A &#946;-ionona (BI) é um isoprenóide que apresenta atividade quimiopreventiva durante a fase de promoção da hepatocarcinogênese. O presente trabalho teve como objetivo avaliar a expressão de genes modulados pela BI envolvidos na quimioprevenção durante a fase de promoção da hepatocarcinogênese induzida pelo modelo do \"Hepatócito Resistente\" (RH). Ratos Wistar machos foram submetidos ao modelo do RH e tratados durante 4 semanas consecutivas com BI (16 mg/100 g de p.c.) ou óleo de milho (OM) (0,25 ml/100 g de p.c.; grupo controle). O perfil da expressão de 1.176 genes foi analisado por macroarray no fígado dos grupos BI, OM e de ratos considerados normais (grupo N). A expressão gênica foi considerada aumentada, quando a razão de expressão foi &#8805; 1,5 ou diminuída, quando &#8804; 0,5. Aplicou-se análise hierárquica de clustering e classificação ontológica dos genes diferencialmente expressos. A expressão gênica foi validada por RT-PCR do tipo \"duplex\", utilizando-se tecido hepático microdissecado de: lesões pré-neoplásicas persistentes (pLPN) ou em remodelação (rLPN) e de regiões ao redor das LPN (surrounding). Um total de 133 e 32 genes foi considerado diferencialmente expresso entre os grupos OM (em relação ao N) e BI (em relação ao OM), respectivamente. Trinta e sete por cento dos genes diferencialmente expressos no grupo BI vs OM referiam-se a receptores celulares. Destes, 4 genes codificantes para receptores nucleares foram identificados como possíveis alvos da BI na quimioprevenção da hepatocarcinogênese: RXR&#945; (receptor X de retinóide &#945;), RAR&#946; (receptor de ácido retinóico &#946;), COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) e Nur77 (nuclear receptor 77). Em comparação ao grupo OM, a expressão de RXR&#945; e RAR&#946; foi maior (p<0,05) especificamente em pLPN e rLPN do grupo &#946;I, respectivamente. Em comparação ao grupo N, Nur77 apresentou maior (p<0,05) expressão no surrounding e nas rLPN do grupo OM. Por outro lado, a expressão de Nur77 em rLPN foi menor (p<0,05) no grupo BI do que no OM. Comparada ao grupo N, a expressão de COUP-TFI foi maior (p<0,05) no grupo OM, tanto no surrounding das LPN como nas pLPN e rLPN. Em comparação ao grupo OM, a expressão de COUP-TFI foi menor (p<0,05) no grupo BI, especificamente nas pLPN e nas rLPN. Os resultados sugerem que os receptores nucleares RXR&#945;, RAR&#946;, Nur77 e COUP-TFI representam alvos moleculares da BI relevantes para a quimioprevenção da hepatocarcinogênese em ratos. / &#946;-ionone (BI) is an isoprenoid which has chemopreventive activity during the promotion phase of hepatocarcinogenesis. This study aimed to evaluate the expression of genes modulated by BI involved in chemoprevention during the promotion phase of hepatocarcinogenesis induced model of \"Resistant Hepatocyte (RH). Male Wistar rats were submitted to the RH model and treated for 4 consecutive weeks with BI (16 mg/100 g bw) or corn oil (CO) (0.25 ml/100 g bw, control group). The expression profile of 1,176 genes was analyzed by macroarray in the liver of groups BI, CO and normal rats (group N). Gene expression was considered increased when the expression ratio was 1.5 or decreased when 0.5. Hierarchical clustering analysis and ontological classification of differentially expressed genes were applied. Gene expression was validated by RT-PCR \"duplex\", using microdissected hepatic tissue from: persistent pre-neoplastic lesions (pPNL) or remodeling pre-neoplastic lesions (rPNL) and regions around the PNL (surrounding). A total of 133 genes and 32 were considered differentially expressed between the two groups (CO to N) and BI (relative to CO), respectively. 37% of differentially expressed genes in group BI vs CO were related to cell receptors. Of these, four genes encoding for nuclear receptors have been identified as possible targets of BI in the chemoprevention of hepatocarcinogenesis: RXR (retinoid X receptor &#945;), RAR&#946; (retinoic acid receptor &#946;), COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) and Nur77 (nuclear receptor 77). Compared to CO group, the expression of RXR&#945; and RAR&#946; was higher (p <0.05) specifically in pPNL and rPNL of BI group, respectively. Compared to the group N, Nur77 showed higher (p <0.05) expression in the surrounding and rPNL of CO group. The expression of Nur77 in rPNL was lower (p <0.05) in BI than the CO group. Compared to N group, the expression of COUP-TFI was higher (p <0.05) in CO group (surrounding, pPNL and rPNL). Compared to CO group, the expression of COUP-TFI was lower (p <0.05) in BI group, specifically in the pPNL and rPNL. The results suggest that the nuclear receptors RXR&#945;, RAR&#946;, Nur77 and COUP-TFI represent relevant molecular targets of BI in the chemoprevention of hepatocarcinogenesis in rats.

