Rozprawy doktorskie na temat „Bile”

The binding of selected bile acids and bile pigments by peptides and quaternary amines has been studied by adsorption and NMR experiments. Novel adsorbents with quaternized peptide-containing functional groups for bile acids have been prepared by solid phase peptide synthesis techniques. The adsorption studies, conducted in aqueous buffer solutions, show that these resins have an enhanced capacity, on a per active site basis, and improved specificity over cholestyramine and colestipol. The interaction between bile acid anions and the pendants is predominantly ionic linkage, although hydrophobic and other interactions are also important. An NMR study of the binding between bile acids and various ligands, including peptides, by the determination of carbon-13 spin-lattice relaxation times, confirms the ionic and hydrophobic interactions which occur cooperatively and simultaneously.<br>New adsorbents for bilirubin have been prepared by covalently coating a water-swellable polyamide resin with polypeptides. These resins have much higher capacities for bilirubin in aqueous buffer solution than cholestyramine and improved capacities over the resins with attached oligopeptide pendants. The binding behavior of the resin coated with poly- sc D-lysine is the same as that of poly- sc L-lysine. The amount of bilirubin adsorbed by these resins is directly proportional to the number of lysine residues on the resin, which is consistent with the formation of an ionic linkage. This is confirmed by a study of the interaction of bilirubin with an oligopeptide, sc L-lysyl- sc L-lysine, by measurements of proton and carbon-13 NMR spin-lattice relaxation times combined with nitrogen-15 NMR experiments. The $ sp{15}$N NMR spectra of bilirubin and some related bile pigments have also been assigned by two-dimensional $ sp{15}$N-$ sp1$H heteronuclear correlation experiments.

Our lab has shown that the bile salt found in the highest concentration in human fecal water, sodium deoxycholate, induces apoptosis in several cell types including Jurkat cells as well as human colonic epithelial cells. We have also found that cells within the normal appearing flat mucosa of patients with a history of colon cancer are relatively resistant to apoptosis induced by NaDOC. The current studies test the hypothesis that sodium deoxycholate induces multiple stress response pathway s that protect against apoptosis. I have tested this hypothesis by developing and analyzing cell lines that are resistant to sodium deoxycholate-induced apoptosis and focusing on two stress-response proteins known to be activated by sodium deoxycholate, poly(ADP-ribose) polymerase (PARP) and the redo-sensitive transcription factor nuclear factor-kappa B (NF-κB). I found that PARP is protective against NaDOC-induced apoptosis, and by independently inhibiting the individual subunits of NF-κB, I found that the p65 subunit is protective, while the p50 subunit is not. Development and subsequent characterization of the NaDOC-resistant HCT-116 cell lines identified several proteins that may be responsible for the development of apoptosis resistance. These proteins will be further tested in future studies.

Bile acids are C24 steroids that are derived in the liver from cholesterol and secreted into the intestinal lumen to aid in emulsification of dietary lipids and lipid-soluble vitamins. The indigenous intestinal microflora modify bile acids, producing up to 20 unique bile acid metabolites. The 7α-dehydroxylation of the bile acids is the most physiologically important bile acid biotransformation. All known intestinal bacteria capable of bile acid 7α-dehydroxylation are anaerobic, gram-positive rods of the genera Clostridium and Eubcicterium. Bile acid 7α-dehydroxylating bacteria often contain bile salt hydrolase, which hydrolyzes the peptide bond in taurine-conjugated bile acids to yield a free bile acid and taurine. Taurine is an organosulfonate containing a sulfite moiety. There have been no published reports indicating whether 7α-dehydroxylating bacteria can utilize taurine. Given that taurine and taurine-conjugated bile acids are found at great concentrations in the intestine, the ability to utilize the compound would confer a competitive advantage to these bacteria. In this study, the ability of 7α-dehydroxylating bacteria to dissimilate taurine and taurine-conjugated bile acids produce hydrogen sulfide was investigated. First, hydrogen sulfide produced by bile acid 7α-dehydroxylating bacteria cultured in tryptic soy broth and semi-defined media from taurine and taurine-conjugated bile acids was qualitatively detected by inclusion of ferric ammonium citrate in the media. The results obtained from trials utilizing anaerobic tryptic soy broth and from semi-defined medium were not consistent, suggesting that qualitative determination of sulfide by inclusion of ferric ammonium citrate is inconclusive. Then hydrogen sulfide produced by bile acid 7α-dehydroxylating bacteria cultured in modified semi-defined medium (not containing a reducing agent) over time in the presence or absence of taurine-conjugated bile acids was quantified using the methylene blue method. Sulfide concentrations in medium cultured with two different strains of bile acid 7α-dehydroxylating bacteria, Eubcicterium sp. 12708 and Clostridium sp. HD-17, in presence of 100 |j.M or 5 mM sulfonates were not significantly higher than those in the absence of sulfonate. In addition, the highest sulfide concentration determined from medium cultured with two different strains of bile acid 7α-dehydroxylating bacteria for a period of five days was not above backgroud level. These data demonstrated that these two bile acid 7α-dehydroxylating bacteria, Eubcicterium sp. 12708 and Clostridium sp. HD-17, are not capable of desulfonating taurine and taurineconjugated bile acids to produce hydrogen sulfide under the conditions tested.

