Le pregnane X receptor (PXR) est un récepteur nucléaire associé à la réponse au stresscellulaire. Des travaux in vitro de notre groupe ont démontré que les activateurs de cerécepteur (spironolactone (SPIR), clotrimazole (CTZ)) inhibent significativement l’apoptosespontanée ou induite, dans des primo-cultures d’hépatocytes de rat ou d’origine humaine.Les données in vitro sont en faveur d’un rôle clé du PXR dans la protection hépatique contreles xénobiotiques et endobiotiques en régulant de façon concertée leur détoxication(transport, métabolisme) et en augmentant leur résistance à l’apoptose. D’autres travauxrécents ont démontré le rôle majeur de ce récepteur dans la régulation de l’homéostasielipidique et glucidique, d’une part en favorisant la lipogenèse, d’autre part inhibant la lipolyseet la néoglucogenèse. L’induction du PXR peut être responsable de l’accumulation de lipidesdans le foie (NAFLD non alcoholic fatty liver disease). La NAFLD est fortement associée àl’obésité. La chirurgie bariatrique est à ce jour le seul traitement efficace à long terme pourl’obésité morbide. L’évolution des lésions hépatiques de la NAFLD après chirurgiebariatrique n’est pas complètement élucidée. L’objectif de ce travail de thèse, a été d’analyser si, in vivo, les agonistes du PXR tels que le CTZ et la SPIR, présentaient chez l’animal les mêmes effets que ceux décrits in vitro, et s’ilspouvaient conduire à une protection similaire du foie, lors d’atteintes pathologiques commeles lésions hépatiques induites par l’ischémie reperfusion. Dans un autre chapitre les effetsde deux procédures bariatriques, le court circuit gastrique (gastric bypass- GBP) et lagastrectomie en gouttière (sleeve gastrectomy- SG), sur le comorbiditées liées à la obésitéont été analysés. Le modèle d’ischémie reperfusion normothermique partielle et totale du foie chez le rongeur(rat et souris) a été utilisé pour étudier le rôle protecteur du PXR contre les lésionsapoptotiques. Les effets du court circuit gastrique sur les comorbiditées associées à l’obésitéont été étudiés dans deux groupes de patients obèses (index de masse corporelle > 50Kg/m2 et < 50 Kg/m2). Les résultats du GBP et de la SG ont été étudiés dans deux groupescomparables de sujets super obèses (IMC > 50 Kg/m2).Résultats 1/ Le traitement par les activateurs du PXR (CTZ et SPIR), chez le rat et chezla souris, provoque l’induction d’expression du CYP 3A1, enzyme sous le contrôle du PXR. Ilest associé à une réduction significative du nombre d’hépatocytes apoptotiques, du niveaudes transaminases, de la caspases activée et de son substrat PARP (poly-ADP-ribosepolymérase).Les mécanismes impliqués comprennent l’induction d’expression de la protéineantiapoptotique Bcl-xL, l’activation de la voie MAP kinase ERK ½, l’inhibition de l’activationde JNK et la sous-expression des heat schock proteins 27, 70, et 90, dans le cas del’ischémie complète du foie. 2/ Un an après l’intervention bariatique, le GBP a montré uneefficacité comparable sur la réversibilité du syndrome métabolique, de l’inflammationsystémique et l’insulino résistance chez les femmes super-obèses et obèses morbides,même si l’IMC moyen des patientes super obèses est resté significativement plus élevé. LeGBP et la SG conduisent à des résultats comparables, 6 mois après la chirurgie, mais lepremier est significativement plus efficace en termes d’amélioration du profil lipidique,d’inflammation à bas grade et de syndrome métabolique.L’activation du PXR est associée à un effet de protection hépatique contre les lésionsd’ischémie reperfusion. Le GBP est efficace sur les comorbiditées chez le super obèseautant que chez l’obèse morbide. ll donne