La ghréline est une hormone de 28 acides aminés, synthétisée principalement par l'estomac. D'abord identifiée comme un sécretagogue de l'hormone de croissance, elle joue également un rôle central dans la prise alimentaire, la glycémie ainsi que dans certains processus liés à l'addiction. Ces effets sont médiés par un récepteur couplé aux protéines G : le GHS-R1a (Growth Hormone Secretagogue Receptor). Ce récepteur possède une activité constitutive élevée et un réseau de signalisation intra-cellulaire relativement complexe via l'activation de β-arrestines et de différentes isoformes de protéines G (Gq, Gi/o, G12/13). Compte tenu de ces multiples effets, les ligands du GHS-R1a présentent un intérêt thérapeutique certain.Cette thèse est consacrée au développement d'antagonistes et d'agonistes inverses du hGHS-R1a, dont la structure est basée sur le motif 1,2,4-triazole 3,4,5-trisubstitué. Grâce à une étude successive des différents substituants de cette plateforme peptido-mimétique nous avons identifié des antagonistes d'affinités nanomolaires ainsi que des agonistes inverses possédant une efficacité significative. Ces composés paraissent donc être des candidats intéressants pour des études in vivo sur des modèles de prise alimentaire ou d'addiction. D'autre part, une étude pharmacologique sophistiquée, menée sur nos composés, a démontré qu'il est possible d'obtenir des ligands biaisés sur la base du motif triazole. Ces résultats fournissent de nouvelles informations sur la sélectivité fonctionnelle du GHS-R1a. Ainsi, associés à des études in vivo complémentaires, ces données pourraient être précieuses pour la conception de nouveaux médicaments possédant des effets secondaires limités. / Ghrelin is a hormone of 28 amino acids, mostly synthesized in the stomach. Firstly identified as a growth hormone secretagogue, this peptide is also involved in food intake, blood glucose and in some processes related to addiction. Ghrelin effects are mediated by a G protein-coupled receptor: GHS-R1a (Growth Hormone Secretagogue Receptor). This receptor has a high constitutive activity and a complex intra-cellular signaling network via the activation of β-arrestin and different isoforms of G protein (Gq, Gi / o, G12 / 13). Given these multiple effects, ligands of GHS-R1a have a therapeutic interest.This thesis is devoted to the development of antagonists and inverse agonists of hGHS-R1a whose structure is based on the 3,4,5-trisubstituted 1,2,4-triazole scaffold. Thanks to a successive study of the various substituents of the peptidomimetic platform we identified antagonists with nanomolar affinity and inverse agonists with a significant efficiency. These compounds appear to be attractive candidates for in vivo studies on food intake or addiction models. On the other hand, a sophisticated pharmacological study, conducted on our compounds, has demonstrated that it is possible to obtain biased ligands based on the triazole motif. These results provide new informations about the functional selectivity of GHS-R1a. Thus, these data, combined with additional in vivo studies, could be useful for the design of new drugs with limited side effects.

