry and medicine. However, there is a lack of quantitative understanding on the role of QS regulatory pathways and rhamnolipid metabolic pathways in rhamnolipid biosynthesis in P. aeruginosa. The gap has been reduced in the final study of the thesis which showed a highly conserved and regulated gene expression profiles for rhamnolipid production. The rhl QS genes are indispensable to produce rhamnolipid while the expression of rhIC only initiates after rhlAB genes have been maximally expressed. This makes the rhlAB the rate limiting factor in rhamnolipid production. The stringent regulation explains why rhamnolipids are synthesized in low yields. Therefore, there is a need to look for associate factors that could be engineered for increasing rhamnolipid production. One of these factors could be the transport proteins that are involved in extracellular movement of rhamnolipid or accumulation of precursors. HPLC-MS/MS chemical characterisation of rhamnolipid from PAO1 with transposon mutation in transport genes revealed that the inactivation does not affect the composition of rhamnolipid with C10-C10 fatty acid congeners being most dominant. However, the yields reported from the mutants were lower than the wild type suggesting these genes may also have a more indirect effect on rhamnolipid production. The thesis therefore suggests that the contribution of QS in P. aeruginosa adaptability, pathogenicity and rhamnolipid production is indeed significant and has contributed novel data which will aid our understanding of these important processes.

Barrett’s metaplasia (BM) is the condition whereby the stratified squamous epithelium at the lower end of oesophagus is replaced by intestinal-like columnar epithelium. BM is associated with chronic cell injury caused by acid and bile salts refluxing from the stomach (so-called Gastro-oesophageal reflux disease (GORD)). BM is important clinically as it is the only known precursor to oesophageal adenocarcinoma. Metaplasia is associated with the aberrant expression of key (master switch) transcription factor(s). It is therefore possible that intestinal transcription factors such as those involved in regulation of intestinal gene expression may be involved in the development of BM. In addition, the development of BM may be linked to exogenous factors such as acid, bile salts and inflammatory mediators, which leads to an inflammatory response and the premalignant condition. Therefore, the aims of the current project are three-fold to investigate (i) the role of HNF4α in the development of BM, (ii) the role of HNF1α, CDX2, FOXA2 and PDX1 in the development of BM and (iii) the effects of exogenous factors involved in the inflammatory response and the Wnt and Notch pathways on HNF4a and CDX2 expression in the development of BM. The data showed that ectopic HNF4α, CDX2 or HNF1α expression induces intestinal gene expression in the human normal oesophageal epithelial HET1A cells, but FOXA2 or PDX1 ectopic expression did not provoke intestinal gene expression. Furthermore, ectopic HNF4α expression in mouse oesophageal epithelial explants induced the squamous-to-columnar cell type conversion as well as VILLIN protein expression. These results suggest that ectopic expression of HNF4α, CDX2 and HNF1α might be involved in the initiation of BM. Finally, exogenous factors involved in the inflammatory and the Wnt and Notch pathways treatment failed to induce intestinal gene expression in the HET1A cells, but further studies such as extending the treatment time and the use of the 3D organotypic models or animal models could be tested.

The aim of this project was to demonstrate the presence of immune complexes (IC) in the sera of patients with Crohn's disease (CD). The integrity of the complement system (C') was also investigated and comparisons were made with similar investigations on patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA). ICs were detected in the sera of 32.2% of patients with CD, 100% of sera from patients with AS and 55% of synovial fluids from patients with RA, as shown by assessment of anticomplementary activity (ACA). The method was believed to detect small ICs. Although total haemolytic complement, as assessed by CH50 assay, was normal both C3 and C4 levels were consistently elevated in the sera of patients with CD. All these factors were depressed in patients with AS and RA. Measurement of C' activity and serum factors C3 and C4 gives a static picture of the involvement of C' in CD. A more dynamic view could be obtained by the demonstration of C3 inactivation products in the sera of patients with CD. This was demonstrated in 32.9% of sera tested. This was taken as evidence of in vivo C' activation by ICs and it was suggested that elevated C3 and C4 levels were due to alterations in catabolism or synthesis. No relationship was found between the presence of ICs and raised C' levels; these factors were not found to be related to duration of disease activity, steroid therapy or disease activity. Since no differences were observed between patients in active or quiescent phases of the disease, it was concluded that the disease is characterized by a continuing immunological process. Gel filtration and immunoglobulin analysis of ACA positive sera revealed that ACA was confined to high molecular weight fractions containing IgG and IgA. It was concluded that ICs in the sera of patients with CD were composed of IgG and possibly IgA complexed with an unknown antigen.