également de meilleurs résultats à un an, sur laperte de poids, l’inflammation systémique et la régression du syndrome métabolique. / The pregnane X receptor (PXR) is a nuclear receptor associated with cellularresponse to xeno and endobiotics. Recently, the involvement of PXR in controlling otherfunctions has been clarified. We previously showed in our laboratory that the PXR activators(spironolactone (SPIR) and clotrimazole (CTZ) protect primoculture of human or rathépatocytes against apoptosis. In vitro data are indicate that the PXR plays a major role inthe protection of the liver against xeno and endobiotics trough the régulation of theirélimination (détoxication) and increasing their resistance against apoptosis. It has also beenshown that the PXR is implicated in the controls of lipid and carbohydrates metabolism. Theinduction of PXR increases lipogenesis, inhibits lipolysis and neoglucogenesis. PXRinduction may be responsible for the accumulations of lipids into the liver (NAFLD, nonalcoholic liver disease). This disease is strongly associated with obesity. To date bariatricsurgery is only effective treatment in the long term for morbid obesity. The evolution ofNAFLD following bariatric surgery has not been fully clarified.Aims The aims of this thesis were to ascertain whether PXR agonists such as CTZ andSPIR resulted in the same effects in the animal model than those observed in vitro andwhether the administration of these drugs was associated with any protection againstnormothermic ischemia reperfusion injury of the liver. In another chapter the effects onobesity related comorbidities of two bariatric procedures such as the gastric bypass and thesleeve gastrectomy were investigated. The animal model used to investigate the role of PXR against apoptosis wasthe partial and total normothermic ischemia reperfusion of the liver in the rodent (rat andmouse). The effects of the GBP and SG on obesity related comorbidities wereinvestigated in two groups of obese women (BMI > 50 Kg/m2 and < 50kg/m2). The results ofGBP and SG in two comparable groups of super obese patients (BMI > 50 Kg/m2) wereinvestigated. Treatment with PXR activators such as (CTZ and SPIR) in the rat and in themouse was associated with the induction of CYP 3A1, an enzyme directly controlled by thePXR, with a significant reduction of the apoptotic hepatocytes, with a significantly lowerlevels of transaminases, activated caspase 3 and its substrate PARP (poly-ADP-ribosepolymérase).The involved mécanismes include the induction of the expression of theantiapoptotic protein Bcl-xL, the activation of the extracellular regulated kinase (ERK ½), theinhibition of JNK and the down régulation of heat shock proteins (hsp 27, 70, and 90) geneexpression in the case of total liver ischemia. One year after surgery GBP showedcomparable results in terms of résolution of metabolic syndrome, systemic inflammation andinsuline resistance in morbdly obese as well as super obese women even if the mean BMI ofsuper obese women remained higher (34.7 Kg/m2 vs 28.1 Kg/m2). GBP and SG areassociated with the same results six months after surgery but GBP is more effective inimproving lipid disturbances, low-grade systemic inflammation and the metabolic syndromeat one year. PXR induction results in a protective effect against normothermic inschemiareperfusion injury in the rodent. Results of GBP in terms of resolution of obesity relatedcomorbidities are comparable in the super obese and morbidly patients. GBP is moreeffective than SG one year after surgery in terms of weight loss, and resolution of lipiddisturbances, low-grade systemic inflammation and metabolic syndrome.

