Conselho Nacional de Desenvolvimento Científico e Tecnológico / Patrick Bateman, o protagonista narrador do romance American Psycho (1991), de Bret Easton Ellis, confunde por ser rico, bonito e educado e, ao mesmo tempo, torturador, assassino e canibal. Mas esta personalidade antagônica não o torna singular. O que o particulariza são as quatro faces que ele apresenta ao longo de sua narrativa: (1) ele consome mercadorias e humanos, (2) compete para ter reconhecimento, (3) provoca horror por suas ações, e (4) não é um narrador confiável. Sendo um yuppie (termo popular usado nos Estados Unidos na década de 1980 para denominar jovens e bem sucedidos profissionais urbanos), Bateman é materialista e hedonista. Ele está imerso em uma sociedade de consumo, fato que o impossibilita de perceber diferenças entre produtos e pessoas. Sendo um narcisista, ele se torna um competidor em busca de admiração. No entanto, Bateman também é um serial killer e suas descrições detalhadas de torturas e assassinatos horrorizam. Por fim, nós leitores duvidamos de sua narrativa ao notarmos inconsistências e ambiguidades. Zygmunt Bauman (2009) afirma que uma sociedade extremamente capitalista transforma tudo que nela existe em algo consumível. Christopher Lasch (1991) afirma que o lendário Narciso deu lugar a um novo, controverso, dependente e menos confiante. A maioria das vítimas de Bateman são membros de grupos socialmente marginalizados, como mendigos, homossexuais, imigrantes e prostitutas, o que o torna uma identidade predatória, segundo Arjun Appadurai (2006). A voz autodiegética e a narrativa incongruente do protagonista, contudo, impedem que confiemos em suas palavras. Estas são as quatro faces que pretendo apresentar deste serial killer / The autodiegetic protagonist Patrick Bateman, in Bret Easton Elliss American Psycho (1991), is a troubling character, for he is highly-educated, wealthy and handsome as well as a torturer, a killer and a cannibal. This antagonistic behavior, nonetheless, does not make him a singular character. The four sides he presents throughout the novel are singular, though: (1) he consumes humans and commodities equally; (2) he competes for recognition and admiration; (3) his acts are horrific; and (4) his narration is unreliable. As a yuppie (a popular term from the 1980s used to define young urban U.S. professionals), Bateman is materialistic and hedonistic. As he lives off the excesses of a consumer society, he is incapable of distinguishing people from products. As a self-absorbed, narcissistic protagonist, he becomes a competitor struggling to get approval from his peers. Nevertheless, Bateman is a serial killer, and his detailed descriptions of tortures and murders are horrifying. Finally, we readers cannot rely on his narrative once we notice ambiguities and divergences. Zygmunt Bauman (2009) posits that an extremely capitalist society forces people to be commodified. Christopher Lasch (1991) asseverates that the old legendary Narcissus gave birth to a new one, paradoxical, dependent and less confident. Most of Batemans victims are socially-marginalized characters, members of minority groups, such as homeless people, homosexuals, immigrants, and prostitutes. As a matter of fact, Bateman may be regarded as having a predatory identity, as defined by Arjun Appadurai (2006). However, this autodiegetic narrator, together with his inconsistent narrative, cannot be entirely trusted. These are the points I want to debate regarding this fourfold serial killer