Les adénomes hypophysaires sont les tumeurs intracérébrales les plus fréquentes. Les adénomes somatotropes hypersécrètent l’hormone de croissance (GH) et sont traités classiquement par des analogues somatostatinergiques. Une petite moitié des patients acromégales est néanmoins résistante à ces traitements. L’on sait depuis, quelques années, que le récepteur de la ghréline possède une forte activité constitutive et joue un rôle majeur dans la sécrétion de GH. Cette activité constitutive est-elle impliquée dans la tumorigenèse des adénomes somatotropes ? Nos travaux ont montré un niveau de transcrits, codant pour le GHS-R, particulièrement élevé dans ces tumeurs, et l’immunocytochimie révèle un marquage punctiforme localisé à la membrane plasmique. La MSP (agoniste inverse du GHS-R) induit une diminution dose-dépendante de la sécrétion de GH des cultures primaires d’adénomes somatotropes. Cette efficacité de la MSP sur la sécrétion de l’hormone de croissance est particulièrement remarquable sur les patients résistants aux agonistes somatostatinergiques chez qui elle démontre une efficacité relative accrue. Des clones, surexprimant le GHS-R humain (lignées MYST-R), ont été générés à partir de lignées somato-lactotropes tumorales de rat (GH4C1). Sur ces cellules, le ligand endogène du GHS-R induit une augmentation d’IP3 intracellulaire. De façon originale, la MSP induit une diminution du niveau d’IP3 intracellulaire. L’inhibition de l’activité constitutive du GHS-R par un agoniste inverse, tel que la MSP, pourrait permettre de réprimer l’hypersécrétion de GH, faisant de cette molécule une alternative pharmacologique aux traitements actuels des adénomes somatotropes. / Pituitary tumors are most usual intracranial tumors. The somatotroph adenomas are characterised by a GH hypersecretion. The current treatments are based on somatostatinergic agonists. Unfortunately, there is steel 50% of patients, which remain insensitive to these treatments. The aim of our work was to find a pharmacological alternative to treat the patients resistant to the current therapies. Ghrelin stimulate pituitary GH release in vivo through GHS-R1a activation. Interestingly, this receptor transduces signal through an unusual high constitutive activity. Noteworthy, human somatotroph adenomas expressed a high level of GHS-R1a at both mRNA and protein level. We actually assess the implication of this constitutive activity in the tumorigenesis of the somatotroph adenomas. Firstly we demonstrated GHS-R1a functionality through its capacity to fixe endogenous ligand. Then we showed that treatment of human somatotroph adenomas primary cultures, with the GHS-R1a inverse agonist (MSP: Modified Substance P), induced a dose dependent decrease of GH secretion. To foremost investigate the transduction mechanisms underlying these results, we developed, from GH4C1 (rat somato-lactotroph tumoral cell line), stable monoclonal cell lines overexpressing human GHS-R1a (named MYST-Rg). Interestingly MYST-Rg cells exhibit relatively high basal activation of the IP3 pathway. GHS-R1a endogenous ligand (ghrelin) strengthens basal IP3 pathway activation of MYST-Rg cells. Noteworthy, the basal IP3 pathway activation can be lessened by MSP treatment. Thus, MSP could be a useful alternative to the current therapies of somatotroph adenomas.

L’adaptation de l’organisme à son environnement est essentielle à sa survie. L’homéostasie énergétique permet l’équilibre entre les apports, les dépenses et le stockage d’énergie. Un surplus calorique important dérègle ce processus et mène au développement du syndrome métabolique caractérisé, entre autres, par une obésité, un diabète de type II, des maladies cardiovasculaires et des dyslipidémies. La ghréline participe au maintien de l’équilibre énergétique durant le jeûne en stimulant la production de glucose par le foie et le stockage lipidique dans le tissu adipeux. Le coactivateur transcriptionnel PGC-1alpha, surexprimé en situation de jeûne, est impliqué dans l’induction de la production de glucose par le foie et l’oxydation des acides gras. Notre hypothèse est que ces deux acteurs clés du métabolisme énergétique constituent un axe de régulation commun. Dans cette étude, nous montrons que la ghréline participe à la régulation de PGC-1alpha. Son récepteur GHS-R1a, possédant une forte activité constitutive, est également impliqué de façon indépendante au ligand. GHS-R1a réduit l’activité transcriptionnelle de PGC-1alpha tandis que l’ajout du ligand inverse modérément cette action. L’effet de GHS-R1a corrèle avec l’acétylation de PGC-1alpha qui est fortement augmentée de façon dose-dépendante. La stabilité de PGC-1alpha est également augmentée par le GHS-R1a indépendamment de l’ubiquitine. La ghréline diminue la capacité de PGC-1alpha à lier PPARbeta, un récepteur nucléaire partenaire de PGC-1alpha. De plus, la ghréline réduit, de façon ligand-dépendante, la capacité de coactivation de PGC-1alpha sur PPARbeta dans les hépatocytes. L’ensemble de ces résultats identifie PGC-1alpha comme cible du signal de la