Cholangiocarcinoma (CCA) is a rare cancer with a poor prognosis. Much of medical research has focused on investigating cancers with a higher incidence and little focus has been devoted to this disease. The aim of this thesis was to perform a protein analysis of CCA and cholangiocyte cell lines. Differences between immortalised cancer and normal cells were sought in order to identify potential therapeutic targets and/or diagnostic tools. A variety of CCA cell lines were used, reflecting both intra and extrahepatic disease. The different subtypes of CCA through the developed and developing world are also represented so differences were also sought between them. Proteomic analysis was performed using DIGE with subsequent spot selection. Identified spots were extracted and processed using mass spectrometry. In addition, available chemotherapy agents were tested in vitro against the same cell lines to check for their action and how this could be enhanced. A benzodiazepine receptor antagonist (PK11195) was used to demonstrate apoptosis promotion in the presence of established cytotoxic agents (gemcitabine, etoposide, 5 fluorouracil and cisplatin). Cytotoxic assays were carried out using the SRB (Sulphorhodamine B) assay. Cell lines were tested for benzodiazepine receptor status using qRTPCR and response was correlated.

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. / 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.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Student: Marie Šimková Supervisor: Mgr. Alena Prašnická Title of diploma thesis: Alternation of bile production due to iron depletion Introduction: Liver has an irreplaceable role in the production and secretion of bile. This body fluid serves as the main excretion way of some endogenous and exogenous substances. Another liver property is the ability to store substances essential for correct functions of the body, e.g. iron. It has been shown that iron could have an impact on the bile production and secretion. Aim: The aim of this diploma thesis was to discover an impact of iron depletion on the bile synthesis and metabolism, especially on bile acids, and the way it affects transporters expression. Methods: Male Wistar rats (n=6 in each group, 250 ± 20 g) were divided into two groups: control group (Chow diet) fed with standard diet and iron depletion group (ID), fed with iron depletion diet for 21 days. To investigate the changes in bile flow, the bile had been collected for 120 min during in vivo clearance study. The analysis of the changes in expression of bile transporters and enzymes responsible for de novo bile acid synthesis was performed at the mRNA (qRT-PCR) and protein (Western blot)...

No description available.

Clostridium scindens VPI 12708 (previously known as Eubacterium sp. VPI 12708) is a bile-acid dehydroxylating bacterium originally isolated from the feces of a colon cancer patient. Many genes required for bile acid 7-a dehydroxylation are found on a large bile acid inducible operon (bai) that has been extensively studied. However the bai promoter, which directs expression of the bai operon, has yet to be characterized due, in part, to a lack of a functional genetic transfer system for this strain. A spontaneous rifampinresistant Clostridium scindens VPI 12708 mutant was used as a recipient to determine the efficacy of conjugation as a method of DNA transfer. The Clostridium perfringens plasmid pECU-001, containing a chloramphenicol acetyl transferase marker, and the Birmingham IncP-a oriT was transformed into Escherichia coli S17-1 which was used as a donor for conjugation. E. coli EM24 (Rif R) served as a recipient to ensure the donor was conjugal. All bacterial cultures were grown to mid-log phase and harvested by centrifugation. Matings between the donor and recipient were carried out on the surface of anaerobic tryptic soy agar slants. hloramphenicol-resistant E. coli EM24 colonies were isolated suggesting E. coli S17-1 was conjugal under the conditions tested. Chloramphenicol-resistant Clostridium scindens VPI 12708 (RifR) colonies were isolated using both 4:1 and 1:4 (vol:vol) donor: recipient ratios. Plasmid preparations with subsequent restriction map analysis from putative Clostridium scindens VPI 12708 transconjugants confirmed the presence of pECU-001. Conjugal transfer of pWKU-001 (which is pECU-001 with the bai promoter insert and a downstream b-glucuronidase reporter gene) was attempted using a similar protocol, but with erythromycin as selection for recipient. The plasmid was successfully introduced into C. scindens, however pWKU-001 was not maintained and lost from the culture. These data suggest DNA can be introduced into Clostridium scindens VPI 12708 via E. coli S17-1-mediated conjugal transfer, which may prove useful for the study of the genetic regulation of the bai promoter. Unfortunately, pWKU-001 appears to be unstable and unsuitable in present form for extensive studies.