L’acide rétinoïque (AR) est un morphogène dérivé de la vitamine A qui contrôle des processus clés au niveau cellulaire et pendant le développement embryonnaire des chordés. Chez les vertébrés les principaux acteurs de la voie de signalisation de l’AR sont les récepteurs de l’acide rétinoïque (RAR) et les récepteurs des x rétinoïdes (RXR). L’évolution de RAR est encore très mal connue cependant, des données récentes indiquent qu’il est également présent chez des animaux non-chordés. Suggérant ainsi, que l’origine évolutive de RAR et de la voie de signalisation de l’AR est plus ancienne qu’initialement prévu. Durant ma thèse j’ai pu retracer l’histoire évolutive de ce récepteur depuis son origine chez Urbilateria jusqu'à sa diversification, suite aux duplications génomiques chez les vertébrés. En caractérisant les RARs d’un annélide, un mollusque et un oursin j’ai démontré que chez l’annélide et l’oursin RAR est activé par l’AR, contrairement au RAR de mollusque qui n’est pas fonctionnel avec l’AR. De plus, des études menées chez l’annélide ont montré que les gènes régulés par l’AR chez cette espèce sont différents de ceux régulés chez les vertébrés. Ces données permettent donc de s’interroger sur la fonction ancestrale probable que ce récepteur avait chez Urbilateria. Finalement, la caractérisation moléculaire des RAR de cyclostomes nous a permis d’étudier l’impact des duplications génomiques sur l’évolution de la poche de liaison au ligand. Ainsi, les résultats obtenus pendant ma thèse permettent de mieux comprendre les relations entre le récepteur et son ligand ainsi que de découvrir de nouveaux aspects sur la fonction de RAR en présence de ligands non-classiques. / Retinoic acid (RA) is a fat-soluble morphogen derived from vitamin A that controls key cellular and developmental processes in chordates. In vertebrates, the major actors of the RA signaling pathway are the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The evolution of RAR is still poorly understood, however, recently RARs have been identified in the genome of non-chordate species, suggesting that RAR and the RA pathway might have a more ancient evolutionary origin than previously thought. The work presented in this manuscript allowed us to retrace the evolutionary history of RAR from its origin in Urbilateria to its diversification following whole genome duplication events in vertebrates. We describe the characterization of the RARs from an annelid, a mollusk and a sea urchin. We showed that the receptors from the annelid and the sea urchin are functional RARs, however, the receptor from the mollusk is not functional with RA. Studies carried out in the annelid revealed that the signaling pathways regulated by RA in this species are different to the ones it regulates in vertebrates. These observations raise questions about the function of RA and RAR on the embryonic development of non-chordate species and their possible function in Urbilateria. Finally, the molecular characterization of cyclostome RARs allowed us to characterize the impact of whole genome duplications on the evolution of the ligand-binding pocket. Altogether, these data will allow us to better understand the relationship between the receptor and its ligand and to reveal novel insights on the function of RAR in response to non-classical ligands.

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...

