Diversas condições durante as etapas de produção dos alimentos podem levar à introdução de micro-organismos patogênicos nas refeições. Esse fato pode ser relevante nos serviços de alimentação localizados em shopping centers pela limitada área física, número de equipamentos insuficientes e volume de refeições servidas. O objetivo do presente estudo foi avaliar esse tipo de estabelecimento quanto ao cumprimento de boas práticas e verificar a presença de matéria orgânica e contaminação bacteriana residual em refrigeradores que haviam sido submetidos à limpeza de rotina. Vinte estabelecimentos localizados em um shopping center de Porto Alegre foram incluídos no estudo. Em cada estabelecimento foi aplicada a Lista de Verificação em Boas Práticas para Serviços de Alimentação prevista na Portaria nº 78 de 28 de Janeiro de 2009 (CEVS) e foram colhidas três amostras com intervalo mensal da parede interna do refrigerador no dia subsequente à limpeza de rotina. As amostras foram avaliadas pelo sistema Clean Trace (3M), quanto à presença de Listeria sp., Coliformes Totais e Escherichia coli. Os dados resultantes da aplicação da Lista de Verificação apontaram que 55% dos estabelecimentos não atingiram a pontuação mínima (>75%) para serem considerados satisfatórios. Em 95% (57/60) das amostras colhidas nos refrigeradores, demonstrou-se a presença de matéria orgânica residual. Já as análises microbiológicas apontaram ausência de Listeria sp., presença de de Coliformes Totais e E. coli em 8,3% e 1,6% das amostras, respectivamente. Não houve associação entre a classificação do estabelecimento (satisfatório/insatisfatório) e a presença de matéria orgânica residual ou Coliformes Totais nos refrigeradores. Estes resultados demonstram a necessidade de constante revisão e monitoramento dos processos de higienização de equipamentos em serviços de alimentação. / Several conditions during all stages of food production can lead to the introduction of pathogenic micro-organisms in food. This fact may be relevant in the food service located in shopping malls by limited physical area, insufficient number of equipment and volume of meals served. The aim of this study was to evaluate this type of establishment for compliance with good practices and verify the presence of organic matter and residual bacterial contamination in refrigerators that had been submitted to routine cleaning. Twenty establishments located in a shopping center from Porto Alegre were included in the study. In each establishment a Checklist for Good Practice for Food Services, provided by Resoltuion No. 78 published on January 28th 2009 (CEVS), was aplied, and three samples were collected at monthly intervals from the inner wall of the refrigerator on the day following the routine cleaning. The samples were evaluated by the Clean Trace system (3M) for the presence of Listeria sp., Total Coliforms and Escherichia coli. The data resulting from application of the Checklist showed that 55% of establishments didn’t attained the minimum score (> 75%) to be classified satisfactory. From de samples taken on the refrigerator’s inner wall, 95% (57/60) demonstrated the presence of residual organic. Nevertheless, microbiological analyzes showed absence of Listeria sp., and isolation of Total Coliforms and E. coli in 8.3% and 1.6% of the samples, respectively. There was no association between the classification of the establishment (satisfactory / unsatisfactory) and the presence of residual organic matter or total coliform in the refrigerator. The results demonstrate the need for constant review and monitoring and improvement of sanitizing procedures of equipment in food service.

