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Ce travail avait pour objectif d'étudier l'intérêt et les limites de l'analyse de deux matrices alternatives que sont la bile et l'humeur vitrée (HV), en toxicologie médicolégale. Pour chacune des deux matrices, une revue de la littérature visait à investiguer les connaissances utiles à leur application en toxicologie médicolégale. Une place importante de ces revues est réservée à l'anatomie et la physiologie de l'HV et du système biliaire ainsi qu'aux mécanismes de distribution des xénobiotiques dans ces matrices. La partie expérimentale décrit trois études: deux menées sur populations autopsiques et une associant expérimentations animales et études de populations autopsiques. Les deux premières ont permis de proposer des outils statistiques d'interprétation des concentrations de méprobamate mesurées dans ces matrices. Ils peuvent être utilisés dans diverses situations à la place ou en complément de l'interprétation des concentrations sanguines: cadavre exsangue, putréfaction avancée du corps, redistribution post mortem des xénobiotiques… La troisième étude concernait six molécules (diazépam, citalopram, cyamémazine, morphine, caféine et méprobamate). Les molécules détectées dans le sang l'étaient systématiquement dans l'HV et la bile aussi bien dans les prélèvements des populations autopsiques que ceux issus des expérimentations animales. Les concentrations vitréennes chez l'animal et chez l'homme étaient systématiquement corrélées aux concentrations sanguines, exceptées celles de cyamémazine et de citalopram chez l'homme. Pour la bile, une corrélation significative était observée pour le méprobamate et la caféine chez l'homme et l'animal. Il ressort de ces résultats, que l'analyse de l'HV et de la bile permettent de disposer d'informations relatives à la nature des molécules absorbées et à leur rôle dans la survenue du décès / The present study sought to assess the interest and limitations of analyzing two alternative matrices, bile and vitreous humor (VH), in forensic toxicology. For each matrix, a literature review established the state of knowledge relating to their forensic application. The review placed special focus on the anatomy and physiology of VH and the biliary system and the mechanisms of xenobiotic distribution within the specific matrix. The experimental sections describe three studies: two performed on autopsy populations, and one associating autopsy populations to an animal model. The first two studies resulted in statistical tools for interpreting meprobamate concentrations in these matrices, which can be used as alternatives or complements to blood concentrations in various situations: exsanguination, advanced putrefaction, postmortem xenobiotic redistribution, etc. The third study focused on 6 molecules: diazepam, citalopram, cyamemazine, morphine, caffeine and meprobamate. Molecules detected in blood were also systematically detected in VH and bile samples from both the autopsy and animal populations. Animal VH and blood levels showed systematic correlation. In autopsy samples, cyamemazine and citalopram showed no such correlation. In bile, significant correlations with blood concentrations were found for meprobamate and caffeine in both the autopsy and animal populations. This study confirmed the interest of postmortem analysis of bile and VH. Results show that analyzing bile and VH sheds light on drugs intake and on their implication in cause of death

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Chapter 1. An Overview of Bile Acid Science This chapter deals with an overview of bile acid science (cholanology) compiling elevant literature review, covering bile acid chemistry, biosynthesis, bile salt evolution, physiology and medicinal values. Figure 1. (a) Digestive system; (b) enterohepatic circulation and (c) cholic acid Bile acids are the end products of cholesterol metabolism, secreted in the liver and stored in the gall bladder (Figure 1). They are normally conjugated with glycine (75%) or taurine (25%). Because of their facially amphiphilic nature, bile salts tend to form micellar aggregates in aqueous solution. They have remarkable ability to transform lamellar array of lipids into mixed micelles. All primary bile acids seem to have three features in common: (1) They are major products of cholesterol metabolism; (ii) they are secreted into the bile largely in a conjugated form and (iii) the conjugates are membrane impermeable, water soluble, amphiphilic molecules. Recent advances in molecular biology have greatly accelerated the knowledge relating to the significance of bile salts in a number of physiological functions. The new role of bile salts as pheromones and ligands for nuclear hormone receptors has been discussed. Chapter 2. Pythocholic Acid: A Major Constituent of Python’s Bile and 16α-Hydroxycholic Acid: A Minor Constituent of Avian’s Bile The first chemical synthesis of pythocholic acid (major constituent of python’s bile) and 16α-Hydroxycholic acid (a minor constituent of avian’s bile) were accomplished starting from cholic acid with overall yields of 5.0% and 5.5%, respectively. A biomimetic remote functionalization strategy was utilized as a key step to achieve the selective chlorination at C-17. Dehydrochlorination of 17-chlorosteroid resulted in the Δ16 olefin. Hydroboration-oxidation of the Δ16 olefin followed by the selective oxidation of the pentol under TEMPO mediated oxidation resulted in an ε-lactone. Hydrolysis of the lactone using 5% KOH in MeOH furnished the 16α-Hydroxycholic acid. On the other hand, selective oxidation of 7-OH of the lactone was achieved using N-bromosuccinimide in acetone/H2O to yield the 7-keto lactone. The ketolactone when subjected to the Huang-Minlon modification of the Wolf-Kishner reduction furnished pythocholic acid. Pythocholic acid showed unusual aggregation behavior and high cholesterol solubilization ability, compared to other trihydroxy bile acids. Chapter 3. 