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No. of bitstreams: 1 LailaDaSilvaAsthFernandes_TESE.pdf: 3019441 bytes, checksum: b9d834e9c1a7ede2d053a98c33bde78e (MD5) Previous issue date: 2016-01-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Introdu??o: Este trabalho investigou os efeitos de dois agonistas parciais pept?dicos, UFP-113 e [F/G]N/OFQ(1-13)NH2, e um agonista parcial n?o pept?dico, AT-090, do receptor NOP no comportamento emocional de camundongos, bem como as vias de transdu??o do sinal decorrentes da liga??o destas mol?culas com o receptor NOP. M?todos: Foram utilizados camundongos machos, das linhagens Swiss e CD-1, al?m dos nocautes para o receptor NOP (NOP(-/-)) e seus controles selvagens NOP(+/+). O labirinto em cruz elevado (LCE) foi utilizado para avaliar o efeito dos compostos sobre a ansiedade. O diazepam e os agonistas do receptor NOP, N/OFQ e Ro 65-6570, foram utilizados como controles positivos no LCE. Os camundongos NOP(+/+) e NOP(-/-) foram utilizados na avalia??o da seletividade de a??o dos compostos com efeito do tipo ansiol?tico. O teste da nata??o for?ada (TNF) foi utilizado a fim de se avaliar os efeitos dos compostos sobre o comportamento do tipo depressivo. A nortriptilina e os antagonistas do receptor NOP, UFP-101 e SB-612111, foram utilizados como controles positivos no TNF. As a??es da N/OFQ, UFP-101, SB-612111, UFP-113, [F/G]N/OFQ(1-13)NH2 e AT-090 foram ainda avaliadas no teste de hiperlocomo??o induzida pelo metilfenidato (HIM), onde o valproato foi utilizado como controle positivo. A influ?ncia do UFP-113 e [F/G]N/OFQ(1-13)NH2 na atividade locomotora foi testada no campo aberto. As vias de transdu??o do sinal (prote?na G e ?-arrestina 2) dos agonistas (N/OFQ e Ro 65-6570), do antagonista (UFP-101) e dos agonistas parciais (UFP-113, [F/G]N/OFQ(1-13)NH2 e AT-090) do receptor NOP foram investigadas por meio da avalia??o da transfer?ncia de energia por resson?ncia de bioluminesc?ncia (BRET). Para isso, foram usadas c?lulas co-expressando o receptor NOP acoplado ? luciferase (doador de energia), e a prote?na verde fluorescente (aceptor de energia) acoplada a uma das prote?nas efetoras: prote?na G ou a ?-arrestina 2. Resultados: Diazepam (1 mg/kg), N/OFQ (1 nmol), Ro 65-6570 (0,1 mg/kg) e AT-090 (0,01 mg/kg) apresentaram efeito do tipo ansiol?tico no LCE. Os efeitos do Ro 65-6570 e do AT-090 foram devidos a ativa??o seletiva do receptor NOP, uma vez que ambos foram inativos em camundongos NOP(-/-) expostos ao LCE. Em contraste, UFP-113 e [F/G]N/OFQ(1-13)NH2 foram inativos no LCE. No TNF, nortriptilina (30 mg/kg), UFP-101 (10 nmol), SB-612111 (10 mg/kg), UFP-113 (0,01 e 0,1 nmol) e [F/G]N/OFQ(1-13)NH2 (0,3 e 1 nmol) apresentaram efeito do tipo antidepressivo, diferentemente do AT-090, que foi inativo neste teste. Os efeitos do UFP-113 e do [F/G]N/OFQ(1-13)NH2 foram devidos a ativa??o seletiva do receptor NOP, uma vez que o pr?-tratamento com N/OFQ preveniu o efeito do tipo antidepressivo de ambos. O metilfenidato (MF, 10 mg/kg) induziu hiperlocomo??o nos camundongos expostos ao campo aberto, que foi prevenida pelo valproato (400 mg/kg). A N/OFQ (1 nmol), assim como UFP-113 (0,01-0,1 nmol) e [F/G]N/OFQ(1-13)NH2 (1 nmol), foram capazes em reduzir a hiperlocomo??o induzida pelo MF, sem alterar a locomo??o per se. O efeito do UFP-113 decorreu da ativa??o seletiva do receptor NOP, uma vez que foi inativo em camundongos NOP(-/-) expostos ao teste da HIM. Em contraste, o UFP-101 (10 nmol), assim como SB-612111 (10 mg/kg) e AT-090 (0,001-0,03 mg/kg) n?o alteraram o aumento na locomo??o induzido pelo MF. Tanto o UFP-113 quanto o [F/G]N/OFQ(1-13)NH2 induziram hipolocomo??o nas maiores doses testadas (1 e 3 nmol, respectivamente). In vitro, tanto a N/OFQ quanto o Ro 65-6570, promoveram a intera??o do receptor NOP com a prote?na G e com a ?-arrestina 2 de forma concentra??o-dependente, comportando-se como agonistas plenos do receptor NOP em ambas as vias de transdu??o. O AT-090, UFP-113 e [F/G]N/OFQ(1-13)NH2 promoveram a intera??o do receptor NOP com a prote?na G com efeitos m?ximos significativamente reduzidos em rela??o a N/OFQ. O AT-090 foi capaz de induzir o recrutamento da ?-arrestina 2 novamente com efeitos m?ximos reduzidos em rela??o a N/OFQ, enquanto o UFP-113 e o [F/G]N/OFQ(1-13)NH2 falharam em induzir o recrutamento da ?-arrestina 2. Portanto, AT-090 se comportou como agonista parcial em ambas as vias de transdu??o, enquanto UFP-113 e [F/G]N/OFQ(1-13)NH2 se comportaram como agonistas parciais e antagonistas, respectivamente, nas vias de transdu??o da prote?na G e da ?