Abstract Fatty acid degradation in living organisms occurs mainly via the β-oxidation pathway. When this work was started, it was known that the hydration and dehydrogenation reactions in mammalian peroxisomal β-oxidation were catalyzed by only multifunctional enzyme type 1 (MFE-1; Δ2-Δ3-enoyl-CoA isomerase/2-enoyl-CoA hydratase 1/(3S)-hydroxyacyl-CoA dehydrogenase) via the S-specific pathway, whereas in the yeast peroxisomes via the R-specific pathway by multifunctional enzyme type 2 (MFE-2; 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl-CoA dehydrogenase). The work started with the molecular cloning of the rat 2-enoy-CoA hydratase 2 (hydratase 2). The isolated cDNA (2205 bp) encodes a polypeptide with a predicted molecular mass of 79.3 kDa, which contains a potential peroxisomal targeting signal (AKL) in the carboxyl terminus. The hydratase 2 is an integral part of the cloned polypeptide, which is assigned to be a novel mammalian peroxisomal MFE-2. The physiological role of the mammalian hydratase 2 was investigated with the recombinant hydratase 2 domain derived from rat MFE-2. The protein hydrates a physiological intermediate (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl-CoA in bile acid synthesis. The sequence alignment of human MFE-2 with MFE-2(s) of different species reveals 12 conserved protic amino acid residues, which are potential candidates for catalysis of the hydratase 2. Each of these residues was replaced by alanine. Complementation of Saccharomyces cerevisiae fox-2 (devoid of endogenous MFE-2) with human MFE-2 provided a model system for examing the in vivo function of the variants. Two protic residues, Glu366 and Asp510, of the hydratase 2 domain of human MFE-2 have been identified and are proposed to act as a base and an acid in catalysis. Mammalian MFE-2 has a (3R)-hydroxyacyl-CoA dehydrogenase domain, whereas the yeast MFE-2 has two dehydrogenase domains, A and B. The present work, applying site-directed mutagenesis to dissect the two domains, shows that the growth rates of fox-2 cells expressing a single functional domain are lower than those of cells expressing S. cerevisiae MFE-2. Kinetic experiments with the purified proteins demonstrate that domain A is more active than domain B in catalysis of medium- and long-chain (3R)-hydroxyacyl-CoA, whereas domain B is solely responsible for metabolism of short-chain substrates. Both domains are required when yeast cells utilize fatty acids as the carbon source.

This dissertation describes the effects of tetrastarch (130/0.4) on serum colloid osmotic pressure and thromboelastography variables in healthy pony mares. Additional variables assessed during this study included markers of haemodilution (PCV, TS) and serum creatinine and bile acid concentrations. Six clinically healthy Nooitgedacht pony mares were utilized in a crossover study design. Tetrastarch (130/0.4) was administered at 10, 20 and 40 ml/kg bwt to each mare in a random sequence with a two week washout period between each of the treatments. Packed cell volume (PCV), plasma total solids (TS), serum colloid osmotic pressure (COP), and platelet count were measured and thromboelastography (TEG) was performed before treatment (baseline), immediately after infusion (time 0), and 1, 6, 12, 24, 48, and 96 h after tetrastarch infusion. All TEG variables remained within reference range in all treatment groups. Administration of tetrastarch at 40 ml/kg bwt resulted in a prolonged K-time at 6 h post-infusion, and decreased maximum amplitude at 0, 1, 6, 24 and 48 h post-infusion compared to baseline. Administration of tetrastarch increased mean COP values above baseline in all three treatment groups, persisting to 24, 6 and 48 h after treatment with 10, 20 and 40 ml/kg of tetrastarch respectively. This study concluded that, although values remained within established reference ranges, the administration of tetrastarch (130/0.4) at 40 ml/kg bwt is more likely to induce changes in TEG variables than doses of 20 ml/kg or less. Tetrastarch increased COP in healthy horses at all evaluated dose rates. / Dissertation (MMedVet)--University of Pretoria, 2014. / gm2014 / Companion Animal Clinical Studies / unrestricted