Orientador: Rejane Maira Góes / Resumo: O câncer de próstata (CaP) é um dos tipos mais recorrentes de câncer em homens e o consumo excessivo de lipídeos saturados favorece o seu desenvolvimento. Ao contrário, dietas ricas em ácidos graxos poli-insaturados (PUFA) tipo ômega-3 (n-3) tem sido associadas com menor incidência do CaP. O presente trabalho avaliou as repercussões do consumo de ácidos graxos PUFAs n-3 da série marinha, ácido docosahexaenoico (DHA) e ácido eicosapentaenoico (EPA), sobre a progressão tumoral na próstata ventral de camundongos transgênicos para o adenocarcinoma de próstata (TRAMP). Camundongos TRAMP foram alimentados com dieta padrão e eutanasiados com 8 (C8), 12 (C12) e 20 (C20) semanas de vida ou então alimentados com uma dieta rica em óleo de peixe (10% óleo de peixe) a partir da oitava semana de vida e então eutanasiados com 12 (T12) ou 20 semanas (T20). Os resultados adquiridos demonstraram o aumento na proliferação celular bem como na expressão tecidual do receptor de andrógeno (AR) e glicocorticoide (GR) e no número de linfócitos T na próstata dos grupos controles conforme o aumento da idade e da agressividade das lesões. A intervenção dietética com PUFAs n-3 proporcionou a preservação do microambiente glandular levando a uma menor frequência de lesões proliferativas, indicando um atraso na progressão tumoral, onde foi observado níveis de proliferação celular, da expressão tecidual de AR e GR e de linfócitos T inferiores que seus controles de mesma idade. Ainda, o consumo de DHA/EPA pro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prostate cancer (PCa) is one of the most frequent cancers among male individual and saturated lipid intake is linked to its development. However, diets rich in omega-3 (n-3) polyunsaturated fatty acid (PUFA) have been associated to lower PCa development risk. The present study assessed the outcome of the marine n-3 PUFA intake, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), upon tumor progression at the transgenic adenocarcinoma of the mouse prostate (TRAMP). TRAMP mice were chow-fed and euthanized at 8 (C8), 12 (C12) and 20th (C20) week of age or fed with a fish oil-enriched diet from 8 to 12 (T12) or 20th (T20) week of age and then euthanized. The results indicated an increase in proliferation rate, tissue expression of androgen receptor (AR) and glucocorticoid receptor (GR) and the number of T-cells at the prostate of the control groups according to the increase of age and aggressiveness of the lesions. Dietary intervention with n-3 PUFA led to the maintenance of the glandular microenvironment, highlighted by a reduction of the frequency of proliferative lesions, indicating a delay in PCa progression whereas the levels of cell proliferation, tissue expression of AR and GR and the number of T-cells were lower than the control groups of the same age. Also, DHA/EPA intake promoted a lowering effect of serum triglycerides and cholesterol, improving the metabolic profile of these animals. Thereby incorporation o DHA/EPA on diet is capable of decrease the severity o... (Complete abstract click electronic access below) / Mestre

Les tumeurs de la vessie sont le quatrième cancer en terme de fréquence chez l’homme dans les pays industrialisés. Elles peuvent schématiquement être séparées en deux sous-groupes principaux : les tumeurs basales, peu différenciées, et les tumeurs luminales, différenciées. PPARγ est un récepteur nucléaire fortement exprimé dans ce second groupe, qui agit en hétérodimère avec un co-récepteur partenaire, RXRα, et possède un rôle essentiel dans la différenciation des cellules de l’urothélium normal. Récemment, un rôle protumorigénique de PPARγ a été mis en évidence dans les tumeurs luminales de vessie, où son activité est augmentée par le biais d’amplifications de PPARγ, ainsi que des mutations activatrices de son partenaire, RXRα. Le premier axe de mon projet a consisté à mieux caractériser ce rôle protumorigénique de PPARγ. J’ai d’abord participé à la mise en évidence de mutations récurrentes activatrices de PPARγ dans les tumeurs luminales de la vessie renforçant le rôle protumorigénique du récepteur dans ces tumeurs. J’ai ensuite cherché à identifier les gènes cibles de l’hétérodimère PPARγ/RXRα pouvant médier cet effet protumorigénique. Grâce à des analyses transcriptomiques après extinction de l’expression PPARγ ou de RXRα et des expériences de ChIP-sequencing de PPARγ et RXRα, j’ai pu identifier 30 gènes candidats. L’étude de l’implication de ces gènes dans la progression tumorale des tumeurs luminales présentant des altérations de la voie PPARγ/RXRα est actuellement en cours. Parallèlement à ce rôle protumorigénique dans les tumeurs luminales, des résultats suggéraient que ce gène pourrait posséder un rôle suppresseur