Diversas condições durante as etapas de produção dos alimentos podem levar à introdução de micro-organismos patogênicos nas refeições. Esse fato pode ser relevante nos serviços de alimentação localizados em shopping centers pela limitada área física, número de equipamentos insuficientes e volume de refeições servidas. O objetivo do presente estudo foi avaliar esse tipo de estabelecimento quanto ao cumprimento de boas práticas e verificar a presença de matéria orgânica e contaminação bacteriana residual em refrigeradores que haviam sido submetidos à limpeza de rotina. Vinte estabelecimentos localizados em um shopping center de Porto Alegre foram incluídos no estudo. Em cada estabelecimento foi aplicada a Lista de Verificação em Boas Práticas para Serviços de Alimentação prevista na Portaria nº 78 de 28 de Janeiro de 2009 (CEVS) e foram colhidas três amostras com intervalo mensal da parede interna do refrigerador no dia subsequente à limpeza de rotina. As amostras foram avaliadas pelo sistema Clean Trace (3M), quanto à presença de Listeria sp., Coliformes Totais e Escherichia coli. Os dados resultantes da aplicação da Lista de Verificação apontaram que 55% dos estabelecimentos não atingiram a pontuação mínima (>75%) para serem considerados satisfatórios. Em 95% (57/60) das amostras colhidas nos refrigeradores, demonstrou-se a presença de matéria orgânica residual. Já as análises microbiológicas apontaram ausência de Listeria sp., presença de de Coliformes Totais e E. coli em 8,3% e 1,6% das amostras, respectivamente. Não houve associação entre a classificação do estabelecimento (satisfatório/insatisfatório) e a presença de matéria orgânica residual ou Coliformes Totais nos refrigeradores. Estes resultados demonstram a necessidade de constante revisão e monitoramento dos processos de higienização de equipamentos em serviços de alimentação. / Several conditions during all stages of food production can lead to the introduction of pathogenic micro-organisms in food. This fact may be relevant in the food service located in shopping malls by limited physical area, insufficient number of equipment and volume of meals served. The aim of this study was to evaluate this type of establishment for compliance with good practices and verify the presence of organic matter and residual bacterial contamination in refrigerators that had been submitted to routine cleaning. Twenty establishments located in a shopping center from Porto Alegre were included in the study. In each establishment a Checklist for Good Practice for Food Services, provided by Resoltuion No. 78 published on January 28th 2009 (CEVS), was aplied, and three samples were collected at monthly intervals from the inner wall of the refrigerator on the day following the routine cleaning. The samples were evaluated by the Clean Trace system (3M) for the presence of Listeria sp., Total Coliforms and Escherichia coli. The data resulting from application of the Checklist showed that 55% of establishments didn’t attained the minimum score (> 75%) to be classified satisfactory. From de samples taken on the refrigerator’s inner wall, 95% (57/60) demonstrated the presence of residual organic. Nevertheless, microbiological analyzes showed absence of Listeria sp., and isolation of Total Coliforms and E. coli in 8.3% and 1.6% of the samples, respectively. There was no association between the classification of the establishment (satisfactory / unsatisfactory) and the presence of residual organic matter or total coliform in the refrigerator. The results demonstrate the need for constant review and monitoring and improvement of sanitizing procedures of equipment in food service.

Diversas condições durante as etapas de produção dos alimentos podem levar à introdução de micro-organismos patogênicos nas refeições. Esse fato pode ser relevante nos serviços de alimentação localizados em shopping centers pela limitada área física, número de equipamentos insuficientes e volume de refeições servidas. O objetivo do presente estudo foi avaliar esse tipo de estabelecimento quanto ao cumprimento de boas práticas e verificar a presença de matéria orgânica e contaminação bacteriana residual em refrigeradores que haviam sido submetidos à limpeza de rotina. Vinte estabelecimentos localizados em um shopping center de Porto Alegre foram incluídos no estudo. Em cada estabelecimento foi aplicada a Lista de Verificação em Boas Práticas para Serviços de Alimentação prevista na Portaria nº 78 de 28 de Janeiro de 2009 (CEVS) e foram colhidas três amostras com intervalo mensal da parede interna do refrigerador no dia subsequente à limpeza de rotina. As amostras foram avaliadas pelo sistema Clean Trace (3M), quanto à presença de Listeria sp., Coliformes Totais e Escherichia coli. Os dados resultantes da