ghréline et suggère un axe de régulation ghréline/PGC-1alpha/PPARbeta.Une meilleure compréhension de cet axe de régulation va permettre la mise en évidence de nouvelles cibles thérapeutiques pour faire face aux pathologies associées au syndrome métabolique. / The adaptation of an organism to its environment is essential to its survival. Energy homeostasis is defined as the balance between intakes, expenses and storage of energy. An excess of calories disrupts this process and leads to the development of the metabolic syndrome that is characterized by obesity, type II diabetes, cardiovascular diseases and dyslipidemia. During fasting, ghrelin participates in the maintenance of energy balance by stimulating hepatic production of glucose and lipid storage in adipose tissue. The transcriptional coactivator PGC-1alpha is overexpressed in the liver during fasting and is involves in the induction of the hepatic glucose production and fatty acid oxidation. Our hypothesis is that these two key performers in the energy metabolism constitute a common axis control. In this study, we show that ghrelin plays a role in the regulation of PGC-1alpha. The ghrelin receptor GHS-R1a is also involved because of its strong constitutive activity in absence of ligand. We found that GHS-R1a inhibited PGC-1alpha transcriptional activity whereas adding ghrelin to cells moderated this effect. PGC-1alpha activation by GHS-R1a correlated with a dose-dependent increase of PGC-1alpha acetylation. The stability of PGC-1alpha was also increased by ghrelin receptor in a manner involving the ubiquitin-independent proteasome pathway. Ghrelin decreased the ability of PGC-1alpha to bind to PPARbeta, one of its nuclear receptor partners. Furthermore, ghrelin decreased the ability of PGC-1alpha to coactivate PPARbeta in a ligand-dependent manner in hepatocytes. Together, these results identify PGC-1alpha as a metabolic target of GHSR-1a signaling and defines a new regulatory axis involving ghrelin/PGC-1alpha/PPARbeta in hepatocytes. A better understanding of this regulation axis will provide novel aspects in therapeutic targeting of diseases associated with the metabolic syndrome.

L’adaptation de l’organisme à son environnement est essentielle à sa survie. L’homéostasie énergétique permet l’équilibre entre les apports, les dépenses et le stockage d’énergie. Un surplus calorique important dérègle ce processus et mène au développement du syndrome métabolique caractérisé, entre autres, par une obésité, un diabète de type II, des maladies cardiovasculaires et des dyslipidémies. La ghréline participe au maintien de l’équilibre énergétique durant le jeûne en stimulant la production de glucose par le foie et le stockage lipidique dans le tissu adipeux. Le coactivateur transcriptionnel PGC-1alpha, surexprimé en situation de jeûne, est impliqué dans l’induction de la production de glucose par le foie et l’oxydation des acides gras. Notre hypothèse est que ces deux acteurs clés du métabolisme énergétique constituent un axe de régulation commun. Dans cette étude, nous montrons que la ghréline participe à la régulation de PGC-1alpha. Son récepteur GHS-R1a, possédant une forte activité constitutive, est également impliqué de façon indépendante au ligand. GHS-R1a réduit l’activité transcriptionnelle de PGC-1alpha tandis que l’ajout du ligand inverse modérément cette action. L’effet de GHS-R1a corrèle avec l’acétylation de PGC-1alpha qui est fortement augmentée de façon dose-dépendante. La stabilité de PGC-1alpha est également augmentée par le GHS-R1a indépendamment de l’ubiquitine. La ghréline diminue la capacité de PGC-1alpha à lier PPARbeta, un récepteur nucléaire partenaire de PGC-1alpha. De plus, la ghréline réduit, de façon ligand-dépendante, la capacité de coactivation de PGC-1alpha sur PPARbeta dans les hépatocytes. L’ensemble de ces résultats identifie PGC-1alpha comme cible du signal de la ghréline et suggère un axe de régulation ghréline/PGC-1alpha/PPARbeta.Une meilleure compréhension de cet axe de régulation va permettre la mise en évidence de nouvelles cibles thérapeutiques pour faire face aux pathologies associées au syndrome métabolique. / The adaptation of an organism to its environment is essential to its survival. Energy homeostasis is defined as the balance between intakes, expenses and storage of energy. An excess of calories disrupts this process and leads to the development of the metabolic syndrome that is characterized by obesity, type II diabetes, cardiovascular diseases and dyslipidemia. During fasting, ghrelin participates in the maintenance of energy balance by stimulating hepatic production of glucose and lipid storage in adipose tissue. The transcriptional coactivator PGC-1alpha is overexpressed in the liver during fasting and is involves in the induction of the hepatic glucose production and fatty acid oxidation. Our hypothesis is that these two key performers in the energy metabolism constitute a common axis control. In this study, we show that ghrelin plays a role in the regulation of PGC-1alpha. The ghrelin