16-Epi-pythocholic acid: An Unnatural Analogue of Pythocholic Acid The synthesis of 16-epi-pythocholic acid, an unnatural analogue of pythocholic acid, was accomplished starting from cholic acid. Cholic acid was converted to Δ8-14) olefin using ZnCl2 in refluxing acetone. Methylation followed by isomerization in CHCl3 by passing dry. HCl at -78 oC resulted in the Δ14 olefin. Allylic oxidation using Na2Cr2O7.2H2O in the presence of N-hydroxysuccinimide in acetone furnished the enone. Selective reduction of the olefin using Pd/C-H2 resulted in 16-Epi-pythocholic acid the 16-keto steroid. NaBH4 reduction of this ketone in MeOH/THF (2:1 v/v) followed by hydrolysis produced the 16-OH bile acid. Analysis of spectral data confirmed that it is a 16β-epimer of pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid). Critical micellar concentration and cholesterol solubilization properties were studied. Chapter 4. Low Molecular Mass Organogelators Derived from Simple Esters of Cholic Acid This chapter begins with an introduction to low molecular mass organogelators and highlights their applications. Serendipitous gelation of a number of organic solvents by allyl cholate and the design of related simple esters of cholic acid are discussed. A series of simple and easily accessible esters of bile acids were prepared. Ethyl cholate and propyl cholate were found to immobilize a variety of organic solvents like benzene, toluene, xylene, mesitylene, 1,2-dichlorbenzene (DCB) and chlorobenzene (Figure 2). The morphology of the xerogels was well characterized using SEM, AFM and polarizing optical microscopy (POM) techniques, Which revealed the presence of highly entangled self-assembled 3D-fibrillar network (SAFINs). The fiber diameter was found to vary between 300-500 nm. Direct imaging of the collapse of this fibrillar network and direct observation of the evolution of nanofibers was achieved for the first time using variable temperature POM techniques. FT-IR studies, X-ray powder diffraction and variable temperature POM studies revealed the resemblance of SAFINs to the bulk solid. Formation of helical fibrillar network was observed in SEM images and the existence of chiral aggregates was confirmed by induced circular dichroism experiment using achiral Reichardt’s dye as the chromophore. Chapter 5. Ambidextrous Gelators Derived from Spacer Linked Bile Acid Derivatives Based on our observation of simple esters of cholic acid as organogelators a rational design of a series of spacer linked dimers and tripodal derivatives were carried out. Some of these molecules formed highly transparent gels in solvents like haloarenes, anisole, xylene and dibromoalkanes. These molecules also showed rapid gelation in DMF/H2O and DMSO/H2O mixtures in varying proportions of water and the co-solvent. These types of gelators are known as ambidextrous gelators. The xerogels were characterized using SEM, TEM and POM techniques and the presence of highly entangled 3D-fibrillar network (Figure 3) was observed. XRPD showed crystalline nature of bulk solid, whereas the xerogels were shown to lose their crystalline nature. (For figures and structural formula pl see the pdf file.)

Author: Nikola Jozefčeková Title: Endoglin and liver Form: Diploma Thesis University: Charles University, Faculty of Pharmacy in Hradec Králové Degree: Pharmacy This diploma thesis concludes the available information about endoglin, its isoforms in the liver and its impact on the liver during various pathological conditions. In the first part of the thesis describes morphology and physiology of the liver, its structure, histology and metabolic functions. Second part contains an information about endoglin, its isoforms and role in TGF-β signaling complex, expression of endoglin and regulation of the expression. In the third part are described liver diseases during which is expression of endoglin changed. This part deals with the significance of endoglin as a diagnostic and prognostic marker. Endoglin (CD105, TGF-β receptor III) is a homodimeric transmembrane glycoprotein and a membrane co-receptor TGF-β with high expression in endothelial cells. Endoglin plays an important role in the vascular development. Endoglin is involved in the processes of angiogenesis, vascular homeostasis and TGF-β signalization. It affects activity of TGF-βRII, ALK1 and ALK5 receptors. Due to the interactions with TGF-β complex, modulation of activity of ALK receptors and Smads, endoglin controls fibrotic and anti-...

