Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anna Giannitsi Supervisor: RNDr. Jakub Hofman, Ph.D Title of diploma thesis: The role of biotransformation enzymes in the resistance of cancer cells against standard cytostatics Drug resistance is currently one of the major problems of chemotherapy. Tumor cells are able to defend themselves against the effect of cytostatic drugs due to various mechanisms which leads to a failure of anticancer therapy. The effort to describe new mechanisms of resistance and to develop new therapeutic methods, which would limit this therapeutic obstacle, is logically the subject of many studies. The activity of drug metabolizing enzymes and the subsequent decrease of intercellular concentration of anticancer drugs belongs to one of the possible mechanisms of pharmacokinetic resistance. Enzymes of I. and II. phase of biotransformation participate in this phenomenon. Cytochromes P450, main enzymes of the I. phase, play a major role in the metabolism of many cytostatic agents producing either pharmacologically active or inactive metabolites. Increased expression in tumors and the involvement of individual isoforms into the overall metabolism of cytostatic, which is deactivated by their activity, seems to be one of the...

A hidroquinona (HQ) é um composto fenólico obtido a partir da metabolização endógena do benzeno, está presente no cigarro, medicamentos, reveladores fotográficos, alimentos e plantas medicinais. Temos demonstrado que a intoxicação experimental de ratos à HQ compromete a migração de leucócitos para o pulmão na vigência de resposta inflamatória alérgica ou inespecífica. O presente trabalho visou avaliar os efeitos da HQ sobre atividades da célula endotelial e de neutrófilos envolvidas na inflamação. Culturas primárias de células endoteliais da rede microcirculatória obtidas do músculo cremaster de ratos Wistar, machos, foram tratadas com HQ (10 ou 100 µM, 2 horas) e posteriormente incubadas ou não com LPS de E. coli (LPS; 2 µg/mL). Neutrófilos obtidos da cavidade peritoneal de ratos Wistar, 4 horas após a injeção local de glicogênio de ostra (10 mL, 1%) , foram tratados com HQ (5 ou 10 µM, 1 hora) e, em seguida, foram incubados ou não com LPS (5 µg/mL). Células controles receberam volumes equivalentes dos veículos da HQ e do LPS. Os dados obtidos mostram que o tratamento com a HQ não induziu necrose ou apoptose em ambos os tipos celulares; reduziu a produção de NO pela célula endotelial e por neutrófilos, por bloqueio das atividades das óxido nítrico sintases; reduziu a expressão gênica e protéica de TNF-α, IL-6 e IL-1β induzida pelo LPS em neutrófilos, possivelmente decorrente de redução da translocação nuclear do NFκB; por outro lado aumentou a expressão gênica e protéica basal de TNF-α, IL-1β, ICAM-1, PECAM-1 e VCAM-1, bem como a translocação nuclear do NF-κB; reduziu a atividade fagocítica e microbicida de neutrófilos frente a Candida albicans; não afetou a expressão gênica da CYP2E1 em ambos os tipos celulares, mas aumentou a expressão gênica de MPO em neutrófilos. Em conjunto, os dados permitem concluir que a HQ atua diferentemente nos dois tipos de células estudadas, ativando e inibindo propriedades inflamatórias na célula endotelial e nos neutrófilos, respectivamente. É possível as ações sobre os neutrófilos possam contribuir, pelo menos em parte, pela redução da migração celular durante a resposta inflamatória observada após exposição in vivo à HQ. / Hydroquinone (HQ) is a fenolic compound obtained after benzene metabolism, it is a component of cigarette, medicines, photographic developer, and it is also finding in some foods and medicinal herbs. Our research group has been shown that rats in vivo exposed to HQ present impaired leukocyte migration into lung during allergic or non-specific inflammation. In the present study, we investigate the effects of HQ on functional activities of neutrophils and endothelial cells (EC) involved in inflammation. Primary cultured EC was obtained from microcirculatory network of male Wistar rats, and treated with HQ (10 or 100 µM, two hours). After the treatments, EC was incubated in presence or absence of lipopolissacharide of E. coli (LPS; 2 µg/mL). Peritoneal neutrophils obtained four hours after local injection of oyster glycogen (10 mL, 1%) were incubated with HQ (5 or 10 µM, one hour) and in sequence it was incubated in presence or absence of LPS (5 µg/mL) .Control cells were cultured with equivalent volumes of HQ and LPS vehicle. Results obtained showed that treatment with HQ did not induce apoptosis or necrosis in both types of cells; impaired NO production by endothelial cells and neutrophils dependent on blockade of Ca+2-dependent and independent NOS activity; decreased gene and protein expression of TNF-α, IL-6 and IL-1β in neutrophils induced by LPS, possibly due to reduced nuclear translocation of the NF-κB. On the other hand, HQ treatment enhanced basal protein and gene expression of TNF-α, IL-1β , ICAM-1, PECAM-1 and VCAM-1 and the nuclear translocation of NF-κB; impaired Candida albicans phagocytic and killing indexes; did not affect the gene expression of CYP2E1 in both types of cell, but increased the gene expression of MPO in neutrophils. Taken together, results obtained show that HQ acts differently in the two types of cells studied, activating and inhibiting inflammatory properties in endothelial cells and neutrophils, respectively. Actions on neutrophils may contribute, at least in part, on the reduced leukocyte recruitment during in vivo HQ exposure.