Le cancer colorectal est marqué par une importante mortalité dans les stades avancés du fait du fort taux de récidives tumorales après chimiothérapie. La prédiction de l'efficacité et de la toxicité des cytotoxiques s'impose comme un des enjeux majeurs de ces prochaines années. Parce que la majorité des anticancéreux sont pris en charge par les enzymes et transporteurs dont l'expression est contrôlée par le niveau d'expression et d'activation des xénosenseurs CAR et PXR, il est fort probable que ces xénosenseurs puissent représenter des facteurs prédictifs à prendre en compte dans la prise en charge des cancers. Notre équipe a récemment montré que les récepteurs des xénobiotiques PXR (NR1I2) (Raynal et al, 2010) et CAR (NR1I3) sont exprimés dans des lignées cellulaires et des tissus coliques humains. Leur surexpression dans les lignées coliques LS174T et T84 entraine leur résistance à l'irinotécan et à son métabolite actif le SN38 alors que leur inhibition antagonise cette résistance. Des dosages intra- et extra-cellulaires du SN38 et du SN38-G, ainsi que la quantification des ARNm des l'UGT1As et du transporteur MDR1, montrent que CAR et PXR augmentent le métabolisme détoxifiant et l'efflux du SN38. L'impact de la surexpression de ces xénosenseurs sur la viabilité des cellules LS174T à différentes classes de cytotoxiques (anti-métabolites, intercalants, inhibiteurs de topoisomérases, poisons du fuseau) a ensuite été évaluée. Nous avons observé que l'expression de CAR ou PXR conduit à une forte chimiorésistance au paclitaxel, au docétaxel et au 4-hydroxy-cyclophosphamide alors que PXR entraîne une sensibilisation marquée au cisplatine et au carboplatine en augmentant la quantité d'adduits de platine sur l'ADN. Les études du transcriptome de nos modèles cellulaires nous ont permis d'identifier les gènes cibles impliqués dans ces variations de cytotoxicité. Des études de confirmation par modulation pharmacologique ou ARNs interférents de ces gènes cibles sont en cours et nous permettront de préciser les mécanismes mis en jeu dans les variations de chimiosensibilité. Ces travaux devraient permettent de mieux appréhender le rôle des xénosenseurs CAR et PXR sur le métabolisme intra-tumoral des cytotoxiques et potentiellement sur la réponse à des chimiothérapies variées. / Colorectal cancer is characterized by high mortality in advanced stages due to the high rate of tumor recurrence after chemotherapy. The prediction of the efficacy and toxicity of cytotoxic drugs represents a major challenge in the coming years. Because the majority of cancer drugs are supported by the enzymes and transporters whose expression is controlled by the level of expression and activation of the xenosensors CAR (NR1I3) and PXR (NR1I2), it is likely that they may represent predictive factors in the management of cancer. Our team has recently shown that xenobiotic receptors PXR (Raynal et al, 2010) and CAR are expressed in cell lines and human colon tissues. Their overexpression in colon cancer cell lines LS174T and T84 leads to resistance to irinotecan and to its active metabolite SN38, while their inhibition reverse this resistance. Irinotecan metabolites detection assays of SN38 and SN38G, and the quantification of UGT1As and MDR1 mRNA, show that CAR and PXR increase the detoxifying metabolism and the efflux of SN38. The impact of overexpression of these xenosensors on LS174T cell viability to different classes of cytotoxic agents (anti-metabolites, DNA intercalators, topoisomerase inhibitors, antimitotic agents) was then evaluated. We observed that the expression of CAR or PXR results in a significant drug resistance to paclitaxel, docetaxel and 4-hydroxy-cyclophosphamide whereas PXR leads to a marked sensitization to cisplatin and carboplatin by increasing the amount of platinum adducts the DNA. Microarray studies of our cell models allowed us to identify the target genes potentially involved in these changes in cytotoxicity. Further studies by pharmacological modulation or interfering RNAs of these target genes are in progress and will allow us to clarify the mechanisms involved in the changes in chemosensitivity. This work should help us to understand the impact of CAR and PXR xenosensors on the intratumoral metabolism of cytotoxic drugs and potentially on the response to various chemotherapies.