Ammonium-containing polyacrylamide resins, ammonium-containing polystyrene resins, and their metal-ion coordinated analogs have been synthesized for the sorption of bile salts (BS). The effects of altering the backbone, the type of ammonium group, the number of ammonium group, the hydrophobicity of the pendant group, and of varying the metal ions on the adsorption of BS's, were studied to elucidate the structure-activity relationship.<br>The binding capacity of the resins for BS's generally increased with increasing number of ammonium groups per pendant group of the ammonium-bearing resins, and with higher charge of the metal complex cation of the metal-ion coordinated resin, demonstrating the primary importance of electrostatic interactions. It was also observed that, as the resin backbone was changed from hydrophilic polyacrylamide to hydrophobic polystyrene, the adsorption capacity increased substantially, demonstrating that hydrophobic interactions (H-H's) between the resin backbone and the BS's also play a significant role in the binding. The incorporation of hydrophobic segments -(CH$ sb2) sb{ rm n}$- into the pendant groups on polyacrylamide resins increased binding capacity, but had no effect for the more hydrophobic polystyrene resins. Primary ammonium-bearing resins often showed higher binding capacities than their quarternary analogs, suggesting that H-bonding reinforces the binding. Isotherms with an S-shape observed for most polyacrylamide resins indicate a positive cooperativity in the binding due to H-H's and H-bonding among BS anions bound at adjacent positions within resin beads. Binding models have been proposed to depict the formation of pendant group-BS mixed reversed micelles and ordinary BS micelles.<br>The studies of the kinetics of the binding of BS anions by the resins showed that a small resin particle size and more intense shaking substantially increase the rate of binding. This indicates that the binding process is mainly controlled by the diffusion of BS anions near and within the beads. (Abstract shortened by UMI.)

Made available in DSpace on 2016-04-25T20:21:22Z (GMT). No. of bitstreams: 1 Fhoutine Marie Reis Souto.pdf: 974083 bytes, checksum: ab742073e878f7eb4d7cf381d4f4a311 (MD5) Previous issue date: 2015-03-27<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>This work takes as a base the hypothesis that terrorism has been fought throughout history by the nations in a way akin to how Western medicine fights cancer, that unpredictable and unbearable disease that still has no definitive cure. Since its emergence in the end of the 19th century, terrorism this being the definition given by the world s states to violent struggles that endanger their wellbeing and existence has been targeted by procedures intent on physically eliminating it. However, just like tumors that strike again even after being extirpated, the diverse terrorist movements do not give u: The States have tried new treatments which, as radiotherapy and chemotherapy do when dealing with cancer, have a wide-ranging effect and intense, long-lasting side effects. When faced with the impossibility of a cure, both medicine and States invest in prevention and meticulous examinations to identify and eliminate potential threats still in formation. After over a century of fighting, both terrorism and cancer continue to manifest themselves. The victories are always individual, since as long as there is a State there will be terrorism just as cancer is a risk to everybody for as long as there s life, which remains ungovernable<br>Este trabalho parte da hipótese de que o terrorismo historicamente tem sido combatido pelos Estados como a medicina ocidental combate ao câncer, enfermidade imprevisível e insuportável, para a qual não há solução geral e definitiva. Desde sua emergência, no fim do século XIX, o terrorismo - definição dada pelos Estados às lutas contundentes que colocam em risco sua saúde e existência é alvo de procedimentos que visam sua eliminação física. Porém, como tumores que renascem mesmo após terem sido extirpados, os terrorismos não cessaram. Os Estados, por sua vez, lançaram mão de novos tratamentos, que semelhantes à rádio e a quimioterapia, tinham ação mais abrangente, efeitos colaterais intensos e prolongados. Diante da impossibilidade da cura, medicina e Estados investem em prevenção e exames minuciosos para identificar e eliminar potenciais ameaças ainda em formação. Após mais de um século de combates, terrorismo e câncer continuam a se manifestar. As vitórias são sempre individuais, pois enquanto houver Estado haverá terrorismo, do mesmo modo que o câncer é um risco para cada um enquanto há vida, que permanece ingovernável

Bile acids have been implicated by epidemiologic evidence as causative agents in colon cancer. Previous studies have indicated that the bile acids damage DNA. However, the conjugated forms of bile acids (eg. tauro conjugates) have not been tested for interaction with DNA. The present study compared the DNA-damaging ability of unconjugated and conjugated bile acids using the E. coli SOS test system. The E. coli tester strain was incubated with the bile acids and conjugated bile acids. Both cell survival and induction of the SOS response was measured. Among unconjugated bile acids, deoxycholate, chenodeoxycholate, and lithocholic acid were confirmed as DNA damaging agents by a decrease in surviving fraction and increase of the fraction of blue colonies undergoing SOS induction with increasing doses. Cholate, however, did not cause DNA damage by these criteria. Among the conjugated bile acids, taurochenodeoxycholate caused as much DNA damage as chenodeoxycholate. Taurodeoxycholate caused DNA damage, but had less of an effect than deoxycholate. Taurocholate and taurolithocholate failed to show a DNA damaging effect.