A hidroquinona (HQ) é um composto fenólico obtido a partir da metabolização endógena do benzeno, está presente no cigarro, medicamentos, reveladores fotográficos, alimentos e plantas medicinais. Temos demonstrado que a intoxicação experimental de ratos à HQ compromete a migração de leucócitos para o pulmão na vigência de resposta inflamatória alérgica ou inespecífica. O presente trabalho visou avaliar os efeitos da HQ sobre atividades da célula endotelial e de neutrófilos envolvidas na inflamação. Culturas primárias de células endoteliais da rede microcirculatória obtidas do músculo cremaster de ratos Wistar, machos, foram tratadas com HQ (10 ou 100 µM, 2 horas) e posteriormente incubadas ou não com LPS de E. coli (LPS; 2 µg/mL). Neutrófilos obtidos da cavidade peritoneal de ratos Wistar, 4 horas após a injeção local de glicogênio de ostra (10 mL, 1%) , foram tratados com HQ (5 ou 10 µM, 1 hora) e, em seguida, foram incubados ou não com LPS (5 µg/mL). Células controles receberam volumes equivalentes dos veículos da HQ e do LPS. Os dados obtidos mostram que o tratamento com a HQ não induziu necrose ou apoptose em ambos os tipos celulares; reduziu a produção de NO pela célula endotelial e por neutrófilos, por bloqueio das atividades das óxido nítrico sintases; reduziu a expressão gênica e protéica de TNF-α, IL-6 e IL-1β induzida pelo LPS em neutrófilos, possivelmente decorrente de redução da translocação nuclear do NFκB; por outro lado aumentou a expressão gênica e protéica basal de TNF-α, IL-1β, ICAM-1, PECAM-1 e VCAM-1, bem como a translocação nuclear do NF-κB; reduziu a atividade fagocítica e microbicida de neutrófilos frente a Candida albicans; não afetou a expressão gênica da CYP2E1 em ambos os tipos celulares, mas aumentou a expressão gênica de MPO em neutrófilos. Em conjunto, os dados permitem concluir que a HQ atua diferentemente nos dois tipos de células estudadas, ativando e inibindo propriedades inflamatórias na célula endotelial e nos neutrófilos, respectivamente. É possível as ações sobre os neutrófilos possam contribuir, pelo menos em parte, pela redução da migração celular durante a resposta inflamatória observada após exposição in vivo à HQ. / Hydroquinone (HQ) is a fenolic compound obtained after benzene metabolism, it is a component of cigarette, medicines, photographic developer, and it is also finding in some foods and medicinal herbs. Our research group has been shown that rats in vivo exposed to HQ present impaired leukocyte migration into lung during allergic or non-specific inflammation. In the present study, we investigate the effects of HQ on functional activities of neutrophils and endothelial cells (EC) involved in inflammation. Primary cultured EC was obtained from microcirculatory network of male Wistar rats, and treated with HQ (10 or 100 µM, two hours). After the treatments, EC was incubated in presence or absence of lipopolissacharide of E. coli (LPS; 2 µg/mL). Peritoneal neutrophils obtained four hours after local injection of oyster glycogen (10 mL, 1%) were incubated with HQ (5 or 10 µM, one hour) and in sequence it was incubated in presence or absence of LPS (5 µg/mL) .Control cells were cultured with equivalent volumes of HQ and LPS vehicle. Results obtained showed that treatment with HQ did not induce apoptosis or necrosis in both types of cells; impaired NO production by endothelial cells and neutrophils dependent on blockade of Ca+2-dependent and independent NOS activity; decreased gene and protein expression of TNF-α, IL-6 and IL-1β in neutrophils induced by LPS, possibly due to reduced nuclear translocation of the NF-κB. On the other hand, HQ treatment enhanced basal protein and gene expression of TNF-α, IL-1β , ICAM-1, PECAM-1 and VCAM-1 and the nuclear translocation of NF-κB; impaired Candida albicans phagocytic and killing indexes; did not affect the gene expression of CYP2E1 in both types of cell, but increased the gene expression of MPO in neutrophils. Taken together, results obtained show that HQ acts differently in the two types of cells studied, activating and inhibiting inflammatory properties in endothelial cells and neutrophils, respectively. Actions on neutrophils may contribute, at least in part, on the reduced leukocyte recruitment during in vivo HQ exposure.