Stargazers (Uranoscopidae) have two specialized characters- large volume of bile and a small-size brain. To explain why stargazers need large volume of bile, two hypotheses were proposed: (1) a function of the bile of stargazers is acting as the sex pheromone, and (2) bile enhances calcium uptake efficiency. The brain volume was measured, and the volumes of each part of the brain were also measured. The elementary investigations indicate that the stargazers have larger bile volume ratio and smaller brain weight ratio than other fishes reported in the present studies. The bile volumes are no significant difference between the female stargazers and the male stargazers, the bile volumes showed no monthly difference. Besides, the number of olfactory epithelium and the neuron density of the telencephalon in stargazers were not more than other fishes, the function of sex pheromone of bile can not be confirmed. The cranium hardness, bone density, and calcium content in stargazers are significantly more than other fishes. Most fish uptake calcium by the gill and the intestine from seawater or food. However, stargazers often hide in the sand and wait for the prey for most of the time, less water flow through the gills are expected. Therefore stargazers possibly uptake calcium through the intestine. Efficiency of the bile to aid calcium uptake has to be examined by experiments. The paraffin histology has been taken in various parts of the brain. Crista cerebelli is the center for sensing mechanical vibration; the volume of stargazer¡¦s crista cerebelli is higher than other fishes. Stargazers have large saccular otolith compare to the whole brain. Studies on the lateral line and acoustic sense of stargazers are new research topics. Furthermore, decrease in brain size in stargazers is for the purpose of reducing the oxygen consumption, so as to hiding in the sand. Stargazers are one of the fisheries species on western Taiwan coast. However there are very few studies on the stargazers in Taiwan; we should understand more about them in order to protect and consume the stargazers in a better way.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 98-116). Also available in electronic version. Access restricted to campus users.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Photochromism is a phenomenon where two isomers with markedly different absorption spectra are interconverted by a reversible photochemical reaction. The photochromism of 1’,3’,3’-trimethyl-6-nitrospiro[2H-1]-benzopyran-2,2’-indoline (NSP) and 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5-hexafluoro-1-cyclopentene) (DAE) was studied in aqueous solutions containing NaCl and the bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) or sodium taurocholate (NaTC). Bile salts are amphiphilic compounds that aggregate in water. These aggregates can solubilize hydrophobic organic compounds in water and affect the reactivity of the bound compounds. NSP and DAE are photochromic compounds that can be switched between a colored and a colorless isomer. The colored isomer of DAE can only be transformed into the colorless form by irradiation of light, while the colored isomer of NSP is also converted into the colorless form by a dark reaction. The dark reaction rate constant of NSP increases at high concentrations of bile salt and NaCl. The bile salt structure also affects the dark reaction rate constant, which is smaller in NaTC and approximately the same in NaC and NaDC. The activation energy for the reaction in all conditions studied is similar to the value reported for polar organic solvents. A method that employs HPLC was developed to determine the molar absorptivity coefficients of photochromic compounds. The values obtained were important to determine the quantum yields for photocoloration (ΦAB) and photodecoloration (ΦBA). Quantum yield values were determined by a photokinetic method that employs irradiation at a single wavelength and numerical analysis. The values of ΦAB and ΦBA for DAE in bile salts are the same as the values in cyclohexane. For NSP, ΦAB is dependent on the structure of the bile salt and increases in the order NaTC < NaC < NaDC. / Graduate

Photochromism is a phenomenon where two isomers with markedly different absorption spectra are interconverted by a reversible photochemical reaction. The photochromism of 1,3,3-trimethyl-6-nitrospiro[2H-1]-benzopyran-2,2-indoline (NSP) and 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5-hexafluoro-1-cyclopentene) (DAE) was studied in aqueous solutions containing NaCl and the bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) or sodium taurocholate (NaTC). Bile salts are amphiphilic compounds that aggregate in water. These aggregates can solubilize hydrophobic organic compounds in water and affect the reactivity of the bound compounds. NSP and DAE are photochromic compounds that can be switched between a colored and a colorless isomer. The colored isomer of DAE can only be transformed into the colorless form by irradiation of light, while the colored isomer of NSP is also converted into the colorless form by a dark reaction. The dark reaction rate constant of NSP increases at high concentrations of bile salt and NaCl. The bile salt structure also affects the dark reaction rate constant, which is smaller in NaTC and approximately the same in NaC and NaDC. The activation energy for the reaction in all conditions studied is similar to the value reported for polar organic solvents. A method that employs HPLC was developed to determine the molar absorptivity coefficients of photochromic compounds. The values obtained were important to determine the quantum yields for photocoloration (AB) and photodecoloration (BA). Quantum yield values were determined by a photokinetic method that employs irradiation at a single wavelength and numerical analysis. The values of AB and BA for DAE in bile salts are the same as the values in cyclohexane. For NSP, AB is dependent on the structure of the bile salt and increases in the order NaTC < NaC < NaDC. / Graduate / 2015-03-31