Ovarian steroid hormones are known to produce several different effects in the brain. In addition to their role in gonadotropin release, ovulation and sexual behaviour they also seem to affect mood and emotions, as shown in women with the premenstrual tension syndrome. Some steroids have the ability to affect brain excitability. Estradiol decreases the electroshock threshold while progesterone acts as an anti-convulsant and anaesthetic in both animals and humans. Several earlier studies have shown a specific uptake of several steroids in the animal brain but only a few recent studies have established the presence of steroids in the human brain. In the present studies, the dissections of rat and human brains were carried out macroscopically and areas that are considered to be related to steroid effects were chosen. Steroid concentrations were measured by radioimmunoassay after extraction and separation with celite chromatography. The accuracy and specificity of these methods were estimated. In the animal studies, immature female rats were treated with Pregnant Mare's Serum Gonadotropin (PMSG) to induce simultaneous ovulations. Concentrations of estradiol and progesterone were measured in seven brain areas pre- and postovulatory. The highest concentration of estradiol, pre- and postovulatory, was found in the hypothalamus and differences between the two cycle phases were detected in most brain areas. The preovulatory concentrations of progesterone were low and the highest postovulatory concentration was found in the cerebral cortex. In one study, the rats were injected with pharmacological doses of progesterone to induce "anaesthesia". High uptake of progesterone was found and a regional variation in the formation of 5&lt;*-pregnane-3,20-dione in the brain with the highest ratio in the medulla oblongata. Concentrations of progesterone, 5a-pregnane-3*20-dione, estradiol and testosterone were determined in 17 brain areas of fertile compared to postmenopausal women. All steroids displayed regional differences in brain concentrations. Higher concentrations of estradiol and progesterone were found in the fertile compared to the postmenopausal women. In summary, these studies show that the concentrations of ovarian steroids in the brain are different at different endocrine states in both rats and humans and that there are regional differences in brain steroid distribution. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1987, härtill 5 uppsastser</p> / digitalisering@umu

The ligand activated transcription factor retinoid X receptor (RXR) forms a DNA binding heterodimer with pregnane X rseceptor (PXR) in response to foreign xenobiotics. In addition to RXR and PXR there are other proteins involved in the RXR/PXR signaling pathway. Many proteins involved in this pathway are still unknown. This study documents the production of RXR and PXR in a bacterial recombinant fusion system. These proteins were expressed in a system that allowed purification with six histidine residues. Once the proteins were expressed and purified from E. coli, they were solublized and tested for function. Different strategies were employed including temperature and inducer studies and denaturing and renaturing techniques to solublize PXR. Following the solubilzation of each protein, all proteins were subjected to a method of functional analysis. RXR function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with PXR. These studies involving RXR and PXR demonstrate that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system. In addition, this study documents various strategies for combating "inclusion body" formation in the overexpression ofPXR. Also, it describes the production of plasmid pCMV-RXR for transfection into the HepG2 cell line to monitor the levels of cellular RXR in various tissue types.

No description available.