Helicobacter pylori is a gram negative, microaerophilic bacteria that plays an important role in chronic gastritis and peptic ulcer disease. Multiple antimicrobial therapies including combinations of clarithromycin, metronidazole or amoxycillin and proton pump inhibitor have been used to eradicate H. pylori. Antimicrobial resistance in H. pylori has been associated with treatment failure. In this study, antimicrobial susceptibility patterns of amoxycillin, ciprofloxacin, clarithromycin, erythromycin, tetracycline and metronidazole were evaluated in 110 H. pylori strains isolated from 454 antral biopsies of patients undergoing endoscopy at the Royal Infirmary, Edinburgh. The MICs were determined by E-test. Resistance to clarithromycin and erythromycin was found in 8.2% (9/110) and 9.1% (10/110) respectively. Metronidazole resistance was demonstrated in 8 isolates (7.3%). Tetracycline resistance was found in one of the isolates (0.9%). Two isolates were resistant to ciprofloxacin (1.8%). Resistance to amoxycillin was not detected. Molecular mechanisms of fluoroquinolone, macrolide and metronidazole resistance in H. pylori were investigated. Resistant to fluoroquinolone has been associated with alterations in the Quinolone Resistance-Determining Region (QRDR) of gyrA gene. Mutation at position 91, leading to an amino acid change from Aspartic acid to Asparagine was found in 2 ciprofloxacin-resistant isolates. One isolate had a mutation at Asparagine-87 to Lysine. Mutations in the 23S rRNA conferring macrolide resistance were investigated. Mutations at position 2143 (A to G) were shown in seven of the ten macrolide-resistant isolates. Two of the seven isolates carried an additional T to C mutation at either position 2182 or 1934. Of the ten macrolide-resistant isolates, two had a single mutation at either position 2182 or 2195. Mutation at position 2182, however, has previously been identified not to be associated with macrolide resistance. The mutations at position at 1934 (T to C) and position 2195 (C to T) have not previously been reported. One of the ten isolates (MIC > 256 mg/L) had no alteration in the 23S rRNA. The results indicate that different mechanisms play a role in macrolide resistance in these H. pylori strains. Metronidazole resistance has been reported to be associated with mutations in the rdxA gene, encoding oxygen-insensitive nitroreductase. To investigate the role of rdxA, sequencing analysis of rdxA of metronidazole-resistant isolates was determined. The results showed that no particular amino acid substitution was associated with metronidazole resistance. One isolate contained a nonsense mutation, generating a stop codon. However, two metronidazole-sensitive strains had alterations in rdxA by insertions of a mini-IS605 sequence. These results suggest that alterations in rdxA are not the sole mechanism of metronidazole resistance and other mechanisms are required in the development of resistance. Since the prevalence rate of metronidazole resistance is variable, ranging from 11 to 70%, it is possible that some variation in reported resistance levels derives from difficulties in the method of sensitivity testing. To set a standard for susceptibility testing for metronidazole in H. pylori, the optimum conditions for sensitivity testing were evaluated. Activation of metronidazole requires an anaerobic environment. It was found that incubation under microaerophilic conditions elevated metronidazole MIC, suggesting that microaerophilic conditions cannot activate metronidazole to its active form. Pre-incubation of H. pylori in anaerobic conditions for 24 hours prior to incubation under microaerophilic conditions for 72 hours was found to be necessary to achieve accurate susceptibility results. This can explain why some centres report high levels of metronidazole resistance. To investigate the development of fluoroquinolone resistance in H. pylori, ciprofloxacin-resistant mutants were selected in vitro by exposing sensitive strains to serial increments of ciprofloxacin in Columbia blood agar plate. The QRDR of gyrA gene was analysed for mutations. Reduced susceptibility to ciprofloxacin was associated with either a single or double amino acid changes in gyrA gene. Mutations at position 85, 87 and 91 were found to be associated with ciprofloxacin resistance. The results also demonstrated that gyrA mutations are not sole contributors for the mechanism of ciprofloxacin resistance in H. pylori. As no parC gene has not yet been identified in H. pylori, any contribution from topoisomerase IV cannot be quantified. To investigate the DNA gyrase activity in H. pylori, gyrA and gyrB were separately cloned and overexpressed by using a T7 promoter vector, which contain a fusion tag of six histidine residues. GyrA and GyrB were purified by affinity chromatography using nickel-chelating resins. The ability of DNA gyrase to supercoil relaxed DNA was determined.