The work in this thesis demonstrates that bile acids and their conjugates are competitive inhibitors of glucocorticoid metabolism by both 11β-HSD1 and 5β-reductase in liver. In vivo, in male Wistar rats, manipulation of hepatic bile acid concentrations by dietary supplementation with chenodeoxycholic acid (CDCA; 1% w/w) suppressed activities of hepatic 5β-reductase and 11βHSD1 activity and caused a reduction in total urinary (mainly 5β-reduced) glucocorticoid metabolites. In response to acute restraint stress bile acid treated animals showed a delay in return to basal corticosterone levels indicative of altered clearance. In addition, adrenal weight and lipid accumulation within the adrenal gland was reduced in bile acid treated animals. Stimulation of bile acid synthesis by cholestyramine (5% w/w) reduced hepatic 5β-reductase activity with animals excreting fewer urinary total (mainly 5β-reduced) glucocorticoid metabolites. Conversely, a reduction in hepatic bile acid content via administration of a fat-free diet, induced hepatic 5β-reductase activity accompanied by an increase in total, principally 5β-reduced, urinary metabolites. Animals fed the fat-free diet had larger adrenal glands, and suppressed circulating corticosterone, again suggesting altered HPA axis regulation. These findings were pursued in rats with bile duct ligation, a pathological model of elevated bile acids. In these animals, reduced activities of 5β-reductase and 11β-HSD1 were also identified. Lastly investigations were made of the impact of bile acids on the renin-angiotensin-aldosterone system (RAAS). Manipulations which increased bile acids concentrations were predicted to reduce the rate of aldosterone inactivation, since it too is a substrate for 5β-reductase. Treatments increasing bile acids were accompanied by a renin-independent increase in aldosterone and associated sodium retention.

Thesis (M.S.)--University of Adelaide, Dept. of Surgery, 1989.<br>The experimental work described was performed in the Department of Surgery of the University of Adelaide during 1982-1983. Typescript (photocopy). Includes bibliographical references (leaves 191-211).

The interactions of cholestyramine with bile acids in aqueous buffer solutions were studied by in vitro adsorption experiments. Application of the Donnan theory, which is based on ion partitioning between two phases, indicates that the adsorption is an ion-exchange process--the bile acid anion displaces the chloride counter-ion of cholestyramine. Donnan considerations indicate that the bile acid in the resin phase exists in two forms, bound and unbound, but at higher Ceq the bound form is increasingly favoured. Since the concentrations of bound bile acid in the resin phase are above the critical micellar concentration, micelle-type ordering is occurring. It is also possible that the unbound bile acid in the resin phase aggregates to form "regular" micelles. The micellization promotes the partitioning of glycocholic acid into the resin phase, explaining the ability of cholestyramine to adsorb glycocholic acid significantly, in vitro.<br>Ion-exchange resins were prepared by solid phase peptide synthesis with active sites chosen to resemble those of cholestyramine. They were produced by coupling 4-(aminomethyl)benzoic acid, 4-aminophenylacetic acid or 4-(aminomethyl)phenylacetic acid to the backbone. The ion-exchange resins were prepared both as primary amines and in the quaternized form. The cholestyramine-like sorbents were synthesized with systematic changes in the structure, to determine which structural parts of cholestyramine are involved in the adsorption process. As compared to cholestyramine, both sets of resins were remarkably ineffective in adsorbing bile acids in vitro. It was found that the nature of the backbone determines the accessibility to the active site; that the resins with the methylene group positioned between the phenyl group and the amino group have higher adsorption capacity for glycocholic acid; and that quaternization increases the adsorption capacity. The two latter observations indicate the importance of the basicity of the active site. Therefore, in cholestyramine, the backbone is such that it permits the transfer of ionic species and the quaternary ammonium group is involved in the interaction with bile acids.<br>Computer modelling showed that the cholestyramine pendants are close to one another and are separated by benzene rings, thus leaving too little space between them to allow a bile acid molecule to interact with the benzene rings. Therefore, the bile acids must interact with the quaternary ammonium group, leaving the bile acid molecule inside the cavity where they interact with one another to form micelles. The possible modes of interactions of bile acids with the synthesized resins are more numerous since the pendants are not as close together. (Abstract shortened by UMI.)

Bile acid malabsorption or diarrhea (BAM or BAD) is a syndrome of chronic watery diarrhoea diagnosed predominantly by a SeHCAT test and less commonly by measuring levels of 7-hydroxy-4-cholesten-3-one (C4) and furthermore responds to bile acid sequestrants (BAS). Primary bile acid diarrhoea (PBAD) is the condition with no definitive cause and is under diagnosed, often being labelled as IBS-diarrhoea with an associated burden of disease for patients. Recently an alternative mechanism for PBAD involving impaired Fibroblast Growth Factor (FGF19) production has been proposed. Aims: The primary aims were to prospectively recruit patients with chronic diarrhea, and define them into groups of BAD, or chronic diarrhoea with normal SeHCAT; characterisation with SeHCAT, C4 and serum FGF19; investigation of genetic differences involved in bile acid metabolism. Methods: 152 patients recruited had routine investigations for chronic diarrhoea including SeHCAT test. Fasting serum FGF19, C4 and bile acids levels were measured from blood samples and relationships examined; FGF19 optimisation, response of BAS on PBAD patients and on FGF19 levels, effect of bowel preparation & short bowel syndrome (SBS) on FGF19 levels were measured. 11 PBAD patients were studied through the day to examine variations in FGF19. 6 SNPs in genes involved in bile acid metabolism were analysed in PBAD patients. Results: Significantly lower fasting levels of FGF19 and increased levels of C4 were seen in patients with BAD with associations with Body Mass Index. Sensitivities and specificities for FGF19 against known tests were calculated. Excellent responses to BAS with no significant effect of bowel preparation on FGF19 and variable patterns during the day were seen. Reduced FGF19 levels were seen in SBS. SNPs studied in PBAD and control patients have yet to show any significant differences in frequencies. Conclusion: This research has confirmed that levels of an easily measurable and stable hormone, FGF19 are reduced in patients with BAD and is not affected by chronic diarrhoea per se. The work provides evidence of a disordered FGF19 production in PBAD patients, failing to reduce bile acid secretion and resulting in excess bile acids entering the colon causing BAD.