receptor GHS-R1a is also involved because of its strong constitutive activity in absence of ligand. We found that GHS-R1a inhibited PGC-1alpha transcriptional activity whereas adding ghrelin to cells moderated this effect. PGC-1alpha activation by GHS-R1a correlated with a dose-dependent increase of PGC-1alpha acetylation. The stability of PGC-1alpha was also increased by ghrelin receptor in a manner involving the ubiquitin-independent proteasome pathway. Ghrelin decreased the ability of PGC-1alpha to bind to PPARbeta, one of its nuclear receptor partners. Furthermore, ghrelin decreased the ability of PGC-1alpha to coactivate PPARbeta in a ligand-dependent manner in hepatocytes. Together, these results identify PGC-1alpha as a metabolic target of GHSR-1a signaling and defines a new regulatory axis involving ghrelin/PGC-1alpha/PPARbeta in hepatocytes. A better understanding of this regulation axis will provide novel aspects in therapeutic targeting of diseases associated with the metabolic syndrome.

A propriedade de auto-cura, em redes inteligente de distribuição de energia elétrica, consiste em encontrar uma proposta de reconfiguração do sistema de distribuição com o objetivo de recuperar parcial ou totalmente o fornecimento de energia aos clientes da rede, na ocorrência de uma falha na rede que comprometa o fornecimento. A busca por uma solução satisfatória é um problema combinacional cuja complexidade está ligada ao tamanho da rede. Um método de busca exaustiva se torna um processo muito demorado e muitas vezes computacionalmente inviável. Para superar essa dificuldade, pode-se basear nas técnicas de geração de árvores de extensão mínima do grafo, representando a rede de distribuição. Porém, a maioria dos estudos encontrados nesta área são implementações centralizadas, onde proposta de reconfiguração é obtida por um sistema de supervisão central. Nesta dissertação, propõe-se uma implementação distribuída, onde cada chave da rede colabora na elaboração da proposta de reconfiguração. A solução descentralizada busca uma redução no tempo de reconfiguração da rede em caso de falhas simples ou múltiplas, aumentando assim a inteligência da rede. Para isso, o algoritmo distribuído GHS é utilizado como base na elaboração de uma solução de auto-cura a ser embarcada nos elementos processadores que compõem as chaves de comutação das linhas da rede inteligente de distribuição. A solução proposta é implementada utilizando robôs como unidades de processamento que se comunicam via uma mesma rede, constituindo assim um ambiente de processamento distribuído. Os diferentes estudos de casos testados mostram que, para redes inteligentes de distribuição compostas por um único alimentador, a solução proposta obteve sucesso na reconfiguração da rede, indiferentemente do número de falhas simultâneas. Na implementação proposta, o tempo de reconfiguração da rede não depende do número de linhas nela incluídas. A implementação apresentou resultados de custo de comunicação e tempo dentro dos limites teóricos estabelecidos pelo algoritmo GHS. / The characteristic of self-healing, in smart grids, consists of finding a proposal for a reconfiguration of distribution system aiming at restoring the power, partially or completely to supply the network clients, in the event of network failure, which compromises the energy supply. The search for a satisfactory solution is a combinatorial problem whose complexity is proportional to the network size. An exhaustive search-based method is a time-consuming process and often computationally not viable. To overcome this difficulty, techniques for generating minimal spanning trees of the graph, which represents the smart grid, are exploited. However, the majority of studies in this area provide centralized implementations, where the solution for reconfiguration is achieved by a central control system. In this dissertation, we propose a distributed implementation, where each of the network switch collaborates in the development of the solution for reconfiguration. The proposed decentralized solution seeks a reduction in terms of the network reconfiguration time, in case of a single or multiple failures, thus increasing network intelligence. In this purpose, the GHS distributed algorithm is used as a basis for developing a self-healing solution to be embedded in the processing elements that are included within the line commutation switches of smart grid. The proposed solution is implemented using robots as processing units, which communicate via the same network, thereby creating a distributed processing environment. The several tested case studies show that, for smart grids that to have a single distribution feeder, the proposed solution allowed for a successful reconfiguration of the network, regardless of the number of simultaneous failures. In the proposed implementation, the network reconfiguration time does not depend on the number of buses and lines included. The implementation presents results of communication cost and time within the theoretical bounds of the GHS algorithm.

