Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...

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Chloroplasts of sugar beet leaves were subjected to treatment by heating, by incubation at pH 8.5 and pH 5.5, by incubation in ion low water, by sonic oscillation, by digestion with enzymes (lysozyme, pancreatin, and lipase), by extraction with digitonin, and by chemical compounds including 8-hydroxy-quinoline, sodium azide, zinc sulfate, phenyl mercuric acetate, potassium cyanide, 1-allylthiourea, 2,4-dinitrophenol, thymol, and p-chloromercuribenzoate. A comparative study was made of the effects of these treatments upon three photochemical reactions (the photoreduction of 2,6- dichlorophenol indophenol, the photoreduction of indigo carmine in the presence of a couple composed of 2,6-dichlorophenol indophenol and ascorbic acid, and the photo-oxidation of the 2,6-dichlorophenol indophenol-ascorbic acid couple) and upon three enzymes (cytochrome oxidase, peroxidase, and catalase) present in the chloroplast preparations used.

Methanosarcina are methanogens capable of growth and methanogenesis from H₂/CO₂, formate, methanol, methylamines, and acetate. Methanosarcina conserve energy by coupling electron transport and methyl transfer to the generation of ion gradients during acetoclastic growth. This work focuses on cytochrome b and heterodisulfide reductase, two proteins involved in energy conservation by electron transport. A procedure was developed for mass cultivation of Methanosarcina thermophila strain TM-1 in 12-liter fermentations which produced up to 10 grams wet weight/liter, in order to facilitate biochemical studies. Cytochromes occurring in Methanosarcina thermophila were characterized spectrophotometrically using chemical and physiological reactants. This analysis revealed two heme centers, one of which was only reduced by Na₂S₂O₄ or carbon monoxide. Partially purified cytochromes were found to be present in a complex and were characterized by electrophoretic and spectrophotometric analysis. The cytochrome-containing protein was found to contain two hemes and had an M_r of 28,000 Da. Heterodisulfide reductase was isolated from the soluble fraction by anion exchange chromatography and assayed using methyl viologen as an artificial electron donor. Electron transport from CO to the heterodisulfide of 2-mercaptoethanesulfonic acid (HS-CoM) and 7- mercaptoheptanoylthreonine phosphate (HS-HTP) was reconstituted using carbon monoxide dehydrogenase, ferredoxin, membranes, and heterodisulfide reductase. Both membranes and ferredoxin were required for reduction of the heterodisulfide. / Master of Science

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

In recent years, the inhibiition of tyrosine kinases,which may incorrectly regulate some singaling pathway has been used to treat cancer as so-called biological therapy. An example of such inhibitors are vandetanib and lenvatinib. These two substances are used to treat thyroid gland tumors because they affect vascular growth factor receptor or endothelial growth factor receptor that can regulate tumor growth and metastasis. Ellipticine, which has anti-tumor effects on lots of tumor disease, has been investigated in this study together with vandetanib and lenvatinib. In this diploma thesis, the effect of mentioned tyrosine kinase inhibitors, ellipticine and their combinations on gene and protein expression of CYP1A1, 1A2, 3A1 and 3A2 in rat liver in vivo was determined. Protein expression was studied using Western blot method with imunodetection. Gene expression was assessed by quantitative PCR. Moreover, the effect of tested substances and their combinations on CYP1A activity (measured as 7-ethoxyresorufin O-deethylation), CYP1A2 activity (measured as 7-methoxyresorufin O-demethylation), CYP1A1 activity (measured as Sudan I oxidation), CYP3A specific activity (measured as testosteron 6β-hydroxylation) and ellipticine, vandetanib, lenvatinib metabolism was determined. It has been confirmed that...