IN ENGLISH LANGUAGE Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmacology and Toxicology Candidate: Mgr. Alžbeta Štefela Supervisor: Prof. PharmDr. Petr Pávek, PhD. Title of the doctoral thesis: Novel bile acid derivatives as promising therapeutic approach Bile acids (BAs) are amphipathic steroidal molecules that are traditionally known to facilitate intestinal digestion and absorption of lipids and fat-soluble substances. On top, the recent findings have revealed that they represent important signaling agents involved in the orchestration of lipid, glucose and energy metabolism and immune response. BAs exhibit these roles by activating intracellular nuclear receptors such as farnesoid X (FXR), pregnane X (PXR) vitamin D receptors. Furthermore, BAs act as endocrine signaling molecules and activate numerous biological cascades via a membrane G-protein-coupled receptor, termed TGR5. Therefore, the extensive modulation of BA scaffold underwent to identify compounds with specific targeting of above-mentioned receptors as a promising therapeutic approach for the treatment of various liver and metabolic disorders including cholestasis, biliary cirrhosis, nonalcoholic steatohepatitis or diabetes. The principal aim of this doctoral thesis was to investigate the structure...

De nombreux insectes s’avèrent être des ravageurs de culture, pouvant de surcroît transmettre des pathogènes aux plantes et ainsi causer des dégâts d’importance économique notables. Leur contrôle repose essentiellement sur l’utilisation d’insecticides, mais cela pose problème en termes de pollution de l’environnement, d’effets non désirés sur les espèces non-cibles, d’apparition de populations d’insectes résistants aux insecticides, sans parler de leur toxicité pour l’homme. L’olfaction joue un rôle clef dans le développement de nombreux comportements chez les insectes, comme la recherche de la plante nourricière ou du partenaire sexuel, le repérage de sites de ponte ou de prédateurs. Leurs organes olfactifs portent les sensilles sensorielles dans lesquelles se déroulent les étapes de la réception du signal : les molécules odorantes pénètrent par les pores et sont transportées à travers la lymphe sensillaire par des "odorant binding proteins" jusqu’aux récepteurs olfactifs (Ors) avec lesquels elles interagissent pour déclencher la cascade de transduction, puis le signal olfactif est inactivé. Si les interactions odeurs/ORs sont largement étudiées et élucidées, il n’en est pas de même pour le transport et l’inactivation du signal. En particulier, les "odorant-degrading enzymes" (ODEs) qui seraient impliquées dans cette dernière étape, en dégradant les molécules odorantes en métabolites inactifs, i.e. ne pouvant plus stimuler les récepteurs. Les ODEs appartiennent à diverses familles d’enzymes de biotransformation, comme les cytochromes P450s (CYPs), les carboxylestérases (CCEs), les glutathion-S-transférases (GSTs), ou les UDP-glucosyltransférases (UGTs). La plupart sont exprimées fortement dans les antennes. Peu d’entre-elles ont été caractérisées fonctionnellement, principalement par des approches in vitro, peu d’études ont été réalisées in vivo avec des approches électrophysiologiques ou comportementales. Au cours de ma thèse, nous avons caractérisé deux CCEs antennaires, l’Estérase6 (Est6) et la « Juvenile Hormone Esterase duplication » (JHEdup), chez Drosophila melanogaster, en combinant des approches transcriptomiques et fonctionnelles. Nous avons montré que ces deux estérases étaient exprimées très fortement dans les antennes et capables de métaboliser in vitro certaines odeurs alimentaires émises par les fruits mûrs. Nous avons aussi démontré que ces deux estérases étaient impliquées in vivo dans les réponses physiologiques et comportementales à ces odorants, et qu’elles s’avèrent donc être des ODEs.. Pour deux ODEs candidates, Ugt35b (UGT) et Cyp308a1 (CYPs), nous avons établi leur patron d’expression dans les antennes afin de préciser les types de sensilles impliquées, en préliminaire à des études fonctionnelles plus ciblées. Enfin, pour ugt35b, cyp308a1 et jhedup, nous avons mis en évidence une expression dans différentes structures gustatives, posant la question de leur rôle possible dans le métabolisme de molécules sapides. Les quatre enzymes antennaires présentées ici ne sont que le début d’une longue liste d’ODEs candidates identifiées lors de l’analyse du transcriptome antennaire de D. melanogaster. Ce travail participe à une meilleure compréhension des mécanismes moléculaires impliqués dans le fonctionnement du système olfactif. Du point de vue appliqué, les ODEs pourraient constituer des cibles d’intérêt (via des inhibiteurs spécifiques par exemple) pour modifier des comportements olfacto-induits, et ce dans un contexte de contrôle de populations d’insectes ravageurs plus respectueux de l’environnement. Par ailleurs, les connaissances acquises sur les ODEs chez cette espèce modèle pourraient contribuer à leur caractérisation chez d’autres espèces, en