The central idea of this work was design, synthesis and study of catalytic activity and enantioselectivity of new organocatalysts containing a chiral cavity mimicking the enzymatic pocket of Aldolase I. Following the longstanding interest of our research group in the use of bile acid derivatives in enantioselective processes, attention was addressed to the development of organocatalysts having bile acid structure, where, because of its concave structure, due to the cis junction of the A and B cyclohexanic rings (Figure 3), the cholestanic backbone and the appended substituents should form a chiral cleft that can help the enantioselection. In addition, the presence of free hydroxyl groups can constitute a further advantage by controlling, via hydrogen bonds, the position of the substrate in the cavity of organocatalyst. In particular in this project were synthesized: · A wide class of monoprolinamide derivatives of bile acids, in order to find the right match between proline and cholestanic backbone. In particular synthesis of proline derivatives in different position of bile acid could throw light on the influence of position of proline moiety in the cholestanic emicavity on selectivity of organocatalyst. · A class of bisprolinamide derivatives of bile acids that could take advantage from the cooperative effect of two proline moieties. In designing synthesis of these derivatives attention has been paid to the use of low cost, easy and fast procedures in order to improve availability of the new organocatalysts. Activity and enantioselectivity of this new organocatalysts have been studied with particular attention to: · Aldol reaction · Michael reaction During the activity studies evaluation of parameters that can improve rate of different reaction (temperature, solvents, catalyst loading, reagents…) was considered. The possibility to carry out reactions in water and with very low catalyst loading was checked, in order to evaluate ecosostenibility of the chemical process. Experimental results were collected and analyzed with the help of conformational and computational studies.

Chapter 1. Introduction to Molecular Tweezers Whitlock and Zimmerman developed a class of molecular hosts, popularly known as molecular tweezers, which sandwich aromatic guests by ii=ii interaction. Chapter 1 summarizes molecular tweezers of various kinds which have recently been synthesized. Chapter 2. Design and synthesis of "Bile Acid-Based Molecular Tweezers" Bile acids have a rigid backbone, and the array of hydroxyl groups separated by 5-7 A provides opportunities for the attachment of binding surfaces such as two extended chromophoric units.

Chapter 1. Introduction to Molecular Tweezers Whitlock and Zimmerman developed a class of molecular hosts, popularly known as molecular tweezers, which sandwich aromatic guests by ii=ii interaction. Chapter 1 summarizes molecular tweezers of various kinds which have recently been synthesized. Chapter 2. Design and synthesis of "Bile Acid-Based Molecular Tweezers" Bile acids have a rigid backbone, and the array of hydroxyl groups separated by 5-7 A provides opportunities for the attachment of binding surfaces such as two extended chromophoric units.

In familial adenomatous polyposis (FAP), inactivation of the APC gene is directly linked to the development of gastrointestinal polyps and cancer. It is likely that other epigenetic factors are involved in the malignant change of polyp to carcinoma. Previous studies have implied an abnormal bile acid profile, both in faeces and bile. In this study, using carefully matched control groups, extraction of bile acids from faeces and bile was performed and analysis was rigorously performed using Gas-liquid chromatography-Mass Spectrometry. No significant differences were found between the two groups in the profile of major bile acids. An increased faecal excretion of two minor bile acids, 5β-cholanoic acid-3α-ol-12-one and 5α-cholanoic acid-3α-ol-12-one and an increased level of 5β-cholanoic acid-3α-01-12-one in bile was found in patients with FAP. A difference in the faecal neutral sterol profile had also been suggested, but this study showed no significant difference between the two groups when matching controls are used. This study does not support the idea that there are significant differences in faecal bile acid, biliary bile acid or neutral sterol profiles between individuals with familial adenomatous polyposis and controls.