A propriedade de auto-cura, em redes inteligente de distribuição de energia elétrica, consiste em encontrar uma proposta de reconfiguração do sistema de distribuição com o objetivo de recuperar parcial ou totalmente o fornecimento de energia aos clientes da rede, na ocorrência de uma falha na rede que comprometa o fornecimento. A busca por uma solução satisfatória é um problema combinacional cuja complexidade está ligada ao tamanho da rede. Um método de busca exaustiva se torna um processo muito demorado e muitas vezes computacionalmente inviável. Para superar essa dificuldade, pode-se basear nas técnicas de geração de árvores de extensão mínima do grafo, representando a rede de distribuição. Porém, a maioria dos estudos encontrados nesta área são implementações centralizadas, onde proposta de reconfiguração é obtida por um sistema de supervisão central. Nesta dissertação, propõe-se uma implementação distribuída, onde cada chave da rede colabora na elaboração da proposta de reconfiguração. A solução descentralizada busca uma redução no tempo de reconfiguração da rede em caso de falhas simples ou múltiplas, aumentando assim a inteligência da rede. Para isso, o algoritmo distribuído GHS é utilizado como base na elaboração de uma solução de auto-cura a ser embarcada nos elementos processadores que compõem as chaves de comutação das linhas da rede inteligente de distribuição. A solução proposta é implementada utilizando robôs como unidades de processamento que se comunicam via uma mesma rede, constituindo assim um ambiente de processamento distribuído. Os diferentes estudos de casos testados mostram que, para redes inteligentes de distribuição compostas por um único alimentador, a solução proposta obteve sucesso na reconfiguração da rede, indiferentemente do número de falhas simultâneas. Na implementação proposta, o tempo de reconfiguração da rede não depende do número de linhas nela incluídas. A implementação apresentou resultados de custo de comunicação e tempo dentro dos limites teóricos estabelecidos pelo algoritmo GHS. / The characteristic of self-healing, in smart grids, consists of finding a proposal for a reconfiguration of distribution system aiming at restoring the power, partially or completely to supply the network clients, in the event of network failure, which compromises the energy supply. The search for a satisfactory solution is a combinatorial problem whose complexity is proportional to the network size. An exhaustive search-based method is a time-consuming process and often computationally not viable. To overcome this difficulty, techniques for generating minimal spanning trees of the graph, which represents the smart grid, are exploited. However, the majority of studies in this area provide centralized implementations, where the solution for reconfiguration is achieved by a central control system. In this dissertation, we propose a distributed implementation, where each of the network switch collaborates in the development of the solution for reconfiguration. The proposed decentralized solution seeks a reduction in terms of the network reconfiguration time, in case of a single or multiple failures, thus increasing network intelligence. In this purpose, the GHS distributed algorithm is used as a basis for developing a self-healing solution to be embedded in the processing elements that are included within the line commutation switches of smart grid. The proposed solution is implemented using robots as processing units, which communicate via the same network, thereby creating a distributed processing environment. The several tested case studies show that, for smart grids that to have a single distribution feeder, the proposed solution allowed for a successful reconfiguration of the network, regardless of the number of simultaneous failures. In the proposed implementation, the network reconfiguration time does not depend on the number of buses and lines included. The implementation presents results of communication cost and time within the theoretical bounds of the GHS algorithm.