Acute pancreatitis is an acute inflammatory process of the pancreas, with variable involvement of other local and remote organs. There are similarities in terms of clinical presentations and manifestations between acute pancreatitis and sepsis. Recent theories from sepsis studies suggested that there is a pro-inflammatory response at the beginning and a subsequent anti-inflammatory response at a later stage of the disease. It has been proposed that it is the uncontrolled pro-inflammatory response which leads to multiorgan dysfunction; whereas the later anti-inflammatory response contributes to an immuno-compromised state, and therefore increases the likelihood for nosocomial infection. The main aim of this project is therefore to characterise the dynamics of the pro- and anti-inflammatory responses during an episode of severe acute pancreatitis, using both lung and peripheral blood as the surrogate markers for remote organ and systemic immune responses respectively during the disease process. Arginine- and caerulein- induced acute pancreatitis rodent models were used to investigate the immune responses. Although there was a trend of more severe acute pancreatitis in the arginine model than the caerulein model, there was no statistical difference in the histological scorings of both acute pancreatitis models. The present studies therefore suggest that: There was a trend of increased alveolar macrophage phagocytic capacity halfway through the disease process. However, the alveolar macrophage phagocytosis was only significantly elevated when the rodents had completely recovered from the episode of severe acute pancreatitis; The overall phagocytic capacities of both monocytes and granulocytes were significantly dampened halfway through the disease process. Granulocytes contributed to the majority of this dampening effect. At the same studied time-point, further analysis on the survival of granulocytes revealed a reduction of apoptosis/necrosis of granulocytes. These observations would therefore suggest a malfunction of bacterial clearance by granulocytes, despite their increased survival. The reason for this malfunction is uncertain. In a similar manner to the alveolar macrophages, monocyte phagocytosis was significantly upregulated in rodents with pancreatitis towards the resolution of the acute pancreatitis episode. Using lipopolysaccharides (LPS) to simulate septic events at different stages of acute pancreatitis in this rodent model, there was a net reduction in granulocyte and monocyte apoptosis halfway through the disease process, but not at any other time-points during an episode of severe acute pancreatitis. This phenomenon suggested that sepsis exerts its most profound effects on leukocyte survival halfway through the recovery from acute pancreatitis, and that this coincided with the reduction in the ability to perform bacterial clearance. In addition, the immune response in the liver, as another remote organ target, during acute pancreatitis was investigated. It was discovered that HO-1 (an anti-oxidant and heat shock protein) was induced within the liver parenchyma at the beginning of the disease process. Its reduction throughout the acute inflammatory process was associated with an increase in oxidation within the liver parenchyma. The results are discussed in the light of current knowledge on the pathophysiology of acute pancreatitis.