de tumeurs dans le sous-groupe basal. La deuxième partie de mon projet a consisté à explorer cette hypothèse. J’ai pu montrer que des délétions hétérozygotes de PPARγ sont associées à ce sous-groupe de tumeurs basales, qui présente également une diminution globale de l’expression de PPARγ. De plus, parmi les mutations non-récurrentes de PPARγ identifiées dans ces tumeurs, certaines sont des mutations inactivatrices, capables d’inhiber l’activité transcriptomique de l’hétérodimère PPARγ/RXRα en favorisant la fixation de co-répresseurs de l’hétérodimère. J’ai également pu montrer un rôle anti-tumoral de PPARG dans les tumeurs basales puisque l’expression de PPARγ dans des lignées cellulaires basales à la suite de leur transfection avec un plasmide codant pour PPARγ est capable d’inhiber leur viabilité. Ce projet a ainsi permis de mettre en évidence un rôle dual de PPARγ dans les tumeurs de vessie, avec un rôle protumorigénique dans les tumeurs luminales, où il est suractivé par des amplifications et des mutations activatrices, et un rôle suppresseur de tumeurs dans les tumeurs basales, où il est réprimé par des délétions et de mutations inactivatrices. / Bladder tumors are the fourth cancer in terms of frequency in men, in industrialized countries. Bladder cancers can be divided into two main subgroups: undifferentiated basal tumors and differentiated luminal tumors. The nuclear receptor PPARγ, which is highly expressed in the second subgroup, forms a heterodimer with its partner, the nuclear receptor RXRα, and plays a key role in normal urothelial differentiation. A protumorigenic role of PPARγ has been established in luminal bladder cancer, with an increased activity through PPARγ genomic amplification or activating mutations of its partner, RXRα. The first part of my project aimed to better characterize this protumorigenic effect. I first participated in the identification of recurrent activating mutations of PPARγ in the luminal subgroup, enhancing its protumorigenic role in those tumors. Then, I tried to identify the PPARγ/RXRα heterodimer target genes that drive its protumorigenic role. By analyzing transcriptomic data following down regulation of PPARγ and RXRα, as well as ChIP-sequencing data for each of them, I identified 30 candidate target genes. The implication of the candidate genes in tumor progression of luminal cancer carrying PPARγ/RXRα pathway alteration is in progress. As opposed to its protumorigenic role in luminal tumors, some results suggested that PPARγ could play a tumor suppressor role in the basal subgroup. The second part of my project explored this hypothesis. I characterized PPARγ deletions associated with the basal subgroup, which presents a global PPARγ downregulation. Moreover, some of the non-recurrent mutations identified in this subgroup are inactivating mutations, able to inhibit the transcriptomic activity of the PPARγ/RXRα heterodimer, by an enhanced affinity of the heterodimer for co-repressors. I also showed an antitumor effect of PPARγ in basal tumors: its expression in basal bladder cell lines after transfection with a plasmid coding for PPARγ was able to inhibit cell viability. This project highlighted the dual role of PPARγ in bladder tumors, with a protumorigenic effect in luminal tumors through amplifications and activating mutations, and a tumor suppressor effect in basal tumors through deletions and inactivating mutations.

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.

Transcriptional regulation of gene expression in eukaryotes has evolved over millions of years. The regulatory pathways of nuclear receptors represent an evolutionarily ancient, but conserved mechanism with associated accessory proteins, many of them forming a functional nexus known as the Mediator complex involved in transcription. Despite the versatility of the pathway, e.g. through the adoption of new regulatory functions in phylogenetically more recent Metazoa, we hypothesise that the intrinsic potential of the NR-Mediator axis to directly translate a stimulus to a biological response is conserved across species, and additional regulation could also be achieved through secondary functions of its essential members. To support the hypothesis, we assessed the ligand-binding capability of retinoic X receptor in Trichoplax adhaerens and provided evidence to support the concept that this capability was already present at the base of metazoan evolution. With regards to the potential secondary functions, we took inspiration from previous research and identified the Mediator subunit 28 (MED28) as the only known member having documented nuclear and cytoplasmic dual roles, and thus possessing the potential to transmit signals from the cellular structural states to the nucleus. Due to the lack of...