Le fipronil, insecticide largement utilisé, est un perturbateur thyroïdien chez le rat modulant le catabolisme hépatique des hormones thyroïdiennes. Ses effets chez le mouton, considéré comme un modèle plus pertinent que le rat pour étudier une régulation de la fonction thyroïdienne chez l'Homme, sont plus limités. Le but de cette thèse était de caractériser au niveau hépatique le mode d'action du fipronil sur la fonction thyroïdienne en s'intéressant 1) au rôle potentiel du principal métabolite du fipronil formé in vivo, le fipronil sulfone, et 2) aux différences interspécifiques de métabolisme du fipronil et/ou de sensibilité à la perturbation thyroïdienne qui peuvent préjuger de la pertinence des différents modèles animaux pour l'analyse du risque du fipronil pour la santé humaine. L'efficacité du fipronil sulfone à induire l'expression et/ou l'activité d'enzymes responsables du métabolisme hépatique des hormones thyroïdiennes ou du fipronil était la même que celle du fipronil autant in vivo chez le rat que in vitro sur hépatocytes. L'utilisation d'un modèle de souris déficientes pour des récepteurs nucléaires xénosenseurs suggérait fortement une implication des récepteurs nucléaires Constitutive Androstane Receptor et/ou Pregnane X Receptor dans la perturbation thyroïdienne induite par le fipronil / The widely used insecticide fipronil is a thyroid disruptor in rat acting on thyroid hormone hepatic metabolism. In sheep, a more relevant species for the human thyroid regulation, fipronil-induced thyroid-disruption is much more limited. The goal of this thesis was to characterize the mode of action of fipronil on thyroid function at the hepatic level focusing on 1) the potential role of fipronil sulfone, the main fipronil metabolite formed in vivo, and on 2) interspecific differences in terms of fipronil metabolism and/or sensitivity to thyroid disruption that can prejudge of the relevance of the different animal models for the risk assessment of fipronil for human health. Fipronil sulfone was as efficient as fipronil to induce the expression and/or activity of enzymes involved in thyroid hormone or fipronil hepatic metabolism both in vivo in rat and in vitro on hepatocytes. The use of knock-out mice for xenosensors nuclear receptors strongly suggested an implication of the nuclear receptor Constitutive Androstane Receptor and/or Pregnane X Receptor on fipronil-induced thyroid disruption

Receptores nucleares controlam a transcrição em células eucarióticas quando ativados por ligantes e, além do sítio de interação com ligantes, há outros sítios alternativos em sua superfície que podem ser alvo de compostos capazes de interferir com as interações proteína-proteína desativando o RN. A ativação do Receptor X de Pregnano (RXP) e do Receptor Constitutivo de Androstano (RCA) resulta na indução do metabolismo e efluxo de fármacos. Portanto, RXP/RCA sao responsáveis por causar reações adversas ou falhar terapias. Uma abordagem combinando a triagem experimental à nível cellular, em uma biblioteca de fármacos, e validação com ensaios in vitro e in silico, conseguimos identificar três novos antagonistas de RXP e cinco novos contra RCA, cada um com um perfil único de interação. / Nuclear receptors can control transcription in eukaryotic cells in a ligand-dependent manner and, besides the ligand-binding pocket there is evidence of the existence of alternative ligand-binding sites on the surface, which can be addressed by small organic molecules that disrupt specific protein-protein interactions and thereby may antagonise NR function. Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) results in the induction of first-pass metabolism and drug efflux. Hereby PXR/CAR may cause adverse drug reactions or therapeutic failure of drugs. Therefore, PXR and/or CAR antagonists can minimise adverse effects or improve therapeutic efficiencies. Combination of cellular high-throughput screen identified CAR and PXR potent antagonists in a library of approved and investigational drugs. Further validated by cellular and in vitro assays, as well as molecular docking, suggesting additional or exclusive binding outside the classical ligand binding pocket. In conclusion, we here have identified three approved drugs as novel potent PXR antagonists and five potential CAR inverse agonists with differential receptor interaction profiles.

