Thèse doctorat : Biologie Moléculaire et Cellulaire : Strasbourg 1 : 2006. / Titre provenant de l'écran-titre. Bibliogr. 15 p.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 126-139).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 133-146).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

My study investigated the evolution of oxidative metabolism in fishes. While intense selection for, or against, non-synonymous point mutations in coding sequence drives the evolution of mitochondrial OXPHOS genes, genome-specific mechanisms such as gene duplication events can play major roles in the evolution of nuclear OXPHOS genes. My thesis focused on the mitochondrial enzyme cytochrome c oxidase (COX), principally in fish because of their evolutionary origins and functional diversity in terms of energy metabolism. In the first part of my thesis, I examined a highly aerobic group of fishes (billfishes and tunas) to study the evolution of mitochondrial COX genes. Though the study began as a structure-function analysis of COX, my approach changed when my preliminary results called into question the accepted phylogenetic relationships of my species of interest. We generated a robust multigene phylogeny of this group to interpret data in a phylogenetically informative context. Phylogenetic analyses in this group provided us with a framework to study the evolution of mitochondrial OXPHOS genes, but unexpectedly revealed that: 1) billfishes are only distantly related to tunas, and share greater evolutionary affinities with flatfishes (Pleuronectiformes) and jacks (Carangidae), and 2) regional endothermy has evolved in a non-scombroid suborder in teleosts. These results collectively imply that regional endothermy has evolved independently at least twice within teleost fish. The second part of my thesis explored the evolution of the nuclear COX subunits, focusing on their origins in fish. Isoform transcription profiles coupled with phylogenetic analyses for each subunit show that vertebrate isoforms arose from a combination of early whole-genome duplications in basal vertebrates or specific lineages (e.g. teleosts), and more recent single gene duplication events. While there is evidence for retained function of some COX orthologues across fishes and mammals, others appear to have diverged in function since their earlier radiation, possibly contributing novel evolutionary functions. Together these two studies provide insight into the evolutionary forces facilitating adaptive change in mitochondrial and nuclear OXPHOS genes. / Thesis (Master, Biology) -- Queen's University, 2009-09-11 11:00:12.562

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyGln) tract in the human androgen receptor (hAR). One mechanism by which polyGln-expanded proteins are believed to cause neuronotoxicity is through aberrant interaction(s) with, and possible sequestration of, critical cellular protein(s). / Our goal was to confirm and further characterize the interaction between hAR and cytochrome c oxidase subunit Vb (COXVb), a nuclear-encoded mitochondrial protein. We had previously isolated COXVb as an AR-interacting protein in a yeast two-hybrid search to identify candidates that interact with normal and polyGln-expanded AR. Using the mammalian two-hybrid system, we confirm that COXVb interacts with normal and mutant AR and demonstrate that the COXVb-normal AR interaction is stimulated by heat shock protein 70 (Hsp70). Also, BFP-tagged AR specifically co-localizes with cytoplasmic aggregates formed by GFP-labelled polyGln-expanded AR in androgen-treated cells. / Mitochondrial dysfunction may precede neuropathological findings in polyGln-expanded disorders and may thus represent an early event in neuronotoxicity. Interaction of COXVb and hAR, with subsequent sequestration of COXVb, may provide a mechanism for putative mitochondrial dysfunction in SBMA.