aplicação da Lista de Verificação apontaram que 55% dos estabelecimentos não atingiram a pontuação mínima (>75%) para serem considerados satisfatórios. Em 95% (57/60) das amostras colhidas nos refrigeradores, demonstrou-se a presença de matéria orgânica residual. Já as análises microbiológicas apontaram ausência de Listeria sp., presença de de Coliformes Totais e E. coli em 8,3% e 1,6% das amostras, respectivamente. Não houve associação entre a classificação do estabelecimento (satisfatório/insatisfatório) e a presença de matéria orgânica residual ou Coliformes Totais nos refrigeradores. Estes resultados demonstram a necessidade de constante revisão e monitoramento dos processos de higienização de equipamentos em serviços de alimentação. / Several conditions during all stages of food production can lead to the introduction of pathogenic micro-organisms in food. This fact may be relevant in the food service located in shopping malls by limited physical area, insufficient number of equipment and volume of meals served. The aim of this study was to evaluate this type of establishment for compliance with good practices and verify the presence of organic matter and residual bacterial contamination in refrigerators that had been submitted to routine cleaning. Twenty establishments located in a shopping center from Porto Alegre were included in the study. In each establishment a Checklist for Good Practice for Food Services, provided by Resoltuion No. 78 published on January 28th 2009 (CEVS), was aplied, and three samples were collected at monthly intervals from the inner wall of the refrigerator on the day following the routine cleaning. The samples were evaluated by the Clean Trace system (3M) for the presence of Listeria sp., Total Coliforms and Escherichia coli. The data resulting from application of the Checklist showed that 55% of establishments didn’t attained the minimum score (> 75%) to be classified satisfactory. From de samples taken on the refrigerator’s inner wall, 95% (57/60) demonstrated the presence of residual organic. Nevertheless, microbiological analyzes showed absence of Listeria sp., and isolation of Total Coliforms and E. coli in 8.3% and 1.6% of the samples, respectively. There was no association between the classification of the establishment (satisfactory / unsatisfactory) and the presence of residual organic matter or total coliform in the refrigerator. The results demonstrate the need for constant review and monitoring and improvement of sanitizing procedures of equipment in food service.

Espécies reativas de oxigênio (EROs) são produzidas normalmente durante o metabolismo de organismos aeróbios. Fungos degradadores de lignina geram estas espécies também no processo de degradação da lignina. As espécies de fungos bioluminescentes Gerronema viridilucens e Mycena lucentipes foram utilizadas para se estabelecer possível dependência entre intensidade de bioluminescência, viabilidade celular e atividades das enzimas de defesa antioxidante e ligninolíticas nos micélios e corpos de frutificação. Verificou-se o efeito de espécies causadoras de estresse químico (metais e fenóis) na emissão de luz, viabilidade celular, defesas antioxidantes e enzimas de respiração celular no fungo bioluminescente G. viridilucens, com o objetivo de conectar a inibição da bioluminescência com danos oxidativos ao organismo. Constatou-se que diferentes espécies de fungos bioluminescentes podem apresentar características diferentes quanto à emissão de luz e proteção antioxidante. Diferenças na intensidade e variação temporal da emissão de luz de diferentes espécies foram observadas nos corpos de frutificação e também no micélio. Isto foi revelado pela irregularidade do perfil de luz reprodutível para a espécie M. lucentipes, ao contrário do observado para G. viridilucens. A viabilidade celular de ambas as espécies varia com o tempo, sendo que no caso de G. viridilucens seu perfil é similar ao da bioluminescência. Os ensaios enzimáticos indicam maior atividade no micélio dos fungos do que nos corpos de frutificação, provavelmente pela função específica reprodutora do corpo de frutificação, enquanto a atividade metabólica do fungo está concentrada no micélio. As enzimas ligninolíticas também apresentam atividade baixa nas culturas estudadas, provavelmente por serem enzimas de degradação extracelulares. O fato de ambas viabilidade celular e bioluminescência do micélio serem reduzidas na presença dos metais (cobre e cádmio) e fenóis (fenol e 2,4,6-triclorofenol) testados atesta a interrelação entre atividade luminogênica e injúria oxidativa aos fungos. Os metais parecem afetar mais negativamente as defesas antioxidantes dos fungos do que os fenóis, os quais possivelmente são eliminados pela atividade da glutationa S-transferase (GST), sem também afetar as demais defesas antioxidantes. No conjunto, estes resultados possibilitam estabelecer uma relação metabólica entre abatimento da bioluminescência e as defesas antioxidantes do organismo. No caso dos metais, os sistemas de defesa antioxidante envolvendo a glutationa são bastante importantes, tanto para eliminar peróxidos produzidos na presença de cobre, como na quelação de cádmio pela glutationa. Sob condições normais, a bioluminescência, defesas antioxidantes e respiração celular do organismo estariam funcionando e o NAD(P)H seria mobilizado por todos estes sistemas. Quando o fungo é submetido ao estresse químico, o fluxo de NAD(P)H seria desviado da bioluminescência para sustentar prioritariamente as defesas antioxidantes e a respiração celular, essenciais para a proteção, manutenção e reprodução do organismo / Reactive Oxygen Species (ROS) are normally produced during the metabolism of aerobic organisms. Ligninolitic fungi also produce these oxidizing species also during the lignin degradation process. The bioluminescent species Gerronema viridilucens and Mycena lucentipes were studied aiming to establish a correlation between the temporal profiles of bioluminescence, cellular viability and antioxidant defense enzymes and ligninolitic enzymes in mycelium and fruiting bodies. Chemical toxicants such as metals and phenols were here found to affect light emission, cellular viability, antioxidant defenses and cellular respiration enzymes when administered to G. viridilucens, thereby attesting a metabolic connection between bioluminescence inhibition and fungal oxidative damage. Different species of fungi exhibit different characteristics linked to light emission and antioxidant defenses. Differences in light emission displayed by different species do not resume to fruiting bodies, but the light profile and intensity can also vary in the mycelia. This may explain the irreproducibility of the light profile from M. lucentipes, differently to that observed with G. viridilucens. The cellular viability of both species varies with time, G. viridilucens profile being similar to the time course of bioluminescence. The enzymatic data pointed to higher activities in mycelium than in fruiting bodies, probably due to a main reproductive function of the latter, whereas the metabolic activities are prevalent in the mycelium. Ligninolytic enzymes exhibit low activities in the extracts of fungus samples, probably because they are extracellular degradation enzymes. The inhibition effect of phenols and metals (copper and cadmium) on mycelium viability reinforces the notion that cellular oxidative damage hampers bioluminescence emission. Redox active (copper) and heavy metals (cadmium) were found to display higher impact on antioxidant defenses than phenols (phenol and 2,4,6-trichlorophenol), which are expected to be promptly metabolized and excluded by principally glutathione S-transferase (GST). Notably, a metabolic correlation between bioluminescence inhibition and antioxidant defenses was unveiled by the present work. Regarding the metals, glutathione was found to be a crucial antioxidant, both to eliminate peroxides when in the presence of copper, and to act as a cadmium chelating agent. Under normal conditions, the bioluminescence system, the antioxidant defenses and the cellular respiration sets cooperate through the common demand of reducing power of NAD(P)H. Under chemical stress, the NAD(P)H flux would be deviated from bioluminescence, to principally sustain the antioxidant defenses and cellular respiration, essential for the protection, maintenance and reproduction of the organism.

Scientific evidence that the ocean plastic crisis is larger in scale and more sinister than previously thought continues to mount, but the rate of plastic production is only rising. What will it take to decisively turn the tide against plastic? We need scientists, politicians, and industry changemakers to continue producing knowledge and positive change in the industry, but we need to go further still. This thesis explores art as an alternative visual communication strategy with the capacity to encourage curiosity, empathy, and positive engagement with the issue of ocean plastics. The series of work explores bacterial bioluminescence as an artistic medium in juxtaposition with objects of found ocean plastic. The photographs in the series build on the concepts of mutualism, illumination, critical densities, and interspecies communication to reimagine how we might further the discourse around ocean plastic.

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradia- tion (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell inva- sion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic.

No description available.