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Indiana University-Purdue University Indianapolis (IUPUI) / Primary Sclerosing Cholangitis (PSC) is a rare and slow progressing cholangiopathy characterized by hepatic inflammation, fibrosis and ductular reaction with liver transplantation as the sole therapeutic option. PSC patients are at high risk of auto-immune comorbidities like irritable bowel disease (IBD), found in up to 80% of PSC patients (PSC-IBD). There are indications of genetic and environmental components for auto-immune development in IBD; however, its etiology remains unclear. Mast cells (MCs) infiltrate the liver and can become activated leading to degranulation and release of mediators, like histamine (HA), which result in increased intrahepatic bile duct mass, biliary senescence, hepatic inflammation, and hepatic stellate cell activation. Similarly, MCs infiltrate the intestine and increase inflammation which alters host-microbiome communication. MCs are necessary for successful liver regeneration and the combat of intestinal pathogens; however, chronic HA signaling exacerbates damage in cholangiopathies and IBD. Bile acid synthesis is tightly regulated by Farnesoid X Receptor (FXR) and its downstream mediator, fibroblast growth factor 15 (FGF15, -19 in humans). Cholangiocytes (i) are the target of cholangiopathies, (ii) modify and recycle bile acids through Apical Sodium Bile Acid Transporter (ASBT)-mediated cholehepatic shunting, which functions outside of enterohepatic circulation of bile acids and (iii) are capable of autocrine HA signaling. The complex relationship between hepatic and intestinal MC infiltration and bile acid signaling has not been established; therefore, identifying MC regulation of bile acid pool and FXR/FGF15 signaling pathway will provide insight into therapeutic treatment of PSC-IBD. Under the rationale that (i) cholestatic liver diseases are positively correlated with auto-immune comorbidities like IBD, (ii) during disease, MCs infiltrate the liver and intestine and release signaling factors like HA, and (iii) MCs express FXR and secrete FGF15/19; we propose the central hypothesis that MC activation regulates bile acid signaling and PSC progression through paracrine crosstalk with cholangiocytes in the liver and intestinal inflammation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19580号 / 医博第4087号 / 新制||医||1013(附属図書館) / 32616 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 木村 剛, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bile acids are physiologically important metabolites, which are synthesized in liver as the end products of cholesterol metabolism and then secreted into intestine. They are amphiphilic molecules which play a critical role in the digestion and absorption of fats and fat-soluble vitamins through emulsification. The concentration of bile acids is an indicator for liver function. Individual suffering from liver diseases has a sharp increase in bile acid concentrations. Hence, the concentration level of bile acids has long been used as a biomarker for the early diagnosis of intestinal and liver diseases. Conventional methods of bile acid detection such as chromatography-mass spectrometry and enzymatic reactions are complex and expensive. It is highly desired to have a simple, fast, and low-cost detection of bile acids that is available for self-testing or point-of-care testing. To achieve this goal, we develop a liquid crystal-based biosensor for the detection of bile acids. The sensor platform is based on the anchoring transition of liquid crystals (LCs) at the sodium dodecyl sulfate (SDS)-laden LC/aqueous interface for the detection of bile acids in aqueous solution. The first part of this dissertation focuses on the detection mechanism of bile acids. Our studies show that the displacement of SDS from the LC/aqueous interface by the competitive adsorption of bile acids induces a homeotropic-to-planar anchoring transition of the LC at the interface, providing an optical signature for the simple and rapid detection of bile acids. The adsorption of bile acids on the interface was found to follow Langmuir-Freundlich isotherm. The adsorption kinetics of different bile acids is compared. We find that both the number and position of hydroxyl groups of bile acids affect their adsorption kinetics. The different optical patterns of LC films formed by the adsorption of bile acids are also discussed. The second part of this dissertation studies the effect of solution conditions, surfactants, and liquid crystals on the detection limit of the LC-based biosensor for bile acids. Low pH and high ionic strength in the aqueous solution can reduce the electrostatic interaction between SDS and bile acids, which leads to a decreased detection limit. Surfactants with smaller headgroup and lower packing density also help to reduce the detection limit. To further reduce the detection limit, we investigate the effect of LC structures and find that LCs with a shorter chain length give lower detection limits. Also, by substituting a phenyl ring with a cyclohexane ring, we find that the detection limit is further reduced due to the decrease of the interaction between the phenyl rings of LCs. By mixing different LCs together, the detection limit can be linearly tuned from 160 μM to 1.5 μM, which is comparable to the traditional