Le tissu adipeux est un organe important pour la régulation de l'homéostasie énergétique de l'organisme. Constitué majoritairement de lipides, ce tissu représente de plus un lieu de stockage pour de nombreux composés environnementaux lipophiles. Les conséquences de cette accumulation, et plus généralement de l'exposition du tissu adipeux à des polluants environnementaux ont été encore peu étudiées. Pourtant, en modulant les fonctions physiologiques ou le développement de ce tissu, ces polluants pourraient jouer un rôle dans le développement de pathologies telles que l'obésité ou le diabète. Le but de cette thèse était d'améliorer notre compréhension des conséquences de l'exposition du tissu adipeux humain à des xénobiotiques environnementaux. Dans un premier temps, nous avons regardé les capacités de métabolisation des xénobiotiques in situ dans le tissu adipeux humain en caractérisant l'expression des 23 isoformes de cytochromes P450 impliquées dans le métabolisme des xénobiotiques. Nous avons montré que seuls les CYP1B1 et CYP2U1 étaient exprimés dans le tissu adipeux humain et que la voie d'induction AhR était fonctionnelle, alors que les voies d'induction CAR et PXR ne semblaient pas l'être. Dans un deuxième temps, nous avons effectué une étude non ciblée par transcriptomique et métabonomique par 1H-RMN des perturbations induites par le traitement de cultures primaires de préadipocytes humains différenciés in vitro avec 2 polluants en particulier : la 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) et le mono-2-ethyl hexyl-phthalate (MEHP). L'intégration de ces données a permis la génération d'un nombre important d'hypothèses quant aux mécanismes de toxicité induits par ces composés. Le MEHP en particulier semble posséder un effet pro-différenciateur sur les préadipocytes humains et induire la glycéronéogenèse dans les adipocyte matures, deux mécanismes par lesquels ce polluant pourrait avoir un effet pro-obésifiant chez l'Homme.

<p><!-- /* Font Definitions */ @font-face {font-family:Garamond; panose-1:2 2 4 4 3 3 1 1 8 3; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:647 0 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --></p><p>Several bacterial species are capable to use perchlorate and/or chlorate as an alternative electron acceptor in absence of oxygen. Microbial respiration of oxochlorates is important for biotreatment of effluent from industries where oxochlorates are produced or handled. One of these species, the Gram-negative <em>Ideonella dechloratans</em>, is able to reduce chlorate but not perchlorate. Two soluble enzymes, chlorate reductase and chlorite dismutase, participate in the conversion of chlorate into chloride and molecular oxygen. The present study deals with the electron transport from the membrane-bound components to the periplasmic chlorate reductase. Soluble <em>c</em> cytochromes were investigated for their ability to serve as electron donors to chlorate reductase. The results show that a 6 kDa <em>c </em>cytochrome serves as electron donor for chlorate reductase. This cytochrome also serves as electron donor for a terminal oxidase in the reduction of oxygen that is produced in the course of chlorate respiration. A gene encoding a soluble <em>c</em> cytochrome was found in close proximity to the gene cluster for chlorate reduction. This gene was cloned and expressed heterologously, and the resulting protein was investigated as a candidate electron donor for chlorate reductase. Electron transfer from this protein could not be demonstrated, suggesting that the gene product does not serve as immediate electron donor for chlorate reductase.</p><p> </p>

Several bacterial species are capable to use perchlorate and/or chlorate as an alternative electron acceptor in absence of oxygen. Microbial respiration of oxochlorates is important for biotreatment of effluent from industries where oxochlorates are produced or handled. One of these species, the Gram-negative Ideonella dechloratans, is able to reduce chlorate but not perchlorate. Two soluble enzymes, chlorate reductase and chlorite dismutase, participate in the conversion of chlorate into chloride and molecular oxygen. The present study deals with the electron transport from the membrane-bound components to the periplasmic chlorate reductase. Soluble c cytochromes were investigated for their ability to serve as electron donors to chlorate reductase. The results show that a 6 kDa c cytochrome serves as electron donor for chlorate reductase. This cytochrome also serves as electron donor for a terminal oxidase in the reduction of oxygen that is produced in the course of chlorate respiration. A gene encoding a soluble c cytochrome was found in close proximity to the gene cluster for chlorate reduction. This gene was cloned and expressed heterologously, and the resulting protein was investigated as a candidate electron donor for chlorate reductase. Electron transfer from this protein could not be demonstrated, suggesting that the gene product does not serve as immediate electron donor for chlorate reductase.