Le récepteur X des farnésoïdes (FXR) fait partie de la superfamille des récepteurs nucléaires et agit comme un facteur de transcription suite à la liaison d’un ligand spécifique. Le récepteur FXR, activé par les acides biliaires, joue un rôle essentiel dans le métabolisme des lipides et du glucose en plus de réguler l’homéostasie des acides biliaires. Notre laboratoire a récemment mis en évidence une nouvelle voie de régulation du récepteur PPARγ en réponse au récepteur de la ghréline. En effet, la ghréline induit l’activation transcriptionnelle de PPARγ via une cascade de signalisation impliquant les kinases Erk1/2 et Akt, supportant un rôle périphérique de la ghréline dans les pathologies associées au syndrome métabolique. Il est de plus en plus reconnu que la cascade métabolique impliquant PPARγ fait également intervenir un autre récepteur nucléaire, FXR. Dans ce travail, nous montrons que la ghréline induit l’activation transcriptionnelle de FXR de manière dose-dépendante et induit également la phosphorylation du récepteur sur ses résidus sérine. En utilisant des constructions tronquées ABC et CDEF de FXR, nous avons démontré que la ghréline régule l’activité de FXR via les domaines d’activation AF-1 et AF-2. L’effet de la ghréline et du ligand sélectif GW4064 sur l’induction de FXR est additif. De plus, nous avons démontré que FXR est la cible d’une autre modification post-traductionnelle, soit la sumoylation. En effet, FXR est un substrat cellulaire des protéines SUMO-1 et SUMO-3 et la sumoylation du récepteur est ligand-indépendante. SUMO-1 et SUMO-3 induisent l’activation transcriptionnelle de FXR de façon dose-dépendante. Nos résultats indiquent que la lysine 122 est le site prédominant de sumoylation par SUMO-1, quoiqu’un mécanisme de coopération semble exister entre les différents sites de sumoylation de FXR. Avec son rôle émergeant dans plusieurs voies du métabolisme lipidique, l’identification de modulateurs de FXR s’avère être une approche fort prometteuse pour faire face à plusieurs pathologies associées au syndrome métabolique et au diabète de type 2. / The farnesoid X receptor (FXR) is a ligand-activated transcription factor within the nuclear receptor superfamily. FXR is activated by bile acids and plays a crucial role in the regulation of glucose and lipid metabolism and in bile acid homeostasis. Our group has recently identified the contribution of the ghrelin receptor in the regulation of the nuclear receptor PPARγ. Indeed, ghrelin triggers transcriptional activation of PPARγ through a concerted signaling cascade involving Erk1/2 and Akt kinases. These results support the peripheral actions of ghrelin in diseases associated with the metabolic syndrome. It is recognized that there is interplay between PPARγ metabolic cascade and FXR. Here, we demonstrate that ghrelin promotes FXR transcriptional activity in a dose-dependent manner and also promotes its phosphorylation on serine residues. By using truncated ABC and CDEF constructs of FXR, we found that ghrelin induces FXR activity through the AF-1 and AF-2 activation domains. The ghrelin-induced FXR activity is additive to the induction by the selective agonist GW4064. Also, we demonstrate that FXR is the target of sumoylation, another post-translational modification. In particular, FXR is modified by SUMO-1 and SUMO-3 in a ligand-independent manner. SUMO-1 and SUMO-3 promote dose-dependent transcriptional activity of FXR. Our results show that lysine 122 is the prevalent site of sumoylation by SUMO-1, though a compensation mechanism seems to exist between the various sumoylation sites of FXR. With its emerging role in several metabolic cascades, identification of FXR modulators represents a promising approach for the treatment of the metabolic syndrome and type 2 diabetes.