Plusieurs types cancéreux prolifèrent par leur capacité à exprimer les marqueurs de régulation négative du système immunitaire, tels que les récepteurs PD-L1 et CD80/86 qui inhibent l’activation et la prolifération des lymphocytes T. L’inhibition de ces voies par des anticorps peut ainsi réactiver la réponse immunitaire chez certains patients. D’autres voies de signalisations sont aujourd’hui explorées, incluant la signalisation purinergique (ATP/adénosine) dans la modulation du microenvironnement tumoral. L’adénosine triphosphate extracellulaire (ATPe) est classifiée parmi les molécules de danger extracellulaire et joue un rôle crucial dans l’activation de l’inflammasome NLRP3, un médiateur important de l’activation des réactions pro-inflammatoires. Les ultrasons sont des ondes mécaniques de haute pression capable d’engendrer la cavitation inertielle des microbulles. Il a été démontré que les microbulles (MB) stimulées par ultrasons (US) libèrent de l’ATP dans le muscle squelettique et dans le muscle cardiaque. Nous posons l’hypothèse selon laquelle le traitement US+MB appliqué sur une tumeur de cancer du sein murin (4T1) in vivo peut libérer de l’ATPe localement dans le but d’activer des réactions pro-inflammatoires pour l’immunothérapie du cancer. Dans ce mémoire, nous présentons la quantification du signal d’ATPe d’une culture de cellules 4T1, puis in vivo dans le muscle et dans une tumeur solide sous-cutanée chez la souris à la suite d’une stimulation par US+MB. Nos études démontrent que la thérapie US+MB libère de l’ATP in vitro et in vivo. En comparant le signal découlant de l’injection IM d’ATP avec celui du muscle et des tumeurs post-US+MB, nous pouvons conclure que le traitement US+MB libère une quantité d’ATPe supérieure à 250 µM, ce qui est supérieur à la quantité d’ATPe dans un microenvironnement tumoral et qui persiste pour une durée d’au moins 60 min dans le muscle et 45 min dans la tumeur. La transfection stable de cellules MC38 (carcinome colorectal) à travers le gène PLenti-PmeLUC, codant la synthèse de luciférase sur la face externe de la membrane cellulaire, est explorée afin d’augmenter le rapport signal sur bruit en bioluminescence (annexe A). L’utilisation de POM-1 (inhibiteur pharmacologique de CD39) et l’utilisation de souris knockout du gène CD39 sont discutées pour la suite du projet afin d’inhiber la dégradation de l’ATP extracellulaire (Annexe B). / Several cancer types proliferate due to their ability to express the negative regulatory markers of the immune system (PD-L1 and CD80/86) which inhibit the activation and proliferation of T cells. Inhibition of these pathways by antibodies (anti-PDL-1, anti-PD-1, anti-CTLA-4) can thus reactivate the immune system in some patients. Other signaling pathways are currently being explored, including purinergic signaling (ATP/adenosine) in the modulation of the tumor microenvironment. Extracellular Adenosine triphosphate (eATP) is classified as danger signal plays a critical role in the activation of the NLRP3 inflammasome, an important mediator of the innate immune response. Ultrasound (US) and microbubbles (MB) have been shown to release ATP in skeletal and cardiac muscle. Thus, we hypothesized that US+MB treatment in 4T1 breast cancer cells could locally activate pro-inflammatory responses by releasing an eATP in tumors for cancer immunotherapy. In this thesis, I present the quantification of the eATP signal after US+MB stimulation in vitro (4T1 cell culture), then in muscle and subcutaneous solid tumors in the mouse. Our studies demonstrate that US+MB treatment releases ATP both in vitro and in vivo. In comparison with the IM injection of ATP, we can conclude that US+MB released a large amount of ATP (>250 µM), which is more than the eATP concentration in the untreated tumor microenvironment, and which persisted for at least 60 min in muscle and 45 min in tumor. The stable transfection of MC38 cells (colorectal carcinoma) through the Plenti-PmeLUC gene, encoding the synthesis of luciferase on the external surface of cell membrane is explored to increase the signal to noise ratio in bioluminescence (see appendix A). The use of POM-1 (pharmacological inhibitor of CD39) and CD39 gene knockout mice to inhibit the degradation of eATP signal are discussed for the continuation of the project.