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

Yes / The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation.

Histone deacetylase inhibitor trichostatin A (TSA) increases cytotoxicity of antineoplastic agent ellipticine to human neuroblastoma cells. Its mechanism of action has not yet been explained. One of the possible mode of action is conformational change in chromatin, which leads to changes in DNA that is more accessible to covalent modification and intercalation. The aim of this work is to study another mode of action, which can explain this phenomenon. The question is, if TSA can increase cytotoxicity of ellipticine to human neuroblastoma cells by modulation of activities and expression of cytochromes P450 and peroxidases. These enzymes are responsible for cytotoxicity of ellipticine to human neuroblastoma cells. TSA has no effect on oxidation of ellipticine mediated by cytochromes P450 leading to metabolites responsible for formation of ellipticine-DNA adducts and detoxication metabolites. TSA increases formation of ellipticine dimer, which is a detoxication metabolite, forming during its oxidation by peroxidases. TSA has no effect on activities of CYP1A1, CYP1A2, CYP3A, which significantly participate in oxidation of ellipticine. TSA modulates expression of enzymes oxidizing ellipticin in human neuroblastoma cells. TSA in the presence of ellipticine increases expression of CYP1A1 a CYP3A4 in...

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and inhibition of topoisomerase II. Ellipticine was also found to form covalent DNA adducts mediated by its enzymatic activation with cytochromes P450 (CYP) and peroxidases. The next study demonstrated increasing formation of these ellipticine-DNA adducts by histone deacetylase inhibitor valproate (VPA) in neuroblastoma cells. This phenomenon correlates with increasing cytotoxicity of ellipticine induced by this histone deacetylase inhibitor. This observation can be explained by several mechanisms. One of them can be loosening the structure of chromatine, which leads to accessing DNA for modification. Another one is the effect of VPA on activities and expression of enzymes metabolizing ellipticine. This study was aimed to test the second hypothesis. Since VPA has been shown to be metabolized by similar enzymes as ellipticine is, we have studied the effect of VPA (i) on oxidation of ellipticine by cytochromes P450 and peroxidases, (ii) on activities of the CYP enzymes, which significantly participate in oxidation of ellipticine (CYP1A, CYP3A) and (iii) on expression of enzymes oxidizing ellipticine (CYP1A1, CYP3A4, lactoperoxidase). Oxidation of ellipticine in vitro by model...

Cytochromes P450 are heme enzymes with very broad substrate specificity, they can oxidize tens to hundreds of different substrates including both eobiotics and xenobiotics, but with varying efficiencies and kinetics. They are responsible for the metabolic activation of many carcinogens resulting in formation of reactive intermediates, these reactive species participate in the formation of DNA adducts and also increase of oxidative stress. Eukaryotic cytochromes P450 are membrane bound enzymes found mostly in the endoplasmic reticulum. In vertebrates, they are expressed in a variety of tissues mostly in the liver, but also in kidney, lung, skin and others. The cytochrome P450 1B1 is an inducible enzyme which occurs mainly in the lung and skin. Its expression is induced predominantly by the presence of polycyclic aromatic hydrocarbons, dioxins and heterocyclic amines. The human cytochromes P450 are typically obtained using heterologous expression and isolated as a C-terminally modified hexa-histaq constructs using immobilized metal affinity chromatography. This thesis focuses on effect of C-terminal modifications on activity of human cytochrome P450 1B1. First, the two expression vectors encoding the human form of cytochrome P450 1B1 were prepared, one contained a classical C-terminal hexa-histaq...

Flavonoids are natural compounds commonly ingested in herbs and vegetables. They are believed to have a positive impact on human organism, in particular by their antioxidant, hepatoprotective and anti-cancer effects. In these days, it is possible to consume high concentrations of these compounds in form of dietary supplements. However it is not clear, whether flavonoids in such unnaturally high concentrations are still beneficial or rather harmful. It has already been proven, that flavonoids can influence the activity of biotransformation enzymes and interfere e.g. with the process of carcinogenesis and drug metabolism. For that reason it is important to investigate the impact of an increased intake of flavonoids. The aim of this thesis was to investigate the influence of dihydromyricetin (a potential drug to cure alcohol use disorder) and its structurally similar flavonoid myricetin on the activity of enzymes, cytochrome P450 2E1 (CYP2E1) and N­acetyltransferases 1 and 2 (NAT1/2). The research included the determination of the impact of a premedication by these flavonoids on the expression and activity of CYP2E1 and NAT1/2. The inhibition capacity of myricetin and dihydromyricetin towards the activity of CYP2E1 and NAT1/2 was also investigated. After the flavonoid premedication of rats the...

