Current evidence indicates synergy between hyperammonaemia and inflammation in the brain with liver failure. Utilising animal and laboratory experiments, this thesis explored a number of concise questions focused on progression to brain oedema and coma with hepatic encephalopathy (HE). STUDY 1: It is unclear whether the background cirrhotic state or hyperammonaemia predisposes to superimposed inflammation. Question 1: Does lipopolysaccharide (LPS)-induced systemic inflammation worsen brain oedema in cirrhotic bile duct-ligated rats? and is this associated with blood-brain barrier disruption? altered brain ammonia and/or inflammatory pathways? Answer 1: LPSinduced pre-coma/coma and exacerbated cytotoxic oedema, indicating synergy between hyperammonaemia and inflammation associated with brain protein nitrosation. STUDY 2: New ammonia-lowering therapies targeting multiple organs are necessary. Question 2: Does combining L-ornithine and phenylacetate (OP), synergistically improve ammonia reduction? Answer 2: L-ornithine and phenylbutyrate synergistically lead to sustained ammonia-lowering and limited oedema; L-ornithine detoxifying ammonia by providing a substrate for glutamine synthesis and phenylacetate renally excreting the glutamine as phenylacetylglutamine. STUDY 3: Despite apparent synergism, therapies for HE target either hyperammonemia or inflammation, not both. Question 3: Can a reduction in ammonia in cirrhotic rats prevent LPS-induced worsening of brain oedema and progression to pre-coma/coma? Does targeting hyperammonaemia and inflammation together provide therapeutic synergy? Answer 3: Ammonia primes the brain to the deleterious effect of LPS, with the ammonia-lowering effect of OP preventing LPS-induced coma and brain edema. STUDY 4: Aquaporin-4 (AQP4), a bi-directional astrocyte water channel, is thought to provoke brain oedema in neuropathic disorders. Question 4: Is AQP4 causally involved in the brain oedema associated with models of liver failure? Answer 4: AQP4 has no causal role in the brain edema associated with hyperammonemia or inflammation, with or without acute liver dysfunction. However in cirrhosis, AQP4 upregulation, with contemporaneous p38MAPK activation is possibly a compensatory response to inhibit edema formation.

Bioartificial Liver Devices (BAL) could provide hepatic function in patients with liver failure awaiting transplantation or liver regeneration. Certain BALs use human hepatoblastoma-derived HepG2 cells that can perform many hepatocyte specific functions, however they cannot adequately detoxify xenobiotics via Cytochrome P450 (CYP) mediated pathways. The aim of this thesis was to identify and develop a cell source able to provide CYP function in a BAL, and focused on CYP1A2, since its inducible function has been well established in HepG2 cells and CYP3A due its large contribution to hepatic CYP expression and function. Firstly, gold standard comparators of freshly isolated primary human hepatocytes (PHH) were established. The influence of BAL culture conditions on CYP function were evaluated, including chemical induction, culture media composition, alginate encapsulation and microgravity 3D culture: HepG2 cells were able to provide sufficient CYP1A2 but limited CYP3A function. Intestinal cell lines LS147T and Caco2 and the hepatocyte derived cell line HC-04 were investigated as potential sources to provide CYP3A BAL function. All three were shown to proliferate in BAL specific medium within a 3D culture system. Under these conditions, increased CYP3A function was demonstrated for each cell line, but their usefulness within our BAL was limited. The effects of increasing HepG2 expression of two nuclear receptors (Constitutive Androstane Receptor and Retinoid X Receptor α) were examined. Within a transient system, increased nuclear receptor expression, in conjunction with chemical induction, resulted in improved function of multiple CYP isoforms; CYP3A function was increased to levels approaching those measured in PHH. Inducible CYP3A function of a stable CAR transfectant was then demonstrated in 3D culture. Lastly HepG2 cell CYP function was also shown to be source-dependent. Overall it was concluded that through adaptation of culture conditions and genetic manipulation, these cells have the potential to provide CYP function within a BAL.