Many Arctic gas hydrate reservoirs such as those of the Prudhoe Bay and Kuparuk River area on the Alaska North Slope (ANS) are believed originally to be natural gas accumulations converted to hydrate after being placed in the gas hydrate stability zone (GHSZ) in response to ancient climate cooling. A mechanistic model is proposed to predict/explain hydrate saturation distribution in “converted free gas” hydrate reservoirs in sub-permafrost formations in the Arctic. This 1-D model assumes that a gas column accumulates and subsequently is converted to hydrate. The processes considered are the volume change during hydrate formation and consequent fluid phase transport within the column, the descent of the base of gas hydrate stability zone through the column, and sedimentological variations with depth. Crucially, the latter enable disconnection of the gas column during hydrate formation, which leads to substantial variation in hydrate saturation distribution. One form of variation observed in Arctic hydrate reservoirs is that zones of very low hydrate saturations are interspersed abruptly between zones of large hydrate saturations. The model was applied on data from Mount Elbert well, a gas hydrate stratigraphic test well drilled in the Milne Point area of the ANS. The model is consistent with observations from the well log and interpretations of seismic anomalies in the area. The model also predicts that a considerable amount of fluid (of order one pore volume of gaseous and/or aqueous phases) must migrate within or into the gas column during hydrate formation. This work offers the first explanatory model of its kind that addresses "converted free gas reservoirs" from a new angle: the effect of volume change during hydrate formation combined with capillary entry pressure variation versus depth. Mechanisms by which the fluid movement, associated with the hydrate formation, could have occurred are also analyzed. As the base of the GHSZ descends through the sediment, hydrate forms within the GHSZ. The net volume reduction associated with hydrate formation creates a “sink” which drives flow of gaseous and aqueous phases to the hydrate formation zone. Flow driven by saturation gradients plays a key role in creating reservoirs of large hydrate saturations, as observed in Mount Elbert. Viscous-dominated pressure-driven flow of gaseous and aqueous phases cannot explain large hydrate saturations originated from large-saturation gas accumulations. The mode of hydrate formation for a wide range of rate of hydrate formation, rate of descent of the BGHSZ and host sediments characteristics are analyzed and characterized based on dimensionless groups. The proposed transport model is also consistent with field data from hydrate-bearing sand units in Mount Elbert well. Results show that not only the petrophysical properties of the host sediment but also the rate of hydrate formation and the rate of temperature cooling at the surface contribute greatly to the final hydrate saturation profiles. / text

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-08-16T15:26:11Z No. of bitstreams: 2 Dissertação - Rafael do Nascimento Ducas - 2015.pdf: 1199090 bytes, checksum: 197e2d02385d6512ad213fe017f881ae (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Olhe a data na citação: DUCAS, R. N. Análise de métodos alternativos ao uso de animais para a classificação da irritação ocular induzida por corantes têxteis. 2015. 52 f. Dissertação (Mestrado em Ciências Farmacêuticas) - Universidade Federal de Goiás, Goiânia, 2018. on 2018-08-17T11:28:42Z (GMT) / Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-08-20T11:47:43Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Rafael do Nascimento Ducas - 2015.pdf: 1199090 bytes, checksum: 197e2d02385d6512ad213fe017f881ae (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Olhe a data da defesa na citação on 2018-08-21T11:19:58Z (GMT) / Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-08-21T11:36:06Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Rafael do Nascimento Ducas - 2015.pdf: 1199090 bytes, checksum: 197e2d02385d6512ad213fe017f881ae (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-22T12:39:57Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Rafael do Nascimento Ducas - 2015.pdf: 1199090 bytes, checksum: 197e2d02385d6512ad213fe017f881ae (MD5) / Made available in DSpace on 2018-08-22T12:39:57Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Rafael do Nascimento Ducas - 2015.pdf: 1199090 bytes, checksum: 197e2d02385d6512ad213fe017f881ae (MD5) Previous issue date: 2015-05-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The evaluation of the toxicity of chemicals still involves the use of animals. However, due to animal welfare and ethical values, research laboratories and regulatory agencies are working together to develop scientifically valid alternative methods. Currently, the Bovine Cornea Opacity and Permeability Test (BCOP) can be used as an alternative methodology to the Draize Test in the determination of ocular irritation and corrosion. In addition, recent data from the literature show that the association between alternative methods represents a useful tool in the evaluation of these parameters. In this context, the objective of this study was to evaluate the efficacy of the BCOP test and the Short-Term Exposure Test (STE) in determining the ocular irritation of Reactive Orange 16 (RO16) and Reactive Green 19 (RG19) based on the United Nations Globally Harmonized System of Classification (UN GHS) and in accordance with the Occupational Safety and Health Administration - Hazard Communication Standard 29 CFR 1910 (OSHA HCS), as Category 2A (eye irritant), using the in vivo test from Draize. The STE test (OECD 491) was used to evaluate cytotoxicity in SIRC cells and the BCOP (OECD 437) assay for opacity, permeability and bovine cornea histopathology. According to the STE test, the RO16 dye obtained cell viability of 47% at 5% concentration and 51% of cell viability at the concentration of 0.05%. The corange RG19 presented cell viability of 41.5% in the concentration of 5% and 44% of cell viability in the concentration of 0.05%. Thus, the dyes were classified as Category 1 of the GHS. The BCOP assay, however, classified only RG19 dye as GHS Category 1 with IVIS: 112,760. The RO16 dye resulted in an IVIS: 12.027, not being classified by this method. To complement this assay, histopathological analyzes were performed, which allowed the observation of significant loss of epithelial cells, intense cytoplasmic vacuolization and damage to the epithelial structure of the corneas exposed to RG19 dye. Although not classified by the BCOP, the corneas exposed to RO16 showed surface coating losses and cytoplasmic vacuolization. Therefore, the STE test alone was not sufficient to define the categorization of the ocular irritation potential of the dyes in relation to the GHS system, but its association with the BCOP test added by the histomorphometric analysis may be an alternative for the prediction of this effect, investigation of other dyes to confirm this fact. / A avaliação da toxicidade de produtos químicos ainda envolve o uso de animais. No entanto, devido ao bem-estar animal e valores éticos, os laboratórios de pesquisas e as agências regulatórias estão trabalhando juntos no desenvolvimento de métodos alternativos cientificamente válidos. Atualmente, o Ensaio de Opacidade e Permeabilidade da Córnea Bovina (BCOP) pode ser utilizado como metodologia alternativa ao Teste de Draize na determinação da irritação e corrosão ocular. Além disso, dados recentes da literatura demonstram que a associação entre métodos alternativos representa uma ferramenta útil na avaliação destes parâmetros. Neste contexto, o objetivo deste trabalho foi avaliar a eficácia da associação do ensaio BCOP com o Teste de Exposição de Curta Duração (STE) na determinação da irritação ocular dos corantes têxteis Reactive Orange 16 (RO16) e Reactive Green 19 (RG19), classificados com base no Sistema Globalmente Harmonizado de Classificação das Nações Unidas (UN GHS) e de acordo com a Occupational Safety & Health Administration - Hazard Communication Standard 29 CFR 1910 (OSHA HCS), como Categoria 2A (irritante para olhos), utilizando o teste in vivo de Draize. Para tanto, foram utilizados o teste STE (OECD 491) para avaliação da citotoxicidade em células SIRC e o ensaio BCOP (OECD 437) para análise da opacidade, permeabilidade e histopatologia da córnea bovina. De acordo com o teste STE, o corante RO16 obteve viabilidade celular de 47% na concentração de 5% e de 51% de viabilidade celular na concentração de 0,05%. Já o corange RG19 apresentou viabilidade celular de 41,5% na concentração de 5% e 44% de viabilidade celular na concentração de 0,05%. Assim, os corantes foram classificados como Categoria 1 do GHS. Já o ensaio BCOP classificou apenas o corante RG19 como Categoria 1 do GHS com IVIS:112,760. O corante RO16, resultou em um IVIS:12,027, não sendo classificado por este método. Para complementar esse ensaio, foram realizadas análises histopatológicas, o que permitiu observar a perda significativa de células epiteliais, a vacuolização citoplasmática intensa e danos à estrutura epitelial das córneas expostas ao corante RG19. Apesar de não ser classificado pelo BCOP, as córneas expostas ao RO16 apresentaram perdas do revestimento superficial e vacuolização citoplasmática. Portanto, o teste STE sozinho não foi suficiente para definir a categorização do potencial de irritação ocular dos corantes frente ao sistema GHS, mas a sua associação com o ensaio BCOP adicionado da análise histomorfométrica pode ser uma alternativa para a predição desse efeito, sendo necessária a investigação de outros corantes para a confirmação desse fato.

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.

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.