The object of this thesis is to investigate the value of analysis of WGLF in the assessment of Gl pathology, specifically in the measurement of blood and protein loss from the gut. The method for haemoglobin measurement was selected on the basis of a need for sensitivity and the need to detect metabolites of haemoglobin as well as the parent molecule. The measurement of haemoglobin in WGLF has been assessed in a series of patients with clinical conditions known to result in anaemia and which may be related to blood loss from the gut. The relationship between the subjective assessment of inflammatory bowel disease activity and the measurement of WGLF proteins arising by leakage from plasma has been examined with a view to establishing more objective activity indices. The specificity of WGLF protein measurements for the diagnosis of active inflammatory bowel disease has been assessed in a large group of patients with a variety of gastrointestinal pathologies. Gut clearance measurements of plasma proteins and haemoglobin have been undertaken to evaluate quantitative protein and blood loss in normal and pathological groups. Many workers have used alpha-1-antitrypsin in faeces as an indicator of inflammatory bowel disease, and different molecular weight forms have been described. The molecular weight of alpha-1-antitrypsin in WGLF has been investigated in controls and patients with inflammatory bowel disease. Hyaluronic acid, a structural glycoprotein found in the submucosa of the intestine has been determined in WGLF in control and patient groups in an attempt to find a direct index of tissue disruption during active disease process in the gut.

The purpose of this thesis was to investigate the regulation and function of 11β-HSD-1 in liver and in the hippocampus. Examination of 11β-HSD-1 activity in a liver perfusion system by measurement of glucocorticoid metabolism across the intact liver confirmed that 11β-reduction is the predominant reaction in this organ and indicated that the route of delivery of substrate is important, with the reactivation of inert substrate increasing as the ratio perfused through the hepatic portal vein: hepatic artery increases. These data suggest that hepatic 11β-HSD-1 activity in liver potentially increases intrahepatic active glucocorticoid levels. Many hepatic enzymes of carbohydrate and fat metabolism are regulated by glucocorticoids, including phosphoenol carboxykinase (PEPCK), the rate-limiting enzyme gluconeogenesis and in humans, 11β-HSD inhibition increases hepatic insulin sensitivity. Therefore I examined the effect of selective and near complete repression of hepatic- 11β-HSD-1 by oestradiol on glucocorticoid-inducible gene expression in liver. Oestradiol treatment resulted in reduced expression of glucocorticoid-inducible genes, including PEPCK. This effect could not be attributed to a direct action of oestradiol. Furthermore, inhibition of whole body 11β-HSD-1 plays an important role in potentiating glucocorticoid action in the liver and illustrates the potential to alter gluconeogenesis/insulin sensitivity by manipulating hepatic 11β-HSD-1 activity. Examination of the regulation of hippocampal 11β-HSD-1 demonstrated that intracerebroventricular administration of growth hormone had no effect on 11β-HSD mRNA expression. This is in direct contrast the periphery, where growth hormone is a major regulator. Examination of 11β-HSD-1 activity in the hippocampus and liver in a well-documented model of chronic psychosocial stress in the tree-shrew showed that chronic stress attenuates hippocampal 11β-HSD-1 activity, whereas excess glucocorticoid administration has no effect.