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

This thesis describes a study of the aggregational behaviour of egg yolk lecithin (EYL), a natural lecithin, and bile salt mixtures especially with respect to an increase of the ionic strength of the solvent. Mixtures of two amphiphiles with very different spontaneous curvature as EYL lecithin and bile salt form mixed micelles and vesicles in aqueous solution. Their properties have been well-studied under physiological conditions, i.e. 150 mM electrolyte concentration and pH 7- 8, while other conditions are still hardly explored. Upon increasing ionic strength the formed structures and the transitional pathways (micelles, coexistence of micelles and vesicles, and vesicles) change the generated structures completely from those observed under physiological conditions. We quantitatively determined these structures formed in a broad range of electrolyte concentrations with various scattering techniques, x-ray, light and neutron scattering and calorimetry. With calorimetry, phase diagrams in the EYL and bile salt concentration phase plane were determined at various ionic strength ranging from physiological salt concentration to up to 1000 mM. Additionally a new electrochemical approach using functionalised electrodes, i.e. sensitive and selective to bile salt, and thus to control the bile salt concentration in solution (concentrations below the critical micellar concentration (cmc)) was attempted, since bile salt removal or injection drives the micelle-to-vesicle or the vesicle-to-micelle transition, respectively, of the mixed aggregational system of EYL/bile salt. Although this control was not achieved within the framework of this thesis, promising results show directions for future experiments.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The collective microbial genomes within our gut(microbiome) represent a powerful metabolic force, leading many authors to call our GI flora an "organ within an organ", and the metagenomic sequencing of our microbiome, "the second human genome project". Bile acids, endogenously produced by the host liver, represent both a strong selective pressure for potential colonizers, aswell as substrates for microbial metabolism. Indeed, microbes have evolved enzymes to deconjugate bile salts, epimerize bile acid hydroxyl groups, and 7alpha-dehydroxylateprimary bile acids. The products of microbial 7alpha-dehydroxylation, secondary bile acids, are suggested by numerous lines of evidence to be involved in promoting colon carcinogenesis. 7alpha-dehydroxylating activity is a multi-step pathway, genes of which have only been identified in a small number of organisms within the genusClostridium. The biochemistry of this pathway has been largely worked out. The third step in the pathway is introduction of a delta-4-double bond; however, the gene product(s) responsible have not been identified. The baiCD and baiH genes were cloned, expressed and shown to have NAD-dependent 3-oxo-delta-4-steroid oxidoreductase activity showing stereospecificity for 7alpha-hydroxy and 7beta-hydroxy bile acid, respectively.In addition, bai genes were isolated from C.hylemonae TN271 by bidirectional genome-walking by PCR. This represents the first report of bai genes from a "low activity" 7alpha-dehydroxylating bacterium. The gene organization and sequence of the baiBCDEFGHI operon was highly conserved between C. hylemonae TN271 and the "high activity" 7alpha-dehydroxylating bacterium C. scindens VPI12708. The baiA gene was located by PCR using degenerate oligonucleotides. Bi-directional genome-walking revealed what appears to be several novel genes involved in bile acid metabolism which were also located in C. scindens VPI 12708. Expression of a 62 kDa flavoprotein and reactionwith [24-14C] 3-oxo-DCA and NADP resulted in a product of greater hydrophilicity than deoxycholic acid. The identity of this product was not determined. A second gene appears to share a common evolutionary origin with the baiF gene. A hypothesis is offered regarding the function of these homologues as Type III CoA transferasesrecognizing 5alpha-bile acids, or 5beta-bile acids (allo-bile acids). A third gene encodes a putative short chain reductase, similar in size and predicted function to the baiA gene, which may be involved in the final reductive step in the pathway. These novel genes also contained a conserved upstream regulatory region with the baioxidative genes. Finally, two genes were identified which may serve as potential drug targets to inhibit bile acid 7alpha-dehydroxylation. The first is an ABC transporter which may be co-transcribed with the other novel bile acid metabolizing genes, and what appears to be a bile acid sensor/regulator similar to the Tryptophan-rich sensory protein (TspO)/mitochondrial peripheral benzodiazepinereceptor (MBR) family of proteins.