This study had 4 aims. The first aim was to explore the relationships between personal control and coping responses in patients with the chronic bowel disorders of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). The second aim examined coping in IBS and IBD patients as a process, by focusing on a specific episode of their primary symptoms. The coping profiles of the IBS and IBD patients were compared to establish whether differences in coping between the 2 groups were present. The third aim examined differences between groups and tested for linear trends across IBS, IBD patients and Non-patient controls in relation to anxiety, depression and self-esteem. A supplementary aim was to evaluate whether anxiety in the 3 groups was considered to be clinically significant (i.e. fell above the threshold for "caseness"). The fourth aim tested differences between the 3 groups in perceived stress, as previous findings have been inconclusive. Fifteen IBS patients and 15 IBD patients who attended a Gastroenterology (G.I.) Clinic were interviewed and completed a battery of self-report measures. Fifteen Non-patient controls completed a battery of self-report measures. The data was analysed using Pearson's product moment correlation coefficients for aim 1; profile analysis using MANOVA for aim 2, and one-way between subjects ANOVA's for aims 3 and 4. The research hypotheses were not met, as the results were not statistically significant. However, two post-hoc findings revealed that patients with bowel disorders used equal proportions of problem and emotion-focused coping and these types of coping were significantly positively correlated. The test of flatness within profile analysis was statistically significant which indicated that patients with bowel disorders do not use a "blanket" approach to coping with an episode of their primary symptoms. A percentage of people in each group met the criterion for "caseness" of clinically significant anxiety. The results were evaluated in relation to the research literature and directions for future research were outlined. It was suggested that there might be a subset of patients with bowel disorders who experience clinical anxiety and/or depression and screening in G.I. clinics was recommended.

This study utilized a qualitative, phenomenological methodology to study obesity. The purpose was to better understand the experiences of obesity, treatment and weight loss from the perspectives of formerly obese individuals, their partners and as couples. The participants consisted of six couples mostly from rural communities. In each couple, at least one the partners had experienced weight fluctuations into the obese range and had achieved at least 50% of his or her weight loss goal. The couples participated in two semi-structured interviews, both individually and as couples. A genogram was constructed with each couple to help identify family of origin influences on their weight individually and as a couple. The participants were considered the experts. The findings of this study were enhanced by use of multiple data sources. Salient meta-themes from the findings included: 1) Fluidity of the experience 2) Family of origin: weight metaphors, childhood stories and obesity fear factors 3) Weight gain, role changes, and social isolation 4) Weight communication: perceived boundaries and inadequacies 5) First-order/second-order changes 6) Body image dysphoria 7) Weight as the third person in the relationship Recommendations for weight loss must include hope about weight loss treatment, and how to begin the journey. Marriage therapists cannot assume that weight loss resolves peripheral issues for the couple; indeed, for some couples, weight loss further complicated their marriages regarding issues of sexuality, social connections and quality of life. Body image dysphoria may persist after weight loss and may require psychotherapy. This study further identified the need to educate and provide support to the partner of the overweight spouse. Some partners are able to provide support; others feel incompetent and intimidated by the sensitivity of weight. Perception of partner support was also identified as an important factor in weight fluctuation. The overweight individual may require guidance in recognizing and receiving partner support. Ostensibly, the importance of support from partners through treatment and weight loss cannot be overemphasized.