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / L'algue unicellulaire Gonyaulax polyedra est notre modèle pour l'étude des mécanismes reliant l'horloge interne aux rythmes circadiens observés. La bioluminescence, présente lors de la phase de nuit, ainsi que la division cellulaire observée au début de la phase de jour sont les deux rythmes sur lesquels porte la présente étude. Chez Gonyaulax, la présence des protéines LBP et luciférase, limitée à la phase obscure, permet la production de lumière. Une régulation au niveau de la traduction par une protéine inhibitrice CCTR se fixant en région 3 de l'ARNm LBP permet l'expression cyclique de la protéine LBP in vivo chez Gonyaulax polyedra . La construction d'une banque d'ADNc dans un vecteur d'expression constitutive (le p425 GPD) représente l'outil nécessaire pour l'isolation d'un ADNc codant pour la protéine CCTR. Cette isolation repose sur un mécanisme moléculaire d'interaction in vivo de la protéine CCTR avec la région régulatrice de l'ARNm lbp annexée à un gène létal inductible (le gène GSP1) dans la construction du plasmide pCS7. La transformation de levure possédant le pCS7 avec la banque d'ADNc permettrait la production de CCTR actif dans la levure et ainsi l'inhibition de la traduction de l'ARNm GSP1 par fixation du CCTR à la région régulatrice de lbp. Le criblage d'une banque d'ADNc comportant 2,8 x 105clones a été effectué. La croissance de 127 colonies a été observée lors du premier criblage de la banque. Chaque colonie a été soumise à une extraction d'ADN plasmidique et les plasmides obtenus ont été utilisés pour une seconde transformation dans les levures porteuses du pCS7. Le second criblage nous a permis de constater qu'aucun de ces positifs ne peut produire une protéine CCTR fonctionnelle. Une contamination par des levures d'une autre souche ou par une source de carbone permettant la croissance (telle que le glucose), une présence de "révertants" ou le clonage d'un ADNc codant pour le facteur de sélection du pCS7 peuvent être à l'origine de ces "faux positifs" obtenus. La production de clones supplémentaires afin d'augmenter statistiquement les chances de représentation d'un gène impliqué dans la génération d'un rythme circadien, ainsi que la transformation répétée de la banque dans les mêmes cellules pour permettre de réunir les différentes composantes qui pourraient être nécessaires à la formation d'un CCTR actif, pourraient permettre d'isoler un ADNc codant pour une protéine CCTR active. Chez la majorité des dinoflagellés on observe la présence d'une phase S dans le cycle cellulaire. De plus, chez Gonyaulax, la division cellulaire est limitée à l'aurore. Il est donc probable que la protéine kinase p34œk2qui régule la formation de l'hétérodimère MPF (impliqué dans l'initiation de la division cellulaire) soit également sujette à une régulation par l'horloge circadienne. L'isolation d'un homologue de la protéine kinase p34ede2repose sur une technique de clonage par compensation d'une mutation. Les levures de la souche cdc28 ne peuvent se diviser normalement à température restrictive de 34°C à cause d'une mutation thermosensible de la protéine kinase p34edc2. La transformation de la banque d'ADNc construite dans des levures de cette souche, puis l'incubation à température restrictive, permet le criblage pour un homologue de la p34cdc2. Le premier criblage de la banque a permis la croissance de sept colonies qui ont toutes été soumises à une extraction d'ADN plasmidique puis à une retransformation dans les cellules de la souche cdc28. Aucun des positifs n'a franchi le seuil de ce second test. Le criblage de clones supplémentaires ou encore le criblage pour un homologue de la cycline (la seconde composante du MPF) pourrait permettre d'isoler directement ou indirectement l'homologue de la p34cdc2.