methods. But the LC-based biosensors have much simpler design and manufacture process. The third part of this dissertation is to apply this LC-based biosensor to the detection of urinary bile acids. We test the influence of several potential interfering species such as urea, creatinine, uric acid and ascorbic acid by conducting experiments in synthetic urine. By adjusting the concentration of SDS, we are able to eliminate the impact of those interfering species, and demonstrate that the LC-based biosensors can selectively detect urinary bile acids in human urine, suggesting its potential for screening liver dysfunctions. The final part of this dissertation is to investigate the application of LC-based biosensors in detecting the lipolysis process by porcine pancreatic lipase (PPL). It has been a long-standing argument over the role of bile salts on the activity of PPL. Thus, we study the time course of the hydrolysis of phospholipid L-dipalmitoylphosphatidylcholine (L-DPPC) by PPL at LC/aqueous interface. The hydrolysis of L-DPPC leads to a homeotropic-to-tilted anchoring transition of the LC at the interface, which allows the hydrolysis process to be monitored by a polarizing optical microscope. The microscopy image analysis reveals a lag-burst kinetics where a lag phase is followed by a burst phase. The effect of bile acids on these two phases is studied. We find that the activity of PPL both in the presence and absence of colipase can be improved by increasing the concentration of bile acids. The improvement becomes more distinct in the presence of colipase.

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No description available.

The gene encoding cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt - 149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139- AGTTCAaggccGGGTAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter- transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.

Chapter 1: Functional and Responsive Supramolecular Gels In this chapter ‘supramolecular gels’ derived from small organic molecules with molecular mass of typically less than 2000 daltons are discussed. Representative examples of various low molecular weight gelators based on their natural availability and also divergent functionalities are mentioned (Scheme 1). Scheme 1 Advances in the recent years have been very rapid in the field of supramolecular chemistry of gels giving rise to ‘Tunable responsive gels’. Control of the gel property in a reversible fashion has been the highlight of responsive gels. A few of the gels which are responsive towards various stimuli such as pH, photoirradiation, cations, anions, neutral species have been discussed. Advances and scope of supramolecular gels in various applications have also been mentioned in detail with respective examples. Utilities of supramolecular gels in synthesis of nanostructures, in biology and medicine, enzyme recognition, catalysis etc are discussed. (Scheme 2). Chapter 2: Charge transfer triggered organogels of bis(bile acid)anthracene conjugates and 2,4.7-trinitrofluorenone. In this chapter the study involves the synthesis of a special class of anthracene based steroidal derivatives. The appending of two amphiphilic bile acid units imparts a unique hydrophobic/ hydrophilic balance on the chromophore. The 2,3-didecyloxyanthracene (DDOA) was reported to be a gelator of various organic solvents but none of the three bile acid derivatives of anthracene synthesized was a gelator on its own. It was also observed that dialkoxy (propyl, heptyl, decyl) derivatives of anthracene formed strong charge-transfer gels in the presence of 2,4,7-trinitrofluorenone (TNF). The addition of electron deficient TNF to the steroidal derivatives of anthracene resulted in the gelation of some specific organic solvents. The driving force behind the gel formation resulted from the charge-transfer (CT) interaction between the electron rich anthracene and electron deficient fluorenone. Figure 1. Chemical structures of 2,3-bis(bile acid)anthracenes and TNF (centre), a scanning electronic microscopy image of xerogels prepared from bis(deoxycholyl)anthracene and TNF (left) and a photograph of the gel of bis(deoxycholyl)anthracene and TNF in n-octanol. Thermochromic property (during sol to gel phase transition), absorption and variable temperature fluorescence measurements supported CT interaction. Thermal stability studies and dynamic rheology experiments confirmed that CT gels were thermally most stable and mechanically stronger with equi-molar amounts of the two components. Stiffness values obtained from rheological experiments also suggested that the gels were viscoelastic solids. Chapter 3(A): Tb(III) sensitization in an organogel matrix: Selective luminescence quenching by an