Les récepteurs nucléaires (RN) sont des facteurs de transcription ligand dépendants qui contrôlent une grande variété de processus biologiques de la physiologie humaine, ce qui a fait d'eux des cibles pharmacologiques privilégiées pour de nombreuses maladies. L'un de ces récepteurs, le récepteur de l’œstrogène alpha (ERα), peut activer la prolifération cellulaire dans certaines sections de l'épithélium mammaire tandis qu’un autre, le récepteur de l'acide rétinoïque alpha (RARα), peut provoquer un arrêt de la croissance et la différenciation cellulaire. La signalisation de ces deux récepteurs peut être altérée dans le cancer du sein, contribuant à la tumorigénèse mammaire. L’activité d’ERα peut être bloquée par les anti-oestrogènes (AE) pour inhiber la prolifération des cellules tumorales mammaires. Par contre, l’activation des voies de RARα avec des rétinoïdes dans un contexte clinique a rencontré peu de succès. Ceci pourrait résulter du manque de spécificité des ligands testés pour RARα et/ou de leur activité seulement dans certains sous-types de tumeurs mammaires. Puisque les récepteurs nucléaires forment des homo- et hétéro-dimères, nous avons cherché à développer de nouveaux essais pharmacologiques pour étudier l'activité de complexes dimériques spécifiques, leur dynamique d’association et la structure quaternaire des récepteurs des œstrogènes. Nous décrivons ici une nouvelle technique FRET, surnommée BRET avec renforcement de fluorescence par transferts combinés (BRETFect), qui permet de détecter la formation de complexes de récepteurs nucléaires ternaires. Le BRETFect peut suivre l'activation des hétérodimères ERα-ERβ et met en évidence un mécanisme allostérique d'activation que chaque récepteur exerce sur son partenaire de dimérisation. L'utilisation de BRETFect en combinaison avec le PCA nous a permis d'observer la formation de multimères d’ERα fonctionnels dans des cellules vivantes pour la première fois. La formation de multimères est favorisée par les AE induisant la dégradation du récepteur des oestrogènes, ce qui pourrait contribuer à leurs propriétés spécifiques. Ces essais de BRET apportent une nette amélioration par rapport aux tests de vecteurs rapporteur luciférase classique, en fournissant des informations spécifiques aux récepteurs en temps réel sans aucune interférence par d'autres processus tels que la transcription et de la traduction. L'utilisation de ces tests nous a permis de caractériser les propriétés de modulation de l’activité des récepteurs nucléaires d’une nouvelle classe de molécules hybrides qui peuvent à la fois lier ERa ou RAR et inhiber les HDACs, conduisant au développement de nouvelles molécules prometteuses bifonctionnelles telles que la molécule hybride RAR-agoniste/HDACi TTNN-HA. / Nuclear receptors (NRs) are ligand-dependent transcription factors that control a wide variety of biological processes in human physiology, which has made them preferred pharmacological targets for many diseases. One such receptor, the estrogen receptor alpha (ERα), can activate cell proliferation in some sections of the mammary epithelium while another, the retinoic acid receptor alpha (RARα), can cause growth arrest and cellular differentiation. Signalling by these receptors can be altered in breast cancer, contributing to tumorigenesis. ERα can be blocked by antiestrogens (AEs) in the clinical setting to inhibit tumor cell proliferation. However, attempts to activate the RARα pathway with retinoids have not proven beneficial in clinical trials. This may result from the lack of specificity of the tested ligands for RARa and/or from their activity only in a subset of breast tumors. Since nuclear receptors form homo- and heterodimers, we sought to develop novel pharmacological assays to study the activity of specific receptor-dimer complexes, their dynamics and quaternary structure. We report here a new FRET technique dubbed BRET with fluorescence enhanced by combined transfers (BRETFect) that can detect the formation of ternary nuclear receptor complexes. BRETFect can monitor the activation of ERα-ERβ heterodimers and highlights an allosteric mechanism of activation that each receptor exerts on its dimer partner. Use of BRETFect in combination with PCA-BRET has allowed us to observe the formation of functional ERα multimers in live cells for the first time. The formation of multimers is favored by AEs which induce receptor degradation, and may underlie their specific properties. These assays are a net improvement over the classical luciferase-reporter experiment as they deliver real-time receptor specific information with no interference by other process such as transcription and translation. Using these BRET assays we developed a new class of NR hybrid ligands that can modulate ER or RAR activity and inhibit HDACs. This has allowed for the development of promising new bifunctional molecules such as the RAR-agonist/HDACi hybrid molecule TTNN-HA. In conclusion, the work presented here brings new insight in NR dynamics and quaternary structure and offers novel tools to study their mechanism of action or design new modulators of NR activity such as hybrid AE-HDACis and Retinoid-HDACis.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...