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de protéines membranaires du génome humain. Ils transmettent les signaux extracellulaires provenant de plusieurs stimuli comme les odeurs, les ions, les hormones et les neurotransmetteurs, à l'intérieur des cellules. En se liant aux RCPGs, ces molécules contribuent à la stabilisation des changements conformationnels activateurs qui se propagent jusqu'au domaine intracellulaire des récepteurs. Ces derniers engagent ensuite un ou plusieurs effecteurs, comme les protéines G hétérotrimériques et les β-arrestines (βarrs), qui activent une cascade d'événements moléculaires menant à la réponse cellulaire.Récemment, la publication de structures cristallines de RCPGs liant des ligands diffusibles a offert une opportunité de raffiner à une résolution atomique les modèles des mécanismes de transduction des signaux. Dans la première partie de cette thèse, nous avons donc exploré les déterminants de la signalisation du récepteur prototypique β2-adrénergique (β2AR), induite par les β-bloqueurs. En ne tenant compte que de leur efficacités sur le β2AR dans les voies de l'adénylate cyclase (AC) et des protéines kinases activées par les facteurs mitogéniques (MAPK), les β-bloqueurs peuvent être classés en 3 groupes distincts (agoniste inverse AC / agoniste MAPK, antagoniste neutre AC / agoniste MAPK et agoniste inverse AC / agoniste inverse MAPK). Afin de déterminer le lien entre leur efficacité et leur mode de liaison, nous avons réalisé des expériences d'arrimages moléculaires in silico entre des β-bloqueurs de chacun des groupes et la structure cristalline du β2AR liée au carazolol. De manière intéressante, les ligands à l'intérieur d'un groupe partagent un mode de liaison, alors que ceux des ligands entre les groupes divergent, suggérant que le mode de liaison des β-bloqueurs pourrait être utilisé pour prédire leur l'efficacité. En accord avec cette hypothèse, nous avons prédit et confirmé l'efficacité agoniste MAPK du carazolol, un inverse agoniste AC du β2AR se liant au récepteur de manière similaire au groupe inverse agoniste AC / agoniste MAPK. De manière intéressante, le groupement aryl des ligands agonistes inverses agonistes AC / agoniste MAPK, le seul groupement chimique variable de ce groupe, est prédite pour lier la région des 3e et 5e hélices transmembranaires (TM3 et TM5). Nous avons donc émis l'hypothèse que cette région pourrait être un déterminant de l'efficacité de ces ligands. En accord avec cette dernière, la mutation de 2 résidus (T118I, S203A) localisés proches du site de liaison des groupements aryls des β-bloqueurs, prévient l'efficacité agoniste inverse de l'ICI-118551 sur la voie de l'AC sans affecter l'efficacité d'un agoniste, indiquant que cette région est importante pour la transmission de l'effet agoniste inverse, du moins sur la voie de l'AC. Les βarrs sont des protéines d'échafaudage qui coordonnent la formation de complexes avec plusieurs dizaines d'effecteurs de signalisation. Originalement identifiées pour leur rôle dans la désensibilisation et l'internalisation des RCPGs, elles sont aussi d'importants effecteurs de la signalisation des RCPGs indépendante des protéines G hétérotrimériques. Cependant, contrairement aux protéines G hétérotrimériques, il n'existe que peu d'outils pour les étudier. Ainsi, la deuxième partie de la thèse est dédiée au développement d'outils pour l'étude des βarrs. À cette fin, nous avons d'abord tenté de transposer une méthode de mesure de l'interaction entre 2 protéines par la technologie de transfert d'énergie de bioluminescence par résonance (BRET) en microscopie et chez des souris transgéniques afin de mesurer de manière subcellulaire et dans un contexte natif l'engagement de la βarr à des RCPGs. Ainsi, nous avons établi les preuves de principe que le BRET peut être utilisé pour localiser l'interaction entre la βarr et le récepteur de la vasopressine de type 2 (V2R) sur une cellule au microscope et pour détecter l'interaction entre la βarr et le β2AR sur des tissus de souris transgéniques exprimant ces protéines fusionnées avec des partenaires BRET. Finalement, il n'existe aucun inhibiteur pharmacologique ciblant les βarrs. Ainsi, grâce à la combinaison d'approches de criblage virtuel sur un modèle de la structure des βarrs et d'essais de validation cellulaire, nous avons développé un inhibiteur pharmacologique des βarrs. À l'aide de cet outil, nous avons confirmé l'implication des βarrs dans l'activation des MAPK par le V2R, mais aussi montré un nouveau rôle des βarrs dans le recyclage du β2AR. Les connaissances et outils développés dans cette thèse permettront de mieux comprendre les déterminants moléculaires de la signalisation des RCPGs et entre autres, grâce à des nouvelles approches pour étudier