Gene trapping in mouse embryonic stem cells has been used to identify and characterise the function of novel genes. Introduction of a gene trap vector into the genome results in the generation of a fusion between the lacZ reporter gene and the endogenous trapped gene. The consequences of this are predicted to be threefold: (i) expression of the reporter gene will be controlled by the promoter and enhancer elements of the endogenous gene and will therefore mirror endogenous gene expression; (ii) the generation of a fusion transcript allows endogenous gene sequence to be cloned by 5'RACE-PCR and (iii) the insertion of the gene trap vector can disrupt the function of the endogenous gene. This work describes the characterisation of one specific gene trap integration, 1114 that has not behaved entirely as predicted but has none-the-less identified an early marker of hepatic specification. The reporter activity profile associated with the 1114 gene trap integration is restricted to the definintive endoderm marking the ontogeny of the foetal liver. Reporter activity is observed as early as the 9 somite stage (8.0-8.5dpc) in endodermal cells of the foregut in the region destined to form the liver diverticulum and is restricted to the hepatic lineage until late gestation. Comparison of 1114 reporter activity with AFP identifies 1114 reporter activity as being the earliest, most specific marker of liver organogenesis identified to date. Breeding of the gene trap integration to homozygosity reveals no overt phenotype but its unique pattern of expression prompted us to clone the endogenous sequence. This has proven to be more complex than predicted as integration of the gene trap vector results in the production of two fusion transcripts. The most abundant (Group I) fusion transcript is ubiquitously expressed. No reporter activity is produced from this fusion transcript as gene trap vector splicing to this sequence places translation of the lacZ gene out-of-frame. The second, less abundant (Group II) fusion transcript is expressed exclusively in the liver during embryogenesis and is predicted to produce reporter activity. The cloning and sequencing of both the genomic sequence and the endogenous gene (gtar - gene trap nkyrin repeat) associated with the Group I and II fusion sequences has revealed that they represent different exons of gtar. Expression of the Group I and Group II sequences independent of the gene trap vector mimics that of the 1114 fusion transcripts. Furthermore, expression of these sequences in 1114 homozygous tissues indicates that the insertion of the gene trap vector has failed to disrupt the expression of gtar. Expression of the liver specific exon of gtar is postulated to be a consequence of either a separate promoter immediately upstream of the exon or alternative splicing.

There is mounting experimental evidence demonstrating protection by free glycine against stress in several cell types. The glycine transporter type 1 (GlyT-1) mediates the high affinity supply of glycine, which together with cysteine is required for the synthesis of the antioxidant glutathione. Previous work in this laboratory has established that GlyT-1 is expressed on the apical and basal membranes of intestinal epithelial cells and that its mRNA levels are regulated by stress. In the present study exactly how stress signals to transcriptional induction of GlyT-1 was investigated. Caco-2 cells transfected with reporter constructs of sequences of the GlyT-1a proximal promoter and 5’UTR cloned upstream of a β-galactosidase coding sequence, showed increased reporter activity following treatment with thapsigargin (Tg), tunicamycin (Tu), amino acid (AA) starvation, tert-Butylhydroquinone (tBHQ) or Diethyl maleate (DEM). Despite no changes in Nrf-2 mRNA levels, a significant increase in total Nrf-2 protein abundance was evident on western-blots following DEM treatment of Caco-2 cells. However, gel shift showed no protein-DNA complexes between Nrf-2 protein and a DNA probe sequence of the putative antioxidant response element (ARE) identified in the GlyT-1a 5’ flank. Despite a significant siRNA mediated knock-down of Nrf-2 mRNA and protein, there was no further effect on GlyT-1a expression. Unlike Nrf-2, the knock-down of Atf-4 diminished the basal and stressed induced expression of GlyT-1a. Atf-4 was detected bound to DNA probes containing a potential amino acid response element (AARE) located in the first exon of the GlyT-1a gene by gel shift and super shift assays. QPCR assays performed on DNA isolated from Caco-2 cells by chromatin immunoprecipitation (ChIP) using antibodies against Atf-4, demonstrated 9, 5 and 2-fold enrichment of the GlyT-1a AARE following Tu, AA starvation and DEM treatment respectively. Site directed mutation of the GlyT-1a AARE showed a 75% reduction in reporter activity as well as attenuated protein-DNA interaction with a representative probe. It is evident from the data presented in this thesis that the direct interaction of Atf-4 at the proposed GlyT-1a AARE contributes to its transcriptional up-regulation following endoplasmic reticulum stress, nutrient stress and oxidative stress.