particulier des ravageurs de cultures. / Insects can be hazardous crop pests that do not only feed on crops but also transmit plant pathogens, causing yearly a great economical damage. Pest control relies mainly on insecticides but an extensive use bears problems such as the pollution of environment, unpredictable effects on non-target species, an increase of insecticide resistant populations and toxicity for humans. Olfaction is fundamental for the implementation of many insect behaviours like host plant and mating partner foraging, identification of suitable oviposition sites and predator avoidance. Insects smell with hairshaped olfactory sensilla, which are located on their antennae and palps. These sensilla are the showplace of early olfactory processing involving several steps: the odor uptake through the sensillar pores and their transport through the sensillar lymph mediated by odorant binding proteins, the detection of odors by olfactory receptors (ORs) and eventually the inactivation of the olfactory signal. Odor/OR interactions have been intensely studied, contrary to odor transport and inactivation that remain not well understood. Odorant-degrading enzymes (ODEs) have been suggested to be responsible for odor inactivation by degrading odorants into inactive metabolites which no longer activate ORs. The ODEs identified to date belong to various biotransformation enzyme families, including cytochrome P450s (CYPs), carboxylesterases (CCEs), glutathione-S-transferases (GSTs), UDP-glucosyltransferases (UGTs). Most of them are highly expressed in insect antennae. To date, only some ODEs have been functionally characterized, most of them in vitro using recombinant protein and showing their ability to efficiently metabolize various odorant molecules. Only a very few studies were investigating ODE function in vivo using electrophysiological and behavioural approaches. In this thesis we functionally characterize two antennal CCEs, Esterase6 (Est6) and Juvenile Hormone Esterase duplication (JHEdup), in the insect model Drosophila melanogaster combining transcriptomic, in vitro and in vivo approaches. We found that both CCEs are highly expressed in antennae and are able to efficiently metabolize certain odors emitted by rotting fruits in vitro. Furthermore, we showed that both are involved in physiological and behavioural responses to these odors. Therefore we propose Est6 and JHEdup as sensillar candidate ODEs. Moreover, we investigated the precise antennal gene expression pattern in toto for two antennal biotransformation enzymes belonging to other classes, Ugt35b (UGT) and Cyp308a1 (CYPs), which will be helpful for further investigations in order to clarify their potential role in olfaction. In case of three ODE candidate genes (ugt35b, cyp308a1 and jhedup) we discovered an interesting expression pattern in various gustatory organs posing new questions about additional functions of these antennal enzymes in taste processing. The antennal enzymes discussed in this thesis are only four of many candidate ODEs that we identified in the antennal transcriptome of the fruit fly. These candidates comprise also enzymes that belong to other classes such as GSTs, aldehyde oxidases, alcohol dehydrogenases or lipases. This work contributes to a deeper understanding of the insect olfactory system including its molecular actors. From an applied point of view ODEs are interesting targets to modify odorant-driven insect behaviours. The identification of specific ODE inhibitors that could interfere with insect ability to respond to environmental olfactory cues, emitted by mating partners or host plants, would contribute to a broader variety of “eco-friendly” olfactory-based insect pest control strategies. In the future the obtained knowledge in the insect model Drosophila will contribute to the characterization of ODEs in different hazardous insect pests which will be the next step to develop new inhibitor-based strategies.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Jan Dušek Supervisor Prof. PharmDr. Petr Pávek, Ph.D. Title of Doctoral Thesis Development of novel Constitutive androstane receptor (CAR) ligands Constitutive androstane receptor is nowadays known as the established nuclear receptor that regulates the expression of several key cytochrome P450 enzymes, predominantly CYP3A4 and CYP2B6. Recently it has been shown that CAR has also essential role in the regulation of endogenous metabolism of glucose, lipids, cholesterole or bile acids. Simultaneously, this receptor is considered to have proliferative effect on human hepatocytes and protective effects against toxic and dietary damage of liver parenchyme. Given the possible therapeutic utilization of CAR, its therapeutic options are being intensively studied. Unfortunately, currently known ligands of human or mouse CAR are either poorly selective or indirect. The aim of my doctoral thesis was to find new ligands of mouse and human CAR, that would enable more detailed study of the receptor.