Cholesterol 7-hydroxylase (CYP7A1) is the rate limiting enzyme of the classical pathway of bile acid (BA) synthesis, which catabolizes approximately half of cholesterol in man. Determination of CYP7A enzymatic activity is a key subject of lipid metabolism research. Direct determination of CYP7A1 activity in hepatic biopsy is mostly not allowed for ethical reasons, so indirect methods are used with serum markers such as 7α-hydroxy-4-cholestene-3- one (C4). The first, methodical aim of the work was to convert the introduced HPLC method for the determination of C4 to LC-MS in order to increase the sensitivity. We focused on the solid phase extraction step, adjusting the composition and volumes of the washing and elution solution. By converting the method from HPLC to LC-MS, the sensitivity was increased approximately 7 times (LD = 1.39 ng/ml). In the second, clinical part of our work, we attempted to confirm the preliminary results of our laboratory on the distribution of C4 in lipoprotein fractions (LPP) in order to find parameter that would correlate with CYP7A1 activity better than C4 level itself. Preliminary results (performed in healthy individuals) showed that most of C4 is carried on HDL, and that the C4 distribution within LPP fractions is similar among examined subjects. We repeated the...

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The physiological role of LCAT has been the subject of a number of recent articles (Glomset, 1979; Nilsson-Ehle et al., 1980). According to most current theories, the enzyme functions in combination with high-density lipoproteins in the reverse cholesterol transport pathway which presumably returns peripheral cholesterol to the liver where cholesterol catabolism takes place. Despite the exciting potential for studies on the catalytic function and the nature of the enzyme-substrate complex, the mechanism of action of LCAT remains largely unexplored. The relatively slow progress in the elucidation of the LCAT reaction mechanism is likely to be due to the difficulties in the isolation of the enzyme in sufficient quantities. Consequently, considerably less is known about the physical and chemical properties of the enzyme. Therefore, the first objective of this investigation was to isolate and purify sufficient amount of enzyme for subsequent characterization studies. The second objective of this investigation was to characterize the physical properties of the enzyme by techniques including analytical ultracentrifugation, ultraviolet spectroscopy, and circular dichroism and fluorescence spectroscopy. The third objective of this investigation was to characterize the chemical properties of the enzyme which deals with the amino acid and carbohydrate composition and with some basic structural features that are related to the chemical composition of LCAT.

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Huian plasma high-density lipoprotein (HDL) were isolated by a procedure employing polyanion precipitation and column chromatography. Lipid and protein composition of the HDL isolated by this method was found to be similar to another HDL preparation isolated by ultracentrifugation. However, minor differences were noted, including a higher phospholipid and apoproteinE content and lower triglyceride content of the HDL isolated by column chromatography. Four subfraction of HDL were obtained following chromatography on an anion exchange column. The subfraction four had the highest esterified to free cholesterol ratio, the second highest phospholipid to unesterified cholesterol, and the lowest molecular weight. In addition it was consistently coincided with lecithin: cholesterol acyltransferase (LCAT) activity and found to be the best substrate for the enzyme.

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L’addiction aux drogues est une maladie comportementale récidivante caractérisée par une recherche et une prise compulsive de drogues, une perte de contrôle sur la prise malgré les conséquences négatives et l’émergence d'un état émotionnel négatif lors de l’absence de la drogue. L’addiction implique des neuroadaptations cérébrales persistantes. Des études récentes montrent que le cholestérol joue un rôle crucial dans le fonctionnement cérébral en participant à divers processus cellulaires et, en particulier, au contrôle de la neurotransmission. L’objectif de ce travail de thèse consistait à étudier l’implication potentielle du métabolisme du cholestérol dans l’addiction et en particulier si ce métabolisme était affecté par les drogues d’abus. Dans ce travail, nous avons étudié l’expression de gènes codant des protéines impliquées dans le métabolisme du cholestérol cérébral après une consommation volontaire chronique d’alcool par des rats et après des injections aigues ou chroniques de cocaïne. Nous avons analysé l’expression de ces gènes dans des structures cérébrales impliquées dans l’addiction, notamment le cortex préfrontal, le noyau accumbens, l’amygdale et l’hippocampe. Nous avons trouvé que l’exposition à l’alcool et à la cocaïne modifient l’expression des protéines impliquées dans la synthèse, le transport et la dégradation du cholestérol de façon spécifique de la drogue, du traitement (aigu/chronique) et de la région cérébrale étudiée. Dans une deuxième partie de la thèse, nous avons utilisé une approche virale permettant de surexprimer la CYP46A1, l’enzyme de dégradation du cholestérol cérébral dans le cortex préfrontal afin d’évaluer l’impact de cette surexpression sur la recherche de cocaïne dans un modèle de rechute. La surexpression de la CYP46A1dans cette structure n’a eu aucun impact sur la recherche de cocaïne. Des études futures seront nécessaires pour déterminer si l’altération de l’expression de cette enzyme dans d’autres structures, comme par exemple le noyau accumbens, pourrait avoir des effets bénéfiques sur la rechute. L’ensemble de ces travaux montrent que l’exposition aux drogues d’abus modulerait le métabolisme cérébral du cholestérol dans certaines structures cérébrales. Ce projet de thèse ouvre de nouvelles perspectives quant au rôle du métabolisme du cholestérol cérébral dans l’addiction, et il pourrait en résulter de nouvelles thérapeutiques dans le traitement de cette maladie psychiatrique coûteuse. / Drug addiction is a chronic brain disease characterized by drug-seeking and compulsive drug taking, a loss of control over drug taking despite the negative consequences and the emergence of a negative emotional state in the absence of the drug. Addiction involves persistent neuroadaptations at the cerebral level. Recent evidences show that cholesterol plays a crucial role in brain function by participating in various cellular processes in particular in the control of neurotransmission. The aim of this thesis was to investigate the potential role of cholesterol in addiction and in particular a potential dysregulation of cholesterol metabolism in response to drugs of abuse. In this work, we investigated the expression of genes encoding proteins involved in the metabolism of cerebral cholesterol after a chronic voluntary consumption of alcohol from the rats and after acute or chronic exposure to cocaine. We analyzed gene expression in brain structures involved in addiction such as the prefrontal cortex, the nucleus accumbens, the amygdala and the hippocampus. We found that exposure to alcohol and cocaine modifies the expression of proteins involved in the synthesis, the transport and the degradation of cholesterol in drug-specific, treatment- specific (acute / chronic) and region-specific manners. In the second part of the thesis, we used a viral approach to overexpress CYP46A1, the cerebral cholesterol degradation enzyme in the prefrontal cortex, in order to evaluate the impact of this overexpression on cocaine-seeking in a model of relapse. The overexpression of CYP in this structure has no effect on drug-seeking for cocaine. Future studies are needed to determine whether altering cholesterol metabolism in other structures, for example the nucleus accumbens, may have beneficial effects on relapse. Altogether these studies show that exposure to drugs of abuse might modulate cerebral metabolism of cholesterol. This thesis project opens new perspectives on the role of cholesterol cerebral metabolism in addiction which may ultimately result in new therapeutic avenues for the treatment of this costly psychiatric disorder.