Intrahepatic Cholestasis of Pregnancy (ICP) is a liver disease that affects approximately 1% pregnancies in the United Kingdom. ICP is characterised by an increase in maternal serum bile acids (BA), as well as a change in bile acid composition, whereby the increase in primary bile acids, taurocholic acid (TC) and taurochenodeoxycholic acid (TCDCA) is most pronounced. ICP is characterised by maternal symptoms and can be complicated by adverse events for the fetus, the latter of which occur more commonly in pregnancies complicated by high maternal serum bile acid levels. Adverse events that may complicate ICP pregnancies include spontaneous preterm labour, fetal distress, fetal heart arrhythmia and even intrauterine death in the third term of pregnancy. The aetiology of the fetal death is still poorly understood and is thought to occur suddenly. This thesis aims to investigate the effect of bile acids on the models of the human fetal heart and elucidate the mechanism of action of the protective role of UDCA. In addition, interaction between cardiomyocytes and fibroblasts will be investigated. Potential mechanisms of protection against bile acid induced fetal arrhythmia were investigated in different fetal heart models. The different models of the fetal heart will be described first, which include previously used models and a novel model using cells isolated from human fetal tissue. The effect of bile acids on these models will be described subsequently, with the focus on prevention of TC induced arrhythmias using Ursodeoxycholic acid (UDCA) and NorUrsodeoxycholic acid (NorUDCA). Human fetal heart tissue was obtained from terminations of pregnancy and for the first time beating cardiomyocytes were obtained. Characterisation of cardiac markers such as α-Actinin in these cells shows that there are similarities between neonatal rat, human fetal cardiomyocytes and the induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) from Cellular Dynamics (CDI). Upon addition of TC to human fetal cardiomyocytes and CDI adverse effects occurred, including decreased spontaneous beating and prolongation of calcium transients and action potentials. Both UDCA and NorUDCA were not toxic to neonatal rat cardiomyocytes and fibroblasts, as used in the model and prevented TC-induced adverse effects. It was previously shown that there was a transient appearance of myofibroblasts and that these myofibroblasts play a role in TC induced arrhythmias. This thesis investigates role of fibroblasts and myofibroblasts in bile acid mediated arrhythmias. Addition of TC to human fetal and neonatal rat fibroblasts led to further depolarisation of these cells which could potentially depolarise connected cardiomyocytes. UDCA and NorUDCA on the other hand hyperpolarised these cells, which may be a potential mechanism of the protection seen in the models. UDCA and NorUDCA may act via modulation of the Sulfonylurea Receptor (SUR), a subunit of the ATP-sensitive inward rectifying potassium channel (KATP channel). This channel is involved in the regulation of the membrane potential of the cell. The interaction between cardiomyocytes and fibroblasts and the role of gap junctions in this was also investigated. Page | 4 Maternal symptoms include pruritus of the skin. Pruritus is defined as itching without a visible rash, which can become very severe in women with ICP. The pruritus usually resolves quickly after delivery of the baby. In addition, high bile acid levels are associated with increased risk of spontaneous preterm labour. The mechanism by which bile acids induce spontaneous preterm labour is not well understood and this thesis aims to further elucidate bile acid associated receptors and labour associated proteins expression levels. Expression levels of nuclear and membrane-bound bile acid receptors were investigated in myometrium tissue of labouring and non-labouring patients at term. FXR, one of the main nuclear bile acid receptors was not expressed. LXRα, LXRβ, PPARα and PPARγ were expressed as were the bile acid G-protein coupled receptor TGR5 and muscarinic receptor M5, while some muscarinic receptors (M1, M3 and M4) were not. It is difficult to obtain human myometrial tissue from earlier gestation, as the tissue is obtained from caesarean sections usually of non-labouring term pregnancies. This makes studies into the causes of preterm labour more difficult, especially those where high bile acid levels are involved, as it is often not possible to obtain consent for tissue collection from patients undergoing an emergency caesarean section due to difficulties whilst in term or preterm labour. Therefore other models could help elucidate the mechanisms behind early labour. For this reason novel protocols using a combinatory method called CombiCult (Plasticell) were developed which led to the differentiation of human embryonic (ES) and induced pluripotent stem (iPS) cells into myometrial smooth muscle cells (SMCs) in collaboration with Plasticell and these protocols will be described in this thesis. The work presented in this thesis provides new insight into the effect of bile acids on the fetal heart, with the focus on the development of novel cellular models of the human heart. The characterisation of human fetal cardiomyocytes and CDI was carried out using immunofluorescence staining of cardiac markers, which show changes upon maturation of the heart. Both the CDI and human fetal cardiomyocytes show an immature phenotype of these markers, similar to the neonatal rat cardiomyocytes. Optical recording of human fetal cardiomyocytes containing naturally occurring myofibroblasts showed that, similar to the neonatal rat model of the fetal heart, TC leads to calcium transient duration prolongation which can be prevented by UDCA. It was shown that Cx43 is involved in this interaction and that the cardiomyocytes and myofibroblasts are functionally coupled by GJs. Furthermore, work presented in this thesis provides further insight into the expression bile acid receptors in the myometrium, by providing evidence of the expression of various bile acid sensitive receptors, including the GPCR TGR5. Finally, for the first time, novel protocols were developed for the differentiation of myometrial smooth muscle cells.