aromatic nitro derivative In this chapter the discovery of metallo organogel formation by mixing methanolic solutions of Tb(OAc)3 and sodium deoxycholate (NaDCh) has been explored. Sensitization of Tb3+ was observed by doping micromolar quantities of 2,3-dihydroxynaphthalene (DHN). Mechanical properties of Tb3+-DCh gels were investigated by rheology at three different ratios of Tb3+ and DCh. It was observed that increasing in the Tb3+ to DCh ratio increased the mechanical property of the gels. Time delayed emission spectra were recorded with increasing concentration of DHN and luminescence increase was noticed in a linear fashion. Importance of gel matrix was demonstrated by measuring the Tb3+ luminescence at fixed concentration (5 mM) with/without DHN in the solution and gel media. Figure 2:: Schematic representation of Tb3+ sensitization by DHN. Photograph (right)) of the Tb3++-DCh (5/15 mM) gels with (a) 50 µM DHHN (b) No DHHN under UVV (365 nm). Sensitization by an electron rich chromophore created interest in us to dope relatively electron deficient compounds into the gel matrix for possible quenching off Tb3+-luminescence. Among the electron deficient analytes screened included 1,5-difluro-2,4-diinitrobenzenne (DFDNB)), 2,4 dinitrophenol (DNPPh), p-nitrobenzaldehydde (p-NB), 2,4,6-trinitrootoluene (TTNT) and 22,4,7¬trinitrofluuorenone (TTNF). Microscopy studies such as AFM, TEMM and SEMM revealed highly entangled fibrous network in the morphology of Tb3+--DCh xerogel. Solid state luminescence experiments suggested that sensiitization was observed in the xerogels and extent of sensitization was comparable to that of the gel state. Xerogel soaking studies inferred the strong adherence of the DHNN to the gel fibres. Chapter 3(B): Anion dependent structural, morphological and mechanical features of Ln(III)-Cholate gels In this chapter the counter anion influence on various aspects of hydrogels has been discussed. It has been reported from our laboratory that mixing of aqueous solutions of sodium cholate (15 mM) and various lanthanide acetates (5 mM) followed by sonication resulted in either transparent or transluscent gels. Unsurprisingly we found that aqueous solutions of lanthanide nitrates and lanthanide chlorides also formed hydrogels upon mixing with sodium cholate (Figure 33). Dried films of Tb3++-cholate and Eu3+-cholate gels prepared from their respective nitrate salts displayed birefringent structures under polarizing optical microscopy (POM). But no significant textures of any type were observed in the case of gels prepared from either chloride or acetate salts. Figure 3:: Photographs of the hydrogels prepared by mixing of aqueous solutions of various salts Tb33+ and Eu3+ with sodium cholate solutions. Scanning electron microscopic images exhibited fibrous structures for all the xerogels in the morphology. Atomic force microscopy and transmission electron microscopy measurements revealed helical morphology for xerogels prepared from nitrate salts where as flat tape-like cross linkage was observed for chloride or acetate based xerogels. Anion effect on mechanical properties was significant in the sense that gels prepared from acetate salts displayed highest mechanical strength followed by nitrate based gels which were stronger than that of chloride based gels. Titration of sodium cholate solution with various lanthanide salt solutions gave the direct evidence of thee pH variation as a function of the anions. Figure 4: TEM images of xerogels prepared from gels of nitrate salts of Tb3+ (left) and Eu3+ (right) Chapter 4: Design, synthesis a nd study of bile acid ‘click’ conjugates of perylene bisimides (PBIs) and naphthalene bisimides (NBIs) In this chapter the synthesis of novel bile acid derivatives of perylene and naphthalene bisimides is discussed. The ‘click’ chemistry procedure was used to link bile acid groups on to the chromophores. Azide derivatives of PBIs and NBIs were prepared inn 3 step methods which were coupled to propargyl esters of bile acids by following standard ‘click chemistry’ protocols to achieve the target molecules (Scheme 3). Scheme 3 The studies conducted mainly focused on Cholic acid (CA) conjugates of PBIs and NBIs. Steady state absorption and emission studies of CA conjugates were performed in 10% MeOH/DCM system. POM and fluorescence images showed red emissive aggregates in case of PBI films. TEM measurements revealed uniform aggregate sizes for both the films of PBI(CA)2 and NBI(CA)2. SEM and AFM (Fig 5) studies exhibited spherical aggregates of diameter around 100-200 nm for PBI(CA)2 films where as aggregates of diameter around 500-700 nm were observed for NBI(CA)2 films. Figure 5: AFM images and their corresponding height profiles of PBI films (left) and NBI films (right)