Cholestatic liver diseases are defined by impairment of bile flow or bile formation. Progressive familial intrahepatic cholestasis (PFIC) is a group of cholestatic disorders, so far associated with three genes encoding canalicular membrane transporters; nevertheless, one third of patients with progressive intrahepatic cholestasis remain without an aetiology. Targeted and/or whole-exome sequencing was undertaken in 83 families, mostly consanguineous, with no mutations in known PFIC genes. Homozygous mutations in tight junction protein 2 (TJP2) were identified in 21 individuals of 15 families. Most were predicted to be protein-truncating, and most patients had severe liver disease requiring liver transplantation. Some also had extrahepatic manifestations. Four individuals were found to carry the same homozygous missense mutation, three had late-onset and remittent cholestasis, one was asymptomatic. TJP2, also known as zona occludens-2 (ZO-2), encodes a cytosolic component of cell-cell junctional structures. Patients with severe disease had no ZO-2 protein. The presence, and distribution of integral tight junction protein, claudin-1, was found to be disrupted. Tight junction structure was abnormal on transmission electron microscopy. The absence of ZO-2 might have been compensated by other junctional components. The expression of tight-junction-related genes was analysed in TJP2 deficiency patients; however significant, biological-relevant, changes were not identified. It appears, therefore, that the complete absence of TJP2 causes disruption of tight junction structures and severe cholestatic liver disease, whilst missense mutations in TJP2 lead to less severe phenotypes. In addition whole-exome sequencing analysis revealed one patient with a novel homozygous missense mutation in the gene α-methylacyl-CoA racemase (AMACR). The change was predicted to be deleterious for the encoded protein. The finding was also supported by the clinical manifestation of this rare metabolic disorder with early-onset cholestasis. A failure in the identification of the causative mutations occurred for the other 5 patients where whole-exome sequencing was performed, possibly due to limitations in the methods used.

Cholesterol is an integral part of the cell membrane and influences both its fluidity and activity. Furthermore membrane lipids are influenced by plasma lipid levels. In familial hypercholesterolaemia (FH) red cell rheology and platelet aggregation is abnormal. These effects could be due to changes in plasma lipids since they are improved by cholesterol reduction. Section A Fasting lipid profile, membrane cholesterol, red cell ghost and platelet fluidity [using diphenyl hexatriene (DPH) and trimethylamino-diphenyl hexatriene (TMA-DPH) fluorescence anisotropy] and Ca2+ -Mg2+ -ATPase activity (using [32P] ATP hydrolysis) in 30 patients with heterozygous FH and 19 controls were compared before and after treatment with colestipol (10g) , simvastatin (10mg) and maxepa (10g). The two groups were generally comparable with respect to age, sex, BMI and blood pressure. In FH plasma cholesterol, membrane cholesterol, TMA-DPH anisotropy and red cell Ca2+ -Mg2+ -ATPase were increased whilst platelet Ca2+ -Mg2+ -ATPase was reduced (p<0.05). Colestipol and simvastatin reversed these changes toward controls. Maxepa increased DPH anisotropy and reduced Ca2+-Mg2+ -ATPase. In FH membrane fluidity and activity is altered and normalised by cholesterol lowering. This may account for the abnormal cell function in this condition. Section B A double blind, randomized, placebo controlled trial in hypercholesterolaemia (cholesterol 6.5-10 mmol/l, triglycerides <3 mmol/l). After 8 weeks of an AHA step I diet, patients were randomized to A: placebo; B: 5g colestipol + 10mg simvastatin; or C: 10g colestipol + 10mg simvastatin for 8 weeks. Patients were assessed 4 weekly. 44 patients were screened, 32 completed the study. Groups were generally comparable (group C patients were older p<0.02). Active treatment resulted in a 38% reduction in LDL cholesterol (p0.8). Mild gastro-intestinal upset was the commonest adverse event. Low dose combinations are effective and well tolerated. There is no apparent advantage of 10g over 5g colestipol when combined with l0mg simvastatin. HDL cholesterol and triglycerides were unchanged.

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Understanding disease ecology is vital in preventing future outbreaks of established infections and to predict the emergence of new pathogens. In recent decades there have been a number of high profile infectious diseases which have swept across countries and in some cases the world. Many of these begin as generalist emerging infections; such microbes are difficult to study in the wild due to their inherently ambiguous life histories and complex associations with numerous hosts and the environment. In this PhD a number of techniques are used to pinpoint and further understand the life history of one such pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer, in the hope that this data can be used to predict and prevent future outbreaks and can be applied to other emerging infections. The results of this study include the first identification of the pathogen in the environment for a whole new continent, South America. Further to this it has led to the discovery of the likely ecological niche of the bacilli by linking its presence to specific functional groups of organisms. In turn the occurrences of these groups have been related to anthropogenic conditions such as deforestation and human mediated land use. Finally complex links between climatic fluctuations and outbreaks of the disease in Southern America and Cameroon, central Africa help complete our understanding of this mysterious disease.