La géosmine est un terpénoïde, provoquant un goût moisi-terreux associée à des flaveurs atypiques dans l'eau et le vin. Chez les bactéries, la voie de biosynthèse de la géosmine est bien caractérisée, mais peu de connaissance sont disponibles au sujet de sa biosynthèse chez les eucaryotes, en particulier dans les champignons filamenteux. L'origine de la géosmine dans la vigne est en grande partie attribuable à la présence de Penicillium expansum sur les raisins. Dans cette thèse, afin de mieux comprendre la voie de biosynthèse de la géosmine chez Penicillium expansum, nous avons décrit la caractérisation et l'analyse de "gpe1", un gène codant pour une cytochrome P450 monooxygénase impliquée dans la biosynthèse de la géosmine. Nous avons démontré que les deux fragments d'ADN: p450-1 et p450-2 appartiennent à un seul gène du cytochrome p450 (gpe1). La séquence d'acides aminés déduite de gpe1 a une identité moyenne de 40 % avec les enzymes PbP450-2 et P450-4 qui ont été trouvées impliquées respectivement dans la synthèse d'indole diterpène et dans la synthèse des gibbérellines. Les amplifications par PCR effectuée sur quatorze espèces de Penicillium ont montré que seules les espèces producteurices de la géosmine ont donné le même fragment de ~1,2 kb que gpe1. L'analyse du gène gpe1 nous a permis d'identifier la présence de certains domaines conservés de cytochromes P450 monooxygénases. Ensuite, la caractérisation fonctionnelle du gène gpe1 chez P. expansum M2230 a été décrite. Nous avons montré que les mutants de gpe1 ont perdus leur pouvoir de produire la géosmine alors que les révertants de gpe1 ont rétablis leur pouvoir de production. Enfin, nous avons démontré qu'une polykétide synthase putative et une putative NRPS sont présentes sur le côté droit du gène gpe1 proposant que le gène gpe1 pourrait être une partie d'un «Cluster» codant pour la biosynthèse de métabolites secondaires. / Geosmin is a terpenoid, an earthy-musty compound associated with off-flavors in water and wine. In bacteria, the biosynthesis pathway of geosmin is well characterized, but little is known about its biosynthesis in eukaryotes, especially in filamentous fungi. The origin of geosmin in grapevine is largely attributable to the presence of Penicillium expansum on grapes. In this thesis, we have described the characterization and analysis of "gpe1", a gene encoding a cytochrome P450 monooxygenase probably involved in the biosynthesis of geosmin in P. expansum M2230, in order to better understand of the biosynthesis pathway of geosmin in this species. We demonstrated that the two DNA fragments i.e. p450-1 and p450-2 belong to a single cytochrome p450 gene (gpe1). We showed that the deduced amino acid sequence of gpe1 has an average identity of 40 % with PbP450-2 and P450-4 enzymes which have been found involved in indole diterpene synthesis and in gibberellin synthesis respectively. Then, the results of PCRs performed on the fourteen Penicillium species showed that only Penicillium species which were producers of geosmin gave the same fragment of ~1.2 kb like gpe1. Analysis of the gpe1 gene enabled us to identify the presence of some conserved domains of cytochromes P450 monoxygenases in the amino acid sequence of gpe1. Then, the functional characterization of the gpe1 gene in P. expansum M2230 was described. We illustrated that the mutants of gpe1 lost their potential to produce geosmin whereas the reverse complements of gpe1 restored their potential to produce geosmin. Finally, we demonstrated that a putative polyketide synthase and a putative NRPS-like enzyme are present on the right side of the gpe1 gene suggesting that gpe1 gene might be the part of a gene cluster encoding the biosynthesis of secondary metabolites.

Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis