Nuclear hormone receptors (NHRs) are ligand-activated transcriptions factors which transduce the effects of various hormones as well as nutritional and other environmental signals. They thus function to maintain physiological homeostasis by integrating the tissue expression of specific target genes to regulate a wealth of biological processes including reproduction, development, metabolism and environmental adaptation. Mounting evidence indicates NHRs are the target of endocrine disrupting compounds (EDCs), exogenous chemicals, often of anthropogenic origin, which disrupt NHRs and thus the processes under their control. EDCs can interfere with NHR signalling by activating receptors (agonists), by inhibiting the actions of the receptor (antagonists), or by disrupting endogenous hormone synthesis, secretion, transport or metabolism. Much of the focus to date has been on the risk of EDCs to reproductive functions, via estrogen and androgen NHRs in humans, and also in aquatic organisms. However environmental pollutants also have the potential to interact with other NHRs, particularly in aquatic environments, and cause dysregulation of other critical physiological processes, including energy homeostasis, immune functions and the stress response. To address this possibility a reporter gene assay was developed, allowing the high-throughput screening of pollutants for their interactions with piscine NHRs with critical roles in energy homeostasis, stress reponse and immune functions, namely the peroxisome proliferator-activated receptors (PPARs) and corticosteroid receptors (CRs) from European plaice (Pleuronectes platessa) and European flounder (Platichthys flesus), respectively. Complementary DNA (cDNA) sequences encoding the ligand-binding domains of PPARs and CRs, critical for receptor-ligand interactions and receptor activation, were ligated to the DNA-binding domain (DBD) of the yeast Gal4 transcription activator protein to create experimental expression plasmid constructs. Co-transfection of these expression plasmids into the fathead minnow (FHM) cell line with an upstream-activating sequence (UAS)-firefly luciferase reporter gene plasmid increased luciferase expression in the presence of known PPAR and CR ligands. Several aquatic pollutants including pharmaceuticals, industrial by-products and biocides were tested for their potential to disrupt PPAR and CR functions by interacting with these receptors in an agonistic or antagonistic manner. Several fibrates, a group of pharmaceutical compounds used to treat dyslipidemia in humans by targeting the PPARs, were able to activate plaice Gal4-PPARα and Gal4-PPARβ in the reporter gene assay, indicative of an interaction with PPAR receptors in non-target species. Fibrates which did not activate Gal4-PPARα were able to inhibit the activation of Gal4-PPARα by the PPARα-specific agonist, Wy14643, suggesting differential effects of fibrates on human and flounder PPARs. In addition some metabolites of widespread phthalate ester pollutants were also agonists of the Gal4-PPARα and Gal4-PPARβ constructs. The Gal4-PPARγ construct was unresponsive to almost all the compounds tested, including the mammalian PPARγ agonist, rosiglitazone. The exception to this was the phthalate metabolite monobenzylphthalate, which induced a small increase in firefly luciferase in Gal4-PPARγ transfected cells. All of the above effects required concentrations of at least 10 µM, which are unlikely to be encountered in the aquatic environment. In contrast bis(tributyltin) oxide (TBTO), a notorious environmental pollutant, inhibited Gal4-PPARα and Gal4-CR constructs at concentrations as low as 1 nM and 100 nM, respectively. These concentrations are lower than those reported in aquatic environments, or in fish tissues, making TBTO a candidate endocrine disruptor in fish by inhibiting PPARα and CR signalling. A European flounder cDNA microarray was used to investigate the trasnscriptional responses of flounder hepatocytes to TBTO (10 nM) exposure. Exposure to TBTO and Wy14643, both alone and in combination, indicated a TBTO-driven downregulation of several potential PPARα-target genes with functions in the immune system, the proteasome, and lipid metabolism, although, based on mammalian comparisons, some potential PPARα-target genes were also upregulated, indicating differences in mammalian and fish PPAR-target genes or reflecting the complexity of organisms at a higher organisational level than cell-based assay systems. However, the microarray-based approach was useful in formulating further hypotheses about the effects of TBTO on PPARα signalling. Overall, these results indicate that exogenous chemicals entering the aquatic environment can interfere with NHRs with functions in energy homeostasis, immune functions and stress, in non-target organisms. The cell-based reporter gene assay is a useful tool for identifying potential endocrine disruptors which target PPARs and CRs and would be a useful method in a first tier testing approach, limiting the use of live animal models and enabling investigation into specific receptors which are targets of endocrine disrupting compounds. Although more work is required to confirm the physiological consequences of TBTO inhibition of PPARα, the results presented here indicate that organisms inhabiting TBTO-polluted environments may experience suppression of the immune system, an increase in non-functional or misfolded proteins through suppression of genes involved in the ubiquitin/proteasome system and a disruption in lipid homeostasis.

The human retinoid X receptor alpha (hRXRalpha) is a member of the nuclear receptor super-family of ligand-activated transcription factors. The Doyle laboratory has previously engineered a variety of functional hRXRalpha variants that activate gene expression in response to synthetic ligands (LG335 and γ-oxo-1-pyrenebutyric acid), compounds that are poor activators of wild-type hRXRalpha. The variants generally no longer respond to the wild-type ligand 9-cis retinoic acid. To enable targeting of these engineered receptors to arbitrary DNA sequences, we developed a program, ESPSearch, for identifying short or specific sequences in DNA or protein. ESPSearch enables identification of combinations of known zinc finger motifs to target arbitrary genes, as well having several other applications. The ability to target any DNA sequence means that the engineered receptors can be directed to control any gene. The ligand binding, self-association, coactivator interactions, and unfolding properties of the ligand binding domain of wild-type hRXRalpha were characterized. Our expression and purification protocol improves upon existing methods, providing high purity protein in a single step with more than twice prior yields. A general fluorescence-based method for measuring ligand affinity with hRXRalpha was developed, and used to determine binding constants for the small molecules. The presence of a peptide containing the binding motif from coactivator proteins (LxxLL) differentially increased the affinity of the receptor for the ligands. Assays to determine the self-association give a Kd for the dimer-tetramer equilibrium of 35 µM. hRXRalpha was found to denature irreversibly when heated, but shifts in apparent Tm due to ligands correlates strongly with the ligand binding affinities. Our results clarify disparities in existing reports and provide a benchmark for comparison. Reliable analysis of our data led to the development of a computer program for rigorous, automated data fitting. Nine functional variants of hRXRalpha were characterized to probe correlations between biophysical properties and the observed functional activity of the receptors, which differ significantly from wild-type. Although the correlation between ligand binding affinity and melting temperature was strong for all variants, there was essentially no correlation between ligand binding and activation of the variants. The mutations, which are all contained within the binding pocket, have significant long-range effects on the protein, causing changes in ligand-LxxLL interactions and oligomerization of the variants. Experimental and computational analysis of selected mutations suggests that they are highly coupled, complicating protein design. However, the large variation in properties amongst the variants also suggests that hRXRalpha can be mutated extensively while still retaining function. The long-range impact of binding pocket mutations will need to be taken into account in future engineering projects, as hRXRalpha is a flexible, dynamic protein.

Nuclear receptors (NRs) are modular ligand-activated transcription factors that control a broad range of physiological processes by regulating the expression of essential genes involved in cell physiology, differentiation, and metabolism. These receptors are implicated in a number of diseases and due to their profound role in development and disease progression and their modularity, much emphasis is being put forth into nuclear receptor based drug discovery and engineering these receptors to bind novel small molecules Chemical Complementation (CC) is a yeast three-hybrid genetic selection system that was developed to aid in the discovery of these engineered receptors by linking the survival of a yeast cell to a small molecules ability to activate the receptor. Due to several advantages, to include faster growth times and higher transformation efficiencies, we have attempted to extend chemical complementation from yeast to E. coli. The bacterial chemical complementation system (BCC) was designed, based on a bacterial two hybrid system, to parallel yeast CC system. However, bacterial chemical complementation did not produce ligand dependent activation due to heterologous protein expression. In a second project designed to further NR based protein engineering and drug discovery, CC was used to evaluate a library of charge reversal variants rationally designed to gain a better understanding of nuclear receptor function and structure and to produce orthogonal ligand receptor pairs. A library of retinoic acid receptor (RARα) variants were developed based on five residues in the binding pocket known to stabilize the natural negatively charged ligand, all-trans retinoic acid (atRA). We altered the binding selectivity of the receptor to bind positively charged retinoid ligands. We were able to engineer two triple variants capable of activating with the positively charged retinoid but not the natural atRA ligand, however they do not activate as well as RARα wild-type does with atRA. In a third project we characterized covalently linked tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. Several dual inhibiting compounds were found to decrease the proliferation of ER positive breast cancer cells better than tamoxifen alone, the HDACi alone, or noncovalently linked HDACi and tamoxifen.

Le foie est un organe clef dans la régulation du métabolisme énergétique de l’organisme. La superfamille des récepteurs nucléaires y joue un rôle primordial de senseur de l’environnement métabolique. Parmi ces récepteurs nucléaires, le récepteur des acides biliaires FXR participe aux mécanismes de régulation de l’activité du foie à travers son action sur les métabolismes des acides biliaires, des glucides et des lipides. FXR est devenu ainsi une cible thérapeutique potentielle dans le traitement de nombreuses maladies impliquant un désordre métabolique comme les cholestases, le diabète de type 2 ou la stéatohépatite non-alcoolique. Malgré des résultats prometteurs dans le traitement de la stéatohépatite non-alcoolique, le traitement de patients avec un agoniste de FXR, le INT747, semble augmenter la concentration plasmatique du LDL-Cholestérol et diminue la concentration du HDL-Cholestérol suggérant un risque accru de développement d’athérosclérose. Ces effets sur le profil lipidique sont le frein majeur du développement clinique de ses agonistes. Les mécanismes moléculaires impliqués dans la régulation par FXR de nombreuses voies comme le métabolisme des lipides et du cholestérol sont peu explorés et peu compris. Compte-tenu de ces informations, il est d’autant plus intéressant d’approfondir les connaissances de ces mécanismes et d’identifier des facteurs ou de nouveaux partenaires capables de moduler l’activité transcriptionnelle de FXR plus spécifiquement dans le cadre du contrôle du métabolisme des lipides et du cholestérol. L’un des facteurs de transcription connu comme régulateur majeur de ces voies métaboliques dans le foie est le facteur de transcription de la famille forkhead FOXA2. Ce facteur de transcription, dont l’activité est dépendante des conditions physiologiques, est activé par le glucagon et inhibé par l’insuline. De plus, c’est également un régulateur du métabolisme des acides biliaires, du cholestérol et des lipides.L’objectif de cette thèse a été d’étudier l’interaction entre les voies de signalisation de FXR et de FOXA2 dans différentes lignées cellulaires d’hépatocytes humains ou murins et dans le foie. Nous avons établi que FOXA2 et FXR sont colocalisés sur la chromatine des cellules HepG2 et dans le foie de souris à proximité de gènes impliqués dans la régulation du métabolisme des lipides et du cholestérol. Ces zones de cofixation de FXR et de FOXA2 présentent très peu de motifs de fixation de FOXA2 suggérant l’implication d’autres motifs de fixation non connus ou un mécanisme de type « tethering ». Nous avons montré que la fixation de FOXA2 à ces zones de cofixation avec FXR est augmentée par l’activation de FXR par son agoniste, le GW4064, évoquant une potentielle interaction entre ces deux facteurs. Nous avons démontré que ces deux facteurs interagissaient physiquement et que FOXA2 est un répresseur de l’activité transcriptionnelle de FXR à travers l’utilisation de différentes approches et modèles cellulaires. Finalement, dans les hépatocytes primaires de souris, FOXA2 est impliqué dans la répression de l’activité transcriptionnelle de FXR par le glucagon sur le gène Shp.L’ensemble de ce travail met en évidence pour la première fois la répression de l’activité de FXR par le facteur de transcription caractéristique du jeûne FOXA2 à travers un mécanisme moléculaire suggérant une transrépression de type «tethering». Ces résultats présentent un mécanisme inédit par lequel l’activité de FXR peut être modulée par le statut nutritionnel de façon gène-spécifique. / The liver is a key regulator of whole-body energy metabolism. The nuclear receptor super-family plays a leading role in the metabolic sensing of the liver. Among the nuclear receptors, the bile acid nuclear receptor FXR contribute to the modulation of liver activity in particular through the regulation of bile acid, lipids and glucose homeostasis. Consequently, FXR became a potential therapeutic target for many diseases implicated metabolic disorder such as cholestasis, type 2 diabete or Non-Alcoholic Steatohepatitis (NASH). Despite promising results especially on NASH, patient treatment with FXR agonist the INT747 seems to increase LDL-Cholesterol plasma concentrations together with a decreased concentration of HDL-Cholesterol suggesting a higher risk to develop atherosclerosis. These effects on plasma lipid profile are the major break against the development of agonists in clinics. Giving the poor understanding and knowledge of the molecular mechanisms which govern FXR regulation of activity on various signaling pathways, it is of major interest to find new partners and regulators of FXR and especially on lipid and cholesterol homeostasis. One of the transcription factor known to be active in the control of these signaling pathways in the liver is the forkhead box transcription factor FOXA2. This transcription factor whose activity is dependent of physiological conditions is activated by glucagon and inhibited by insulin. In addition, this factor is known to regulate bile acid, cholesterol and lipid metabolism, functions very close from FXR activities in the liver.The objective of this PhD was to study the interaction between FXR and FOXA2 signaling pathways in different hepatic cells lines from human or mouse origin and in the liver. We established that FOXA2 and FXR are colocalised in HepG2 cells and liver chromatin near genes implicated in the lipid and cholesterol metabolism. These FXR/FOXA2 cobinding zones present few consensus FOXA2 response elements suggesting the implication of non consensus binding motifs or a “tethering” mechanism. We show that FOXA2 binding to FXR/FOXA2 cobinding zones is increased when FXR is activated and/or more present in the chromatin evoking a potential interaction between these two factors. We demonstrate that FXR and FOXA2 interact physically and that FOXA2 is a repressor of FXR transcriptional activity using different approaches and cellular models. Finally, we show that FOXA2 is implicated in glucagon-induced repression of FXR transcriptional activity on Shp gene.To conclude, our results show for the first time that the fasting key regulator of lipid and cholesterol homeostasis FOXA2 is a repressor of FXR transcriptional activity through a plausible mechanism involving “tethering” process. This work gives a novel mechanism by which FXR activity can be modified by nutritional status in a gene-specific manner.

In our modern society we are exposed to many natural and synthetic chemicals. The assessment of chemicals with regard to human safety is difficult but nevertheless of high importance. Beside clinical studies, which are restricted to potential pharmaceuticals only, most toxicity data relevant for regulatory decision-making are based on in vivo data. Due to the ban on animal testing of cosmetic ingredients in the European Union, alternative approaches, such as in vitro and in silico tests, have become more prevalent. In this thesis existing non-testing approaches (i.e. studies without additional experiments) have been extended, e.g. QSAR models, and new non-testing approaches, e.g. in vitro data supported structural alert systems, have been created. The main aspect of the thesis depends on the determination of data quality, improving modelling performance and supporting Adverse Outcome Pathways (AOPs) with definitions of structural alerts and physico-chemical properties. Furthermore, there was a clear focus on the transparency of models, i.e. approaches using algorithmic feature selection, machine learning etc. have been avoided. Furthermore structural alert systems have been written in an understandable and transparent manner. Beside the methodological aspects of this work, cosmetically relevant examples of models have been chosen, e.g. skin penetration and hepatic steatosis. Interpretations of models, as well as the possibility of adjustments and extensions, have been discussed thoroughly. As models usually do not depict reality flawlessly, consensus approaches of various non-testing approaches and in vitro tests should be used to support decision-making in the regulatory context. For example within read-across, it is feasible to use supporting information from QSAR models, docking, in vitro tests etc. By applying a variety of models, results should lead to conclusions being more usable/acceptable within toxicology. Within this thesis (and associated publications) novel methodologies on how to assess and employ statistical data quality and how to screen for potential liver toxicants have been described. Furthermore computational tools, such as models for skin permeability and dermal absorption, have been created.

Os receptores ativadores da proliferação de peroxissomos (PPARs) são fatores de transcrição dependentes da ligação de ligantes e possuem um papel chave no controle do metabolismo dos lipídios e da glicose. Existem três isotipos desse receptor: PPARα, PPARβ e PPARγ. O PPARγ é alvo molecular para os compostos TZDs, os quais são fármacos usados clinicamente no controle da diabetes do tipo 2, aumentando a sensibilidade à insulina. Enquanto que os fibratos são os fármacos que atuam no PPARα e são utilizados para diminuir os níveis de triglicerídeos. A maioria dos pacientes que sofrem com a diabetes do tipo 2 apresentam desordens no metabolismo de lipídios. Mesmo com a existência de fármacos capazes de controlar estas desordens metabólicas, a busca de um agonista dual para os PPARα e PPARγ é um grande desafio no controle da síndrome metabólica, uma vez que este composto pode combinar os dois efeitos terapêuticos em uma única molécula. O GL479 é um agonista dual que foi sintetizado com dois grupos farmacóforos, ligando-se tanto ao PPARα quanto ao PPARγ. Dentro desse contexto, este estudo apresenta as bases estruturais de interação do agonista dual GL479 aos PPARs por meio da determinação estrutural dos complexos PPARα-LBD:GL479 e PPARγ-LBD:GL479. A análise detalhada desses complexos revelou diferentes modos de interação do ligante em cada receptor, porém em ambos os casos o GL479 interage com a Tyr da H12. Na estrutura do PPARα-LBD, o ligante adquiriu a característica de um agonista total e no caso do PPARγ-LBD, o GL479 adotou características de um agonista parcial dependente da interação com a H12. Além das analises do agonista dual, 16 compostos foram identificados por docking como ligantes do PPARγ. Três desses ligantes (8, 10 e 15) foram caracterizados por ThermoFluor e fluorescência de polarização com valores de IC50 menor que 10 µM. Adicionalmente, um dos compostos identificados no docking (16) foi cocristalizado com PPARγ-LBD. A conformação adotada pelo ligante não permitiu que ele interagisse diretamente com a H12, sugerindo que este composto possa atuar como um agonista parcial independente da H12. Todas estas descobertas podem ser exploradas no desenho de novos moduladores dos PPARs com menores efeitos adversos ou até mesmo na busca de agonistas duais PPARα ⁄γ, que combine os efeitos terapêuticos no tratamento da diabetes do tipo 2 e da dislipidemia. / Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism and they play key roles in the control of glucose and lipid metabolism. There are three different PPAR isotypes: PPARα, PPARβ e PPARγ. PPARγ is a molecular target of TZD agonists, which are clinically used drugs in the control of type 2 diabetes by increasing insulin sensitivity. Whereas fibrates are drugs that act on PPARα and are used to lower serum triglyceride levels. The most patients who have type 2 diabetes also display lipid metabolism disorders. Even with the existence of drugs that can control these metabolic disorders, the search of dual agonist for PPARα and PPAR γ is a major challenge in the control of metabolic syndrome, because this compound could combine both therapeutic effects in a single molecule. GL479 is a dual agonist that was synthesized based on a combination of two key pharmacophores, with the ability to bind in the both PPARs, α, and γ. Thus, this study reveals the structural basis for this dual agonist GL479 by structural determination of the complexes PPARα-LBD:GL479 and PPARγ-LBD:GL479. The detailed analysis of these complexes showed different ligand binding modes for each receptor, however, in the both cases the GL479 interacted with the Tyr of H12. In the PPARα-LBD structure the ligand acquired the features of full agonist and in the case of PPARγ-LBD, GL479 adopted features of a partial agonist dependent of H12 interaction. In addition to the dual agonist analysis, sixteen compounds were identified as PPARγ ligand by docking. Three of these ligands were characterized by ThermoFluor and fluorescence polarization, which resulted in IC50 values smaller than 10 µM. Additionally, one of the compounds, identified by docking, was co-crystallized with PPARγ. The ligand conformation adopted would not allow it a direct interaction with the H12. These contacts were mediated by one water molecule, suggesting this compound might also act as a partial agonist, independent of H12 interaction. All these findings may be explored for the design of PPARs novel modulators with lower side effects, as well, in the exploration of dual agonists PPARα ⁄ γ that combines the therapeutic effects in the treatment of type 2 diabetes and dyslipidemia.