The gastric pathogen Helicobacter pylori is linked to gastritis, leading to gastric cancer but the mechanisms are unclear. Gastritis is characterised by remodelling of the gastric mucosa, which requires degradation of the extracellular matrix (ECM). Proteases such as matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), as well as proteases inhibitors such as plasminogen activator inhibitor (PAI)-1 play an important role in this process. Since members of the uPA system (namely PAI-2, uPA and its receptor, uPAR) have been shown to be increased by H pylori, it was hypothesised that PAI-1 expression is also increased in the gastric mucosa during infection, which could playa role in the tissue remodelling observed during the development of gastritis. The aim of this study was to investigate the effect of H pylori on PAI-1 expression in the gastric mucosa, to examine the consequences of this protease inhibitor on human primary gastric epithelial cell proliferation and to determine its initial contribution to gastric mucosal protection during epithelial injury. Real-time quantitative polymerase chain reaction (Q-PCR) of biopsies from gastric corpus, but not antrum, showed significantly increased PAl-1 expression in H pylori positive patients which was further increased in gastric cancer patients compared to controls. Using immunohistochemistry, it was found that H pylori significantly increased the expression of PAI-1 in primary gastric epithelial cells, particularly in the acid-secreting parietal cells. Expression of PAI-I in gastric cancer patients was also found in epithelial cells. A transgenic mouse model of gastric remodelling (INS-GAS) infected with H felis showed an increase in PAI-1 expression six months post-infection. Proliferation assays showed that both H. pylori and exogenous uPA stimulated human primary gastric epithelial cell proliferation, which was further increased after PAI-I knockdown using anti-sense oligonucleotides treatment, consistent with PAI-l inhibition of endogenous uPA. Transfection of human primary gastric epithelial cells with uPA, PAl-lor uPAR promoters, in luciferase reporter constructs, revealed expression of all three in H+, K+-ATPase- and VMAT-2-expressing cells; uPA-Iuciferase was also expressed in pepsinogen-containing cells and uPAR in TFF-l-expressing cells. In each case, expression patterns of the luciferase reporter constructs were similar to that of the endogenously expressed proteins, and all were increased in response to H. pylori. However, the virulence factor CagE was required for the stimulation ofuPA, but not PAI-lor uPAR. The production and use of uPA system transgenic. mice in an acute model of gastric mucosal injury revealed that PAI-l plays a protective role during gastric epithelial damage. Mice in which this gene is deleted displayed a more severe phenotype after challenge compared to wild-type mice, as did uPA overexpressing mice. However, this phenotype was reversed when PAI-l was overexpressed, consistent with the idea that expression of PAl-1 is vital for the restraint of uPA in order for the initial formation and stabilisation of a haemostatic fibrin plug at the site of epithelial injury to occur. Together these data indicate that the initial induction of PAI-l expreSSIOn 111 acute H. pylori infection is part of a defensive mechanism as it has been demonstrated that PAI-l plays a pivotal role in the protection of the gastric epithelium after injur y. However, PAI-l also acts to restrain uPA, therefore controlling epithelial cell proliferation which IS a key characteristic of a preneoplastic, H pylori infected epithelium. During prolonged H pylori infection it can be suggested that sustained increased levels of anti-fibrinolytic PAI-I contribute to the process of fibrosis, increased proliferation favours the acquisition of mutations which influences tissue remodelling leading to gastric cancer.

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Lecithin-cholesterol acyltransferase (LCAT) is an enzyme principally secreted by the liver into the circulation where it esterifies cholesterol and plays a key role in high- density lipoprotein (HDL) metabolism. In familial and acquired (liver disease) LCAT deficiency, the failure to esterify cholesterol causes many cellular and metabolic disturbances. Here, I describe the purification of recombinant LCAT and assess two approaches to treat LCAT deficiency. Human LCAT cDNA was cloned into a selectable expression vector and used to generate a stably–transfected Chinese hamster ovary (CHO) cells secreting human LCAT tagged with 6 histidine residues. Productive clones were selected, monitoring LCAT activity by a modification of a radioactive enzymic assay for plasma, and the enzyme purified from culture medium by immobilised cobalt affinity chromatography. The pure LCAT, as judged by SDS- PAGE, was used to raise monoclonal antibodies in LCAT knockout mice for future development of a sensitive immunoassay. For therapy, I evaluated injection of pure LCAT into the peritoneal cavity of LCAT knockout mice using single and repeat dose regimes. LCAT activity was measurable in plasma post-injection and the percentage of esterified cholesterol increased, while agarose gel electrophoresis confirmed a rise in HDL levels. In a second approach, I encapsulated the recombinant CHO cells in biocompatible and semipermeable alginate-polylysine microcapsules using a syringe pump extrusion method. A study in vitro showed that, after an initial lag phase, LCAT was secreted for over 90 days with the capsules remaining intact. These microencapsulated cells were implanted into peritoneal cavities of LCAT-deficient mice. LCAT activity was detected in mice plasma one week post-implantation; the relative amount of esterified cholesterol was increased and lipoprotein profile was improved. I conclude that injection of recombinant enzyme or of encapsulated LCAT-secreting cells are feasible therapies for familial and acquired LCAT deficiency.

No description available.

No description available.