For more than 80 years vitamin E has been in the focus of scientific research. Most of the progress concerning non-antioxidant functions, nevertheless, has only arisen from publications during the last decade.<br> Most recently, the metabolic pathway of vitamin E has been almost completely elucidated. Vitamin E is metabolized by truncation of its side chain. The initial step of an omega-hydroxylation is carried out by cytochromes P450 (CYPs). This was evidenced by the inhibition of the metabolism of alpha-tocopherol by ketoconozole, an inhibitor of CYP3A expression, whereas rifampicin, an inducer of CYP3A expression increased the metabolism of alpha-tocopherol. Although the degradation pathway is identical for all tocopherols and tocotrienols, there is a marked difference in the amount of the release of metabolites from the individual vitamin E forms in cell culture as well as in experimental animals and in humans. Recent findings not only proposed an CYP3A4-mediated degradation of vitamin E but also suggested an induction of the metabolizing enzymes by vitamin E itself.<br> In order to investigate how vitamin E is able to influence the expression of metabolizing enzymes like CYP3A4, a pregnane X receptor (PXR)-based reporter gene assay was chosen. PXR is a nuclear receptor which regulates the transcription of genes, e.g., CYP3A4, by binding to specific DNA response elements. And indeed, as shown here, vitamin E is able to influence the expression of CYP3A via PXR in an in vitro reporter gene assay. Tocotrienols showed the highest activity followed by delta- and alpha-tocopherol. An up-regulation of Cyp3a11 mRNA, the murine homolog of the human CYP3A4, could also be confirmed in an animal experiment. The PXR-mediated change in gene expression displayed the first evidence of a direct transcriptional activity of vitamin E. PXR regulates the expression of genes involved in xenobiotic detoxification, including oxidation, conjugation, and transport. CYP3A, e.g., is involved in the oxidative metabolism of numerous currently used drugs. This opens a discussion of possible side effects of vitamin E, but the extent to which supranutritional doses of vitamin E modulate these pathways in humans has yet to be determined. <br><br> Additionally, as there is arising evidence that vitamin E's essentiality is more likely to be based on gene regulation than on antioxidant functions, it appeared necessary to further investigate the ability of vitamin E to influence gene expression. Mice were divided in three groups with diets (i) deficient in alpha-tocopherol, (ii) adequate in alpha-tocopherol supply and (iii) with a supranutritional dosage of alpha-tocopherol. After three months, half of each group was supplemented via a gastric tube with a supranutritional dosage of gamma-tocotrienol per day for 7 days. Livers were analyzed for vitamin E content and liver RNA was prepared for hybridization using cDNA array and oligonucleotide array technology. A significant change in gene expression was observed by alpha-tocopherol but not by gamma-tocotrienol and only using the oligonucleotide array but not using the cDNA array. The latter effect is most probably due to the limited number of genes represented on a cDNA array, the lacking gamma-tocotrienol effect is obviously caused by a rapid degradation, which might prevent bioefficacy of gamma-tocotrienol.<br> Alpha-tocopherol changed the expression of various genes. The most striking observation was an up-regulation of genes, which code for proteins involved in synaptic transmitter release and calcium signal transduction. Synapsin, synaptotagmin, synaptophysin, synaptobrevin, RAB3A, complexin 1, Snap25, ionotropic glutamate receptors (alpha 2 and zeta 1) were shown to be up-regulated in the supranutritional group compared to the deficient group. The up-regulation of synaptic genes shown in this work are not only supported by the strong concentration of genes which all are involved in the process of vesicular transport of neurotransmitters, but were also confirmed by a recent publication. However, a confirmation by real time PCR in neuronal tissue like brain is now required to explain the effect of vitamin E on neurological functionality. The change in expression of genes coding for synaptic proteins by vitamin E is of principal interest thus far, since the only human disease directly originating from an inadequate vitamin