Alterations in glucocorticoid (GC) biosynthesis and metabolism are associated with a variety of pathophysiological disorders including cholestasis, diabetes and other metabolic disorders. Bile acids (BA) are also important modulators of metabolic functions and regulate cholesterol, triglyceride and glucose homeostasis as well as being critical for dietary fat digestion, enterohepatic function, and postprandial thermogenesis. In intact cells and in vivo, the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme converts inactive GC precursors (cortisone in humans, and 11-dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively) thereby amplifying local intracellular GC levels. Interconversion by 11β-HSD1 of other sterols has also been described. These include conversions of 7keto-cholesterol to 7β-hydroxycholesterol, 7-oxodehydroepiandrosterone (7-oxo-DHEA) to 7α-hydroxy- and 7β-hydroxy DHEA, 7- oxo-lithocholic acid (LCA, a bile acid; BA) to chenodeoxycholic acid (CDCA, a 7α- hydroxylated BA) and ursodeoxycholic acid (UDCA, a 7β-hydroxylated BA) in human liver microsomes. In the liver, BA inhibit 11β-HSD1 but whether 11β-HSD1 regulates BA homeostasis is unclear. Evidence of molecular regulation of the enterohepatic recycling of bile acids by liver glucocorticoid receptor (GR) in mice does suggest a role for 11β-HSD1. It was therefore hypothesised that disruption of 11β-HSD1 expression in mice would impair BA recycling and might affect the relative concentrations of BA within the enterohepatic circuit. The primary objective of the current work was to investigate the impact of altered 11β-HSD1 on BA homeostasis. This was achieved using genetically modified mouse models with altered 11β-HSD1 expression, either globally or restricted to hepatocytes. BA are stored in the gall bladder and are released postprandially, to aid digestion. It was hypothesised that 11β-HSD1 deficiency might the affect the process of postprandial gall bladder emptying/refilling. Mice with global 11β-HSD1 knockout (Hsd11b1-/-) and age-matched control mice (C57Bl/6) were either fasted for 4h and culled or fasted for 4h and re-fed for another 4h before culling. Their response to fasting and re-feeding was assessed with specific focus on organs associated with BA recycling in the enterohepatic circuit (liver, gall bladder, serum and small intestine). Gall bladders of fasted Hsd11b1-/- and C57Bl/6 mice had similar volumes of bile but in fasted Hsd11b1-/- mice, BA concentrations were higher in serum and liver. As expected, re-feeding caused gall bladder emptying in C57Bl/6 mice with consequent increased serum and liver bile acid concentrations. In Hsd11b1-/- mice, the gall bladder did not empty and serum and liver BA concentrations were similar to the fasted state. To explore possible reasons for this, levels of mRNA encoding proteins known to be involved in hepatic BA transport were quantified using real-time q-PCR. Levels of mRNA encoding NTCP/ SCL10A1/ SCL10A1, the transporter responsible for most hepatocyte BA uptake, were increased in livers of fasted Hsd11b1-/- mice whereas levels of Slc51b mRNA, encoding the OST- transporter that facilitates BA removal from liver to the systemic circulation, and levels of Mrp2 and Atp8b1/FIC1 mRNAs (both encoding proteins which transport BA from liver into gall bladder) were decreased. This suggests that in fasted Hsd11b1-/- mice, BA transporter expression is altered to increase BA influx into hepatocytes and decrease efflux, to compensate for reduced levels of liver BA. These data together imply that bile acid recycling is controlled by 11β-HSD1 activity which regulates gall bladder emptying, hepatic BA concentration and BA transporter activity to ensure continuity of BA recycling within the enterohepatic circuit compartments. These changes may also affect digestion of lipids and fat-soluble micronutrients. Because 11β-HSD1 can directly metabolise secondary BA, it was predicted that 11β-HSD1 deficiency would lead to changes in the BA profile. Profiling of BA in the gall bladder was performed using mass spectrophotometry. In Hsd11b1-/- mice, 7α-hydroxylated BA predominated (cholic acid [CA]>α-muricholic acid [α- MCA]>CDCA>others), in contrast to C57Bl/6 mice in which 7β-hydroxylated BA predominated (ω-MCA>β-MCA>UDCA>others). The ratio of 7α:7β acids was therefore >100-fold greater in Hsd11b1-/- mice. This suggests that 11β-HSD1 either directly or indirectly controls the epimerisation of 7α- to 7β- hydroxylated BAs. Measurement of mRNAs encoding proteins important for hepatic BA biosynthesis in livers of fasted Hsd11b1-/- mice showed decreased expression of Scarb1/SR-B1, Cyp39a1 and Cyp27a1 (though with no change in levels of CDCA, the product of CYP27A1, in liver or bile fluid), compared to fasted control mice. Hepatic levels of Gpbar1/TGR5/GPBAR1 and Cyp3a11 mRNAs, encoding proteins important in BA detoxification, were increased and decreased, respectively. This suggests that Gpbar1/TGR5/GPBAR1, encoding G-protein coupled bile acid receptor (also called TGR5/GPBAR1) and an FXR target, could be induced to detoxify 7α-hydroxylated BA whereas expression of Cyp3a11, which catalyses the conversion of LCA to hyodeoxycholic acid (HDCA) is decreased; bile fluid of Hsd11b1-/- mice contained lower levels of LCA and little to no HDCA, though LCA and HDCA levels in liver were unaltered. Currently, the functional differences between 7α- and 7β- hydroxylated BA are not clear. However, these findings could have significant implications for bile acid-mediated transcription which, in turn, might affect lipid and sterol metabolism. Also, alterations in BA composition may have other physiological consequences via other pathways. Because cholesterol is the precursor of BA synthesis, it was hypothesised that western diet (WD) (containing cholesterol) would exacerbate and/or alter the phenotype of Hsd11b1-/- mice. Gall bladder weights of fasted Hsd11b1-/- and control C57Bl/6 mice did not change with western diet compared to chow diet. In control C57Bl/6 mice, the total BA concentration in the gall bladder increased in response to WD in comparison to chow diet. In contrast, Hsd11b1-/- mice showed no change in total BA concentration when fed on WD in comparison to chow. These data indicate that 11β-HSD1 is required by mice for the normal increase in total BA concentration in bile in response to dietary cholesterol. BA profiling of bile from control mice fed on WD showed no difference in the relative amounts of 7β-hydroxylated BA and 7α-hydroxylated BA to littermates fed on chow diet with the exception of β–MCA which increased, and α–MCA which decreased. Like chow-fed Hsd11b1-/- mice, BA profiling of bile from WD-fed Hsd11b1-/- mice showed a significant decrease in relative levels of 7β-hydroxylated BA (UDCA < β-MCA < others) and an increase in percentage of 7α-hydroxylated BAs (CA>α-MCA>CDCA>others) compared to C57Bl/6 controls. These data show that Hsd11b1-/- mice fail to show the normal increase in 7β-hydroxylated BA and decrease in 7α-hydroxylated BA observed in control mice in response to a cholesterol containing diet, suggesting 11β-HSD1 deficiency blunts the influence of cholesterol on BA composition. Measurement of hepatic mRNAs encoding BA transporters suggest that hepatocyte uptake of BA is decreased in C57Bl/6 on WD compared to those mice on chow diet, whereas this was not the case in Hsd11b1-/- mice where hepatic expression did not change with diet. Thus, Hsd11b1-/- mice failed to increase expression of Ntcp/ Scl10a1/ Scl10a1 appropriately, suggesting impaired hepatic BA uptake, while Slc51b (encoding OST-β) expression was increased, compared to control mice, possibly to reduce hepatic BA concentration by transporting BA out of hepatocytes into the systemic circulation. Therefore, Hsd11b1-/- mice may adapt to a cholesterol-induced increase in hepatic BA by blunting hepatic BA uptake via NTCP/ SCL10A1/ SCL10A1 and increasing hepatic efflux via OST-β. The effects of 11β-HSD1 deficiency upon BA recycling and BA profile and concentration within the enterohepatic circuit, could reflect 11β-HSD1 action within the liver or could be due to actions in other tissues.<br>To investigate the role of hepatic 11β-HSD1 specifically, 11β-HSD1 liver-specific knockout (Hsd11b1LKO), 11β- HSD1 liver-specific over-expressors (Hsd11b1LOE) and control mice with exon 3 of the Hsd11b1 gene “floxed” (Hsd11b1F) were studied. Findings from this study indicate a role for 11β-HSD1 in adaption to dietary cholesterol and suggest that hepatic 11β-HSD1 (as opposed to 11β-HSD1 in extra-hepatic tissues) is the main factor regulating BA metabolism. Also, work from this thesis demonstrates 11β-HSD1 is an important regulator of gall bladder emptying and filling, an important component of enterohepatic bile acid recycling. Based on these findings it is anticipated that therapeutic use of 11β-HSD1 inhibitors will result in BA imbalances within the enterohepatic circuit and therefore BA homeostasis. Care must therefore be observed when implementing therapeutic use of 11β-HSD1 inhibitors, with particular focus on patients with cholestasis, Addison’s disease and critically ill patients who already have known BA imbalances in their enterohepatic system.