There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question since a proportion of individuals with steatosis progress to steatohepatitis. This thesis is focused on defining i) the effect of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function ii) the contribution of triglyceride, ROS and subsequent mitochondrial impairment to the metabolism of energy substrates iii) the effect of the intracellular antioxidant, N-acetylcysteine, on hepatic mitochondrial function and metabolic alterations associated with human steatohepatitis iv) the effect of different free fatty acids species on ROS production and subsequent metabolic response in hepatocytes. To address these questions, we designed in vitro models using human hepatoblastoma C3A cells treated with various combinations of oleate, octanoate (O), lactate (L), pyruvate (P) and ammonia (N) acutely or for 72 hours, before measurements of triglyceride concentration, cell respiration, ROS production, mitochondrial membrane potential, ketogenesis and gluconeogenesis, metabolomics analyses, confocal and electron microscopy. Acutely, LPON treatment enhanced mitochondrial respiration and ROS formation. After 72 hours, despite the similarities in triglyceride accumulation, LPON treatment, but not oleate, dramatically affected mitochondrial function as evidenced by decreased respiration, increased mitochondrial membrane potential and enhanced ketogenesis. Importantly, this was associated with increased markers of oxidative stress and enhanced gluconeogenesis. Furthermore, reduction in triglyceride with metformin did not improve mitochondrial function. By comparison, respiration and ROS formation remained unperturbed with oleate. The addition of the antioxidant N-acetylcysteine prevented mitochondrial dysfunction and reversed metabolic changes seen with LPON, strongly suggesting ROS involvement in mediating mitochondrial impairment. Our data indicate that increased ROS formation, rather than cellular steatosis per se, impairs mitochondrial function. Thus, reduction in cellular steatosis may not always be the desired outcome without concomitant improvement in mitochondrial function and/or a decrease in ROS formation.

Background: Wireless capsule endoscopy (CE) was introduced in clinical practice just over a decade ago; it has since established a new era in the investigation and diagnosis of small-bowel diseases. Nevertheless, the detection of small-bowel pathology can be limited by issues related to the current level of CE technology. Furthermore, the clinical validity of the use of surrogate markers of diagnostic yield such as the ampulla of Vater (AoV), or that of various prokinetics, to increase the completion rate - and theoretically, the diagnostic yield - have not been clearly established. Other factors that could optimize the rate of detection of small-bowel pathology in CE are: 'speedy' video sequence review, chromoendoscopy and/or the application of three-dimensional (3-D) image-reproduction software. Threedimensional imaging in CE is not currently feasible due to hardware limitations. However, software algorithms (shape-from-shading, SfS) that enable 3-D reconstruction in CE are available. Methods: The database of capsule endoscopy examinations of our centre includes procedures performed with two different models of capsule endoscopes. The detection rate of the duodenal papilla was examined in the largest - to date - cohort of small-bowel capsule endoscopy videos obtained with two different capsule endoscopy systems. Using meta-analysis software, the impact of various prokinetics was analysed. Furthermore, the validity and safety of QuickView pre-read was examined. In regard to proprietary chromoendoscopy software, Blue Mode filter offers better image enhancement when compared with Fujinon Intelligent Chromoendoscopy (FICE). Out of four publicly available SfS algorithms, Tsai's method is the one that gives the better results. Tested on still-capsule endoscopy images, the application of a 3-D reconstruction software leads to image enhancement for a significant proportion of vascular, but less so for inflammatory and protruding, lesions. Furthermore, the adjunct of 3-D reconstruction to the standard two-dimensional video reading software significantly improves the performance of novice small-bowel CE readers in distinguishing masses from mucosal bulges, thus potentially shortening their learning curve. Results: This thesis demonstrates that the selective and judicious use of prokinetics - and specifically that of metoclopramide with purgative and/or real-time viewer - in capsule endoscopy improves the completion rate. My results also show that the persistently low rate of AoV detection using two different smallbowel CE systems underlines the weakness of non-steerable CE. Although the benefits of QuickView are outweighed to some extent by a decrease in the overall detection rate, this mode can be used confidently in overt obscure gastrointestinal bleeding in an urgent inpatient setting and in outpatients with occult obscure gastrointestinal bleeding or suspected Crohn's disease. FICE (especially I) and Blue Mode is useful for the characterization of small-bowel findings. Conclusion: There are limitations in the current commercially available software for CE review. The inclusion of a 3-D representation algorithm may be of training and diagnostic benefit. Until optics technology allows hardware-enabled threedimensional reconstruction, it seems a plausible alternative. Further clinical and development work is required in order to optimize the currently available reading software.