E status is ataxia with isolated vitamin E deficiency. Therefore, with the results of this work, an explanation for the observed neurological symptoms associated with vitamin E deficiency can be presented for the first time. / Chemisch handelt es sich bei Vitamin E um acht lipophile Derivate des 6 Chromanols mit einer Seitenkette. Nach dem Sättigungsgrad der Seitenkette lassen sich die Derivate in die Tocopherole (gesättigte Seitenkette) und die Tocotrienole (ungesättigte Seitenkette mit drei Doppelbindungen) einteilen. Entsprechend der Methylierung des Chromanrings lassen sie sich in alpha-, beta-, gamma- und delta-Tocopherol, bzw. Tocotrienol unterscheiden. Davon besitzt alpha-Tocopherol, das gleichzeitig die im Plasma dominierende Form darstellt, die höchste biologische Aktivität. Aufnahme wie auch der Transport von Vitamin E im Körper sind vergleichsweise gut erforscht. Die Kenntnisse zu Metabolismus und Elimination waren jedoch bis vor kurzem sehr lückenhaft. Lange Zeit waren nur Vitamin E-Metabolite mit geöffnetem Chromanring, die sogenannten Simon-Metabolite Tocopheronsäure und Tocopheronolacton bekannt. Diese Metabolite können nur aus oxidativ gespaltenem Vitamin E entstehen und galten daher auch als Beweis für die antioxidative Wirkung von Vitamin E. Mit verbesserter Analytik wurde vor einigen Jahren gezeigt, dass die Simon-Metabolite größtenteils Isolierungsartefakte sind. Stattdessen wurden Metabolite mit intaktem Chromanring identifiziert. Tocopherole wie auch Tocotrienole werden im Körper durch eine Verkürzung der Seitenkette abgebaut. Die Endprodukte sind in jedem Fall CEHCs (Carboxyethyl Hydroxychromane). Die Seitenkettenverkürzung startet mit einer omega-Hydroxylierung gefolgt von 5 Schritten &#61472;beta-Oxidation. Die omega Hydroxylierung der Seitenkette durch Cytochrom P450 (CYP) Enzyme wurde indirekt bestätigt. CYP3A4 gilt dabei als eines der wahrscheinlichsten Enzyme im Abbau von Vitamin E, die Beteiligung weiterer CYPs wird jedoch gleichfalls angenommen. Auffällig ist, dass nicht alle Vitamin E-Formen in gleichem Ausmaß abgebaut werden. Die Ausscheidung von CEHCs aus alpha-Tocopherol ist, verglichen zu andern Vitamin E-Formen, in kultivierten Zellen wie auch in vivo sehr gering. Die Art der Seitenkettenverkürzung von Vitamin E spricht für einen Abbau über das Fremdstoff-metabolisierende System, welches auch eine Vielzahl von Medikamenten verstoffwechselt.<br> Im ersten Teil der vorliegenden Arbeit konnte mittels Reportergenassay in HepG2 Zellen gezeigt werden, dass Vitamin E einen nukleären Rezeptor, den Pregnan X Rezeptor (PXR), zu aktivieren und die Expression von PXR-regulierten Genen zu beeinflussen vermag. PXR reguliert eine Reihe von Genen für Fremdstoff-metabolisierende Enzyme wie z.B. Cytochrom P450 3A4 durch Bindung an sein responsives Element im Promotor der Zielgene. Die untersuchten Vitamin E-Formen unterschieden sich deutlich hinsichtlich ihrer PXR-Aktivierung. Die Tocotrienole zeigten die höchste PXR-Aktivierung - vergleichbar mit Rifampicin, einem bekannt guten PXR-Aktivator - gefolgt von delta / alpha- und gamma-Tocopherol. Im Tierversuch an Mäusen konnte die erhöhte Expression von Cyp3a11, dem Homolog des humanen CYP3A4 in Abhängigkeit von der alpha-Tocopherol-Zufuhr bestätigt werden. Somit konnte erstmals gezeigt werden, dass Vitamin E die Expression von Genen direkt beeinflussen kann. Darüber hinaus unterstreicht diese Beobachtung die Möglichkeit einer Wechselwirkung von pharmakologischen Dosen Vitamin E mit dem Abbau von Medikamenten. Eine genregulatorische Funktion von Vitamin E ist auf den ersten Blick überraschend. Denn wenngleich Vitamin E vor über 80 Jahren als Fertilitätsfaktor bei Ratten entdeckt wurde, steht die erst später beschriebene antioxidative Eigenschaft von Vitamin E bis heute im Fokus der meisten Publikationen. Die molekularen Mechanismen der Essentialität von Vitamin E wurden dagegen wenig untersucht. Erst in den letzten Jahren finden Funktionen von Vitamin E Interesse, die über seine antioxidative Wirkung hinausgehen. Dabei konnte gezeigt werden, dass Vitamin E in vitro die Expression von Genen wie dem Scavenger Rezeptor CD36, dem Connective Tissue Growth Factor oder dem Peroxisomen-Proliferator aktivierten Rezeptor gamma beeinflussen kann.