Bile acids are physiological detergent molecules with a function to absorb dietary lipids and hydrophobic molecules in the gastrointestinal tract. It has emerged that bile acids are also agonists for the FXR nuclear receptor and the TGR5 membrane-bound receptor, and are key in regulating metabolism via both genomic and non-genomic factors. It is also apparent that bile acids could play an important role in the treatment of Parkinson’s disease and some cancers. A number of pharmaceutical companies have developed selective BA receptor agonists, with some progressing through clinical trials for a variety of metabolic disorders. Fluorine is used extensively in property optimisation due to its ability to modify a plethora of physicochemical effects. By selectively introducing fluorine into the bile acid skeleton, it is possible to modify hydrogen bonding properties, and thus improvements in receptor binding are conceivable. This thesis describes the synthesis of a number of fluorinated bile acid analogues, along with a discussion of some early biological results. Two interesting cases of an intramolecular C-F•••H-O hydrogen bond within the bile acid skeleton will also be presented.

Sodium is an essential electrolyte necessary to keep multiple systems in the human body functional. Knowledge of sodium-proton exchanger will give rise to multiple clinical applications to diseases that affect our current population, which is why I personally am interested in conducting research in this field. In order to understand GW 4064 effect on NHE8 gene expression, protein and RNA expression were investigated via treatment at different concentrations and different time intervals through Western blotting and Real-Time PCR Analysis. To find the mechanism behind GW 4064 effects on NHE 8 expression, the PGL3 FXR promoter was utilized to see if GW 4064 inhibitory mechanism was through transcription or translation. Expression, stimulation, and inhibition were determined via numerical calculations. From the resulting data, we can conclude that GW 4064 inhibition of NHE8 protein and RNA expression occurs via transcription.