Os cânceres de próstata e de mama estão entre as neoplasias mais comuns diagnosticadas na população ocidental. No Brasil, estes dois tipos de neoplasia são as principais causas de morte cuja incidência continua crescendo anualmente, sendo mais comum na população acima de 40 anos. As terapias utilizadas para os tratamentos de ambas as neoplasias estão baseadas principalmente nos receptores de hormônio (andrógeno e estrógeno). Embora muitos fármacos tenham sido desenvolvidos para os tratamentos destas patologias ao longo do tempo, eles perdem eficácia em caso de neoplasias resistentes, que apresentam mutações nas macromoléculas alvo. Assim, novas substâncias bioativas estão sendo investigadas a partir dos alvos biológicos consolidados e também para novos alvos. Neste trabalho, ensaios in vitro foram utilizados para avaliar as atividades farmacológicas e citotóxica de novas substâncias bioativas desenvolvidas no Grupo de Química Medicinal (NEQUIMED), a partir de duas linhagens celulares hormônio-dependentes para o estudo do câncer de próstata (LNCaP) e de mama (MCF-7). A partir das triagens realizadas, duas substâncias foram as mais potentes (Neq0502 e Neq0504) que levaram a morte das linhagens LNCaP e MCF-7 com IC50 na ordem de 20 a 30 µmol/L, respectivamente. No ensaio de ciclo celular, Neq0502 apresentou um perfil semelhante a enzalutamida (fármaco usado como referência), sem perturbações substanciais no ciclo. No entanto, Neq0504 teve um perfil bem distinto do raloxifeno (fármaco usado como referência) para a perturbação do ciclo celular. Finalmente, o índice de seletividade estabelecido a partir dos ensaios com as células de fibroblasto (Balb/C 3T3 clone A31) demonstrou que Neq0502 foi uma substância com a maior seletividade e baixa citotoxicidade em relação à célula não tumoral dentre toda a série estudada. A partir destes dados as novas substâncias poderão ser otimizadas usando Neq0502 como matriz em estudos futuros. / Prostate and breast cancers are among the most common cancers diagnosed in the western population. In Brazil, these two types of cancer are the leading causes of death whose incidence continues to increase annually and is more common in older population than 40 years. The therapies used for the treatment of both cancers are mainly based on the hormone receptors (androgen and estrogen). Although many drugs have been developed for the treatment of these pathologies over time, lose efficacy in case of resistant cancers which have mutations on the target macromolecules. Thus, new bioactive substances are being investigated based on stable biological targets and for new targets. In this study, in vitro assays were used to evaluate the pharmacological and cytotoxic activities of new bioactive substances developed in Medicinal Chemistry Group (NEQUIMED) from two hormone-dependent cell lines for the study of prostate (LNCaP) and breast (MCF-7) cancer. From trials screenings carried out, two compounds were found the most potent (Neq0502 and Neq0504) leading to death of LNCaP and MCF-7 lines with IC50 in the range of 20 to 30 µmol/L, respectively. In the cell cycle assay, Neq0502 made a similar profile to enzalutamide (drug used as a reference), without substantial disruption in the cycle. On the other hand, Neq0504 had a very different profile from raloxifene (a drug used as a reference) to the perturbation of the cell cycle. Finally, the selectivity index established from tests with fibroblast cells (Balb/C 3T3 clone A31) demonstrated that Neq0502 was a substance with high selectivity and low cytotoxicity in order to non-tumor cell from all the substances on the screening. From these data, new substances can be optimized using Neq0502 as a template in future studies.

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.

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