-arrestina 2. O UFP-101 n?o promoveu o recrutamento da prote?na G, nem da ?-arrestina 2, se comportando como antagonista do receptor NOP em ambas as vias de transdu??o. Conclus?o: Ligantes do receptor NOP que produzem o mesmo efeito na intera??o do receptor NOP com a prote?na G (agonismo parcial), s?o capazes de induzir efeitos opostos no recrutamento da ?-arrestina 2 (agonismo parcial vs antagonismo). Essas diferen?as no recrutamento da ?-arrestina 2 podem promover efeitos distintos sobre a ansiedade e o humor, como foi verificado nos testes comportamentais. Este trabalho corrobora o potencial do receptor NOP como uma ferramenta farmacol?gica inovadora no tratamento de transtornos emocionais. / Introduction: This study aimed to investigate the effects of the two peptide NOP partial agonists (UFP-113 and [F/G]N/OFQ(1-13)NH2) and the non peptide NOP partial agonist (AT-090) in the mouse emotional behavior as well as in the intracellular transduction pathways following the receptor binding. Methods: Male Swiss or CD-1 mice were used in this study together with NOP(+/+) and NOP(-/-) mice. The elevated plus maze (EPM) was used to evaluate the effects of compounds on anxiety-like behaviors. Diazepam and the NOP agonists, N/OFQ and Ro 65-6570, were used as positive controls in the EPM. NOP(+/+) and NOP(-/-) mice were used to evaluate the selectivity of those compounds that induced anxiolytic-like behaviors. The forced swim test (FST) was used to evaluate the effects of compounds on depressive-like behaviors. Nortriptyline and the NOP antagonists, UFP-101 and SB-612111, were used as positive controls in the FST. The effects of N/OFQ, UFP-101, SB-612111, UFP-113, [F/G]N/OFQ(1-13)NH2, and AT-090 were assessed in the methylphenidate-induced hyperlocomotion (MIH) test; in this assay valproate was used as positive control. The G protein and ?-arrestin 2 transduction pathways of NOP receptor agonists (N/OFQ and Ro 65-6570), antagonist (UFP-101), and partial agonists (UFP-113, [F/G]N/OFQ(1-13)NH2, and AT-090) were also evaluated using an innovative assay that measures a bioluminescence resonance energy transfer process. For this, cell lines permanently co-expressing the NOP receptor coupled to luciferase (energy donor), and green fluorescent protein (energy acceptor) coupled to one of the effector proteins (G protein or ?-arrestin 2) were used. Results: Diazepam (1 mg/kg), N/OFQ (1 nmol), Ro 65-6570 (0.1 mg/kg), and AT-090 (0.01 mg/kg) induced anxiolytic-like effect in mice in the EPM. The effects of Ro 65-6570 and AT-090 were selective to NOP receptor. UFP-113 (0.01-1 nmol) and [F/G]N/OFQ(1-13)NH2 (0.1-3 nmol) were inactive in the EPM. In the FST, nortriptyline (30 mg/kg), UFP-101 (10 nmol), SB-612111 (10 mg/kg), UFP-113 (0.01 and 0.1 nmol), and [F/G]N/OFQ(1-13)NH2 (0.3 and 1 nmol) induced antidepressant-like effects, while AT-090 (0.001-0.1 mg/kg) was inactive in this assay. The effects of UFP-113 and [F/G]N/OFQ(1-13)NH2 were selective to NOP receptor. Valproate (400 mg/kg) counteracted methylphenidate (MPH, 10 mg/kg)-induced hyperlocomotion in mice in the open field. N/OFQ (1 nmol), UFP-113 (0.01-0.1 nmol), and [F/G]N/OFQ(1-13)NH2 (1 nmol) were also able to reduce the MPH-induced hyperlocomotion, without changing the locomotor activity per se. The effect of UFP-113 was selective to NOP receptor. The UFP-101 (10 nmol), SB-612111 (10 mg/kg), and AT-090 (0.001-0.03 mg/kg) did not change the hyperlocomotor effect of methylphenidate. In vitro, N/OFQ and Ro 65-6570 behaved as NOP full agonists for G-protein and ?-arrestin 2 pathways. AT-090 behaved as NOP receptor partial agonist for both transduction pathways, while UFP-113 and [F/G]N/OFQ(1-13)NH2 behaved as partial agonists and antagonists of NOP receptor for NOP/G protein and NOP/?