le rôle cellulaire et physiologique des βarrs. / G Protein-Coupled Receptors (GPCR) are members of the largest family of membrane protein in the human genome. They transduce the signal from a variety of stimuli like odors, ions, hormones and neurotransmitters, inside the cells. By binding directly to the receptors, these molecules stabilize activating conformational changes that are allosterically propagated through transmembrane to intracellular domains. Effectors like heterotrimeric G protein and β-arrestins (βarrs) are then engaged by activated receptors and trigger a cascade of signalling events leading to a cellular response. Recently, the resolution of the crystal structure of GPCR that bind to freely diffusible ligands provided the opportunity to refine at an atomic level the models describing the mecanisms of receptor signal transduction. In the first section of this thesis, we have explored the determinants of the prototypical β2-adrenergic receptor (β2AR) signalling induced by β-blockers. Given their efficacy on Adenylate Cyclase (AC) and Mitogen-Activated Protein Kinase (MAPK) pathways, β-blockers can be classified within 3 signalling groups (AC inverse agonist / MAPK agonist, AC neutral antagonist / MAPK agonist and inverse agonist for AC and MAPK). In order to gain insight on the relation between their efficacy and binding mode, we performed in silico binding experiments between β-blockers from each group and the β2AR crystal structure bound to carazolol. Interestingly, ligands within a group share similar binding mode in contrast to those of different groups, suggesting that β-blockers binding mode could be used to predict their efficacy. In accordance to this hypothesis, we have predicted and confirmed that carazolol, an AC inverse agonist that bind to β2AR in a similar way than the AC inverse agonist / MAPK agonist group, is indeed an agonist for MAPK pathway. Moreover, aryl chemical function from AC inverse agonist / MAPK agonist ligands, barely the only variable structure feature of this group, was predicted to bind β2AR nearby the transmembrane helices 3 and 5 (TM3 and TM5). We thus have predicted that this region would be a determinant of the AC inverse agonist / MAPK agonist ligand efficacy. Accordingly, we found that mutation of 2 residues (T118I, S203A) close to the aryl moiety binding site prevents inverse agonist efficacy of ICI-118551 on AC pathway, without affecting agonist efficacy, indicating that this receptor region is important for the efficacy of these group of β-blockers, at least on AC inverse agonism.βarrs are scaffolding proteins that coordinate protein complex formation with dozen of signalling effectors. First identified for their role on GPCR desensitization and internalization, βarrs are also an important heterotrimeric G protein independent GPCR signalling effectors. However, in contrast to heterotrimeric G protein, only a few tools are available for their study. Thus, the second section of this thesis aim at developing tools for the study of βarrs. For this purpose, we had attempted to transpose a method to measure protein-protein interaction that use Bioluminescence Resonance Energy Transfer (BRET) technology, in microscopy and in transgenic mice, in order to detect subcellular localization and in a native context the engagement of βarr to RCPGs. Thus, we have established a proof of principle that BRET can be combined with microscopy to locate an interaction between βarr and the type 2 vasopressin receptor (V2R) within a cell. Moreover, we have established a second proof of principle that we can detect βarrs recruitment to β2AR on cells extracted from tissues of transgenic mice expressing these proteins fused to BRET partner. Finally, there is no pharmacological inhibitor of βarrs. Thus, using a combination of virtual screening and cellular validation approches, we have developed the first pharmacological βarrs inhibitor. With this novel tool, we have confirmed the implication of βarrs in V2R-mediated MAPK activation, but also showed a new role of βarrs in β2AR recycling.The finding and the tools presented in this thesis should allow to better understand the molecular determinants of GPCR signalling, and among other things, by proposing new tools to study βarrs cellular and physiological roles.