The biliary epithelium is organized as a single layer of biliary epithelial cells (BECs) lining the biliary tree. BECs have three major biological functions: protection, secretion and proliferation. These functions may all be controlled by bile salts, the main constituents of bile. We therefore determined if human gallbladder-derived BECs exhibited bile salt transport activities that affect their secretory functions. We could show that the apical bile acid sodium- dependent transporter (ASBT) is expressed and functional in primary cultures of human BECs. Once transported inside BECs, bile salts stimulate chloride and mucin secretion through intracellular pathways linked to calcium. In BECs, the secretion process is however mainly elicited by membrane receptors that signal through the intracellular second messenger cAMP. We could show in a subsequent study that bile salts potentiate cAMP-regulated secretion in BECs by stimulating adenylyl cyclase activity through PKC α and δ. One of the most potent bile secretagogues is the vasoactive intestinal peptide, which exerts its effects through the vasoactive intestinal peptide receptor-1 (VPAC1). We have shown that VPAC1 is expressed in all major cell types participating in bile formation in humans (i.e. hepatocytes, intrahepatic and gallbladder biliary epithelial cells). Moreover, VPAC1 displays a gradient of expression along the human biliary tree, the gallbladder showing the highest level of expression. Interestingly, we could show that bile salts regulate VPAC1 expression in BECs through the farnesoid X receptor (FXR), indicating that bile salts may also regulate BECs secretion through transcriptional control. The secretory activities of BECs may be seen as a protective mechanism avoiding excessive exposure to toxics, such as endotoxins. We have shown that bile salts may also exert protection by controlling the expression of the antimicrobial peptide cathelicidin. Bile salts induced cathelicidin expression through two distinct nuclear receptors, FXR and the vitamin D nuclear receptor (VDR). Taken together, our results indicate that bile salts control BECs biological functions through post-traductional and transcriptional control. The involvement of VDR in the control of BECs biology may be of particular interest because VDR hepatic expression is restricted to non-parenchymal liver cells, such as BECs or resident hepatic macrophages (i.e. Kupffer cells).

Chapter One begins with an introduction to enzymology and leads into a discussion on BSSL beginning with the physical properties and the source of the enzyme. The literature concerning the kinetic properties of BSSL is reviewed, followed by a discussion on how the structure of enzymes influences their catalytic activity and specificity. The use of X-ray crystallographic techniques is addressed as a means of elucidating the three dimensional structure of enzymes. The chemicals, apparatus and standard methodologies used in this present investigation are described in Chapter Two. The means by which kinetic data measured for an enzyme-substrate system are analysed and compared are also discussed. Chapter Three describes the purification of bile salt-stimulated human milk lipase (BSSL) from whole human milk. A detailed study of the activity of the purified protein has been conducted against both lipid and ester substrates in order to monitor the progress of the purification. A further determination of the physical properties of the protein has also been conducted. Results from these studies have identified the protein as BSSL. In Chapter Four the methods used for determining the partial amino acid sequence of the enzyme are described. This study has revealed interesting homologies with enzymes from other species. The sequence of that part of the enzyme which includes the active site has been determined and has been found to be identical to the consensus sequence found in the active site of pancreatic lipase, serine proteases and cholinesterases. It may therefore be postulated that the similarity observed for some of the kinetic behaviour of enzymes arises from homology in their amino acid sequences and, in particular, those portions of the protein comprising the active site. The use of kinetic isotope effects, to gain insight as to the mechanism of BSSL catalysed hydrolysis of lipid substrates, is the subject of Chapter Five. A mechanism has been proposed which explains the observed effects and takes into account information from the literature. The mechanism also incorporates findings made from the amino acid sequence study and the literature reports on the residues involved in catalysis. Chapter Six begins within a literature survey of detergent less microemulsions and continues with an account of the kinetic properties of BSSL in this new and novel medium in which enzymic reactions involving substrates of low water solubility may be conducted. The advantage of this medium is that it allows one to monitor the reaction by spectrophotometric means. In more traditional methods of assay of lipid substrates this is not normally possible. This advantage has also been exploited in the study of the kinetics of BSSL against triolein in reversed micelles, which is the subject of Chapter Seven. A detailed description is given of an FT-IR technique which allows one to monitor the course of reaction of biologically important substrates.

Previous in vitro binding studies for bile acids by polymeric ion-exchange resins indicate that the binding process involves three types of interaction; a predominant electrostatic interaction, a secondary hydrophobic interaction and a seemingly less important hydrogen bonding effect. To further investigate the nature of the sorption process, a family of ammonium-bearing polystyrene resins have been synthesized for the in vitro sorption of bile acid anions. A correlation was made between the structure of the resin pendant group and sorption efficiency. The extent to which the binding process is affected by bile acid anion by hydrophobicity was also studied.<br>The primary importance of the ionic interactions was demonstrated by a marked increase in the sorption capacity of the resins with an increase in the number of ammonium groups per resin pendant group. Varying the length of the pendant hydrophobic spacer had a minimal effect on the binding, since, it is postulated, the hydrophobic backbone of the sorbents provides a degree of hydrophobicity sufficiently high to promote the binding of bile acid anions even at low C$ sb{ rm eq}$'s. In the competitive binding studies, the sorption of bile acid anions increased with increasing bile acid anion hydrophobicity and, in some cases, resin selectivity increased with an increase in the number of pendant ammonium groups and the length of the pendant hydrophobic spacer, as reflected by the relative binary separation factors of the resins.