-arrestin 2, respectively. UFP-101 behaved as NOP receptor antagonist for both transduction pathways. Conclusion: NOP ligands producing same effects on NOP/G protein interaction (partial agonism), but with opposite effects on ?-arrestin 2 recruitment (partial agonism vs antagonism), can promote different in vivo effects on anxiety and mood as it was observed in the behavioral tests. This work corroborates the potential of NOP receptor as an innovative pharmacological target for the treatment of emotional disorders.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Patrick Bateman, o protagonista narrador do romance American Psycho (1991), de Bret Easton Ellis, confunde por ser rico, bonito e educado e, ao mesmo tempo, torturador, assassino e canibal. Mas esta personalidade antagônica não o torna singular. O que o particulariza são as quatro faces que ele apresenta ao longo de sua narrativa: (1) ele consome mercadorias e humanos, (2) compete para ter reconhecimento, (3) provoca horror por suas ações, e (4) não é um narrador confiável. Sendo um yuppie (termo popular usado nos Estados Unidos na década de 1980 para denominar jovens e bem sucedidos profissionais urbanos), Bateman é materialista e hedonista. Ele está imerso em uma sociedade de consumo, fato que o impossibilita de perceber diferenças entre produtos e pessoas. Sendo um narcisista, ele se torna um competidor em busca de admiração. No entanto, Bateman também é um serial killer e suas descrições detalhadas de torturas e assassinatos horrorizam. Por fim, nós leitores duvidamos de sua narrativa ao notarmos inconsistências e ambiguidades. Zygmunt Bauman (2009) afirma que uma sociedade extremamente capitalista transforma tudo que nela existe em algo consumível. Christopher Lasch (1991) afirma que o lendário Narciso deu lugar a um novo, controverso, dependente e menos confiante. A maioria das vítimas de Bateman são membros de grupos socialmente marginalizados, como mendigos, homossexuais, imigrantes e prostitutas, o que o torna uma identidade predatória, segundo Arjun Appadurai (2006). A voz autodiegética e a narrativa incongruente do protagonista, contudo, impedem que confiemos em suas palavras. Estas são as quatro faces que pretendo apresentar deste serial killer / The autodiegetic protagonist Patrick Bateman, in Bret Easton Elliss American Psycho (1991), is a troubling character, for he is highly-educated, wealthy and handsome as well as a torturer, a killer and a cannibal. This antagonistic behavior, nonetheless, does not make him a singular character. The four sides he presents throughout the novel are singular, though: (1) he consumes humans and commodities equally; (2) he competes for recognition and admiration; (3) his acts are horrific; and (4) his narration is unreliable. As a yuppie (a popular term from the 1980s used to define young urban U.S. professionals), Bateman is materialistic and hedonistic. As he lives off the excesses of a consumer society, he is incapable of distinguishing people from products. As a self-absorbed, narcissistic protagonist, he becomes a competitor struggling to get approval from his peers. Nevertheless, Bateman is a serial killer, and his detailed descriptions of tortures and murders are horrifying. Finally, we readers cannot rely on his narrative once we notice ambiguities and divergences. Zygmunt Bauman (2009) posits that an extremely capitalist society forces people to be commodified. Christopher Lasch (1991) asseverates that the old legendary Narcissus gave birth to a new one, paradoxical, dependent and less confident. Most of Batemans victims are socially-marginalized characters, members of minority groups, such as homeless people, homosexuals, immigrants, and prostitutes. As a matter of fact, Bateman may be regarded as having a predatory identity, as defined by Arjun Appadurai (2006). However, this autodiegetic narrator, together with his inconsistent narrative, cannot be entirely trusted. These are the points I want to debate regarding this fourfold serial killer

Selon le modèle classique, le signal reçu par les récepteurs couplés