<br> Um weitere Zielgene von Vitamin E in vivo identifizieren zu können, wurden im zweiten Teil der vorliegenden Arbeit Mäuse in drei Fütterungsgruppen mit einer a) defizientem b) adäquatem sowie c) mit einer supranutritiven alpha Tocopherol-Versorgung über 3 Monate gefüttert. Zusätzlich erhielt die Hälfte der Tiere aus jeder Gruppe während der letzten Lebenswoche eine supranutritive Dosis gamma-Tocotrienol pro Tag. Aus den Lebern der Tiere wurde die RNA präpariert und die differentielle Genexpression mittels a) cDNA und b) Oligonukleotide enthaltenden GenChips analysiert.<br> Eine signifikante Änderung in der Genexpression zwischen den verschiedenen Fütterungsgruppen fand sich jedoch nur in den Analysen der Oligonukleotid GenChips. Dies kann auf die begrenzte Anzahl von Genen zurückzuführen sein, die auf den cDNA GenChips repräsentiert waren. Auch ein signifikanter Effekt von gamma-Tocotrienol auf die Genexpression konnte nicht beobachtet werden. Wahrscheinlich ist die hohe Ausscheidung von gamma-CEHC, dem Abbauprodukt von gamma-Tocotrienol, die im Urin der Tiere gemessen wurde und die damit womöglich verringerte Bioverfügbarkeit von gamma-Tocotrienol dafür verantwortlich.<br> Mit Hilfe der Oligonukleotid GenChips konnte jedoch ein signifikanter Effekt von alpha-Tocopherol auf die Expression einer Vielzahl von Genen beobachtet werden. Herausstechend war dabei die erhöhte Expression von für den vesikulären Transport essentiellen Genen, die für den synaptischen Signaltransfer benötigt werden. So wurden z.B. Synapsin, Synaptotagmin, Synaptophysin, Synaptobrevin, RAB3A, Complexin 1, Snap25, die ionotrophen Glutamat Rezeptoren alpha 2 und zeta 1 in Abhängigkeit von der alpha Tocopherol-Versorgung über die Diät erhöht exprimiert. Die Beobachtung, dass Vitamin E bei neurologischen Prozessen eine Rolle zu spielen scheint ist jedoch nicht neu. Bei Patienten mit einem Mangel an funktionellem alpha-Tocopherol-Transfer-Protein (alpha-TTP) kann es zu stark verringerten Plasmakonzentrationen an Vitamin E kommen, da alpha-TTP eine zentrale Rolle in der Aufnahme und Verteilung von Vitamin E im Körper einnimmt. An diesen Patienten können charakteristische Vitamin E-Mangelzustände beobachtet, die durch eine Reihe von neurologischen Störungen wie Ataxien, Hyporeflexie sowie eine verringerte propriozeptive und vibratorische Sensitivität gekennzeichnet sind. Mit den vorliegenden Ergebnissen kann nun erstmals eine mechanistische Erklärung für diese Symptome diskutiert werden. Eine Bestätigung der vorliegenden Ergebnisse via RT-PCR und Western Blot, z.B. in neuronalem Gewebe wie dem Gehirn, sowie anschließende funktionellen Untersuchungen ist daher dringend geboten.

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated calcium permeable ion channels that play a key role in excitatory synaptic transmission and plasticity, and their dysfunction underlies several neuropsychiatric disorders. The overactivation of NMDA receptors by tonically increased ambient glutamate can lead to excitotoxicity, associated with various acute and chronic neurological disorders, such as ischemia, Alzheimer and Parkinson's disease, epilepsy or depression. On the opposite, NMDA receptor hypofunction is thought to be implicated in autism, schizophrenia, or intellectual disability. Recent DNA screening for neurological and psychiatric patients revealed numerous mutations in genes encoding for NMDA receptor subunits. The activity of NMDA receptors is influenced by a wide variety of allosteric modulators, including neurosteroids that could both inhibit and potentiate the activity of NMDA receptors, which makes them promising therapeutic targets. In this thesis, we describe new classes of neurosteroid analogues which possess structural modifications at carbons C3 and C17 of the steroidal core, and analogues without D-ring region (perhydrophenanthrenes). We evaluated the structure-activity relationship (SAR) for their modulatory effect on recombinant GluN1/GluN2B receptors. Our results...

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第19649号 / 薬博第819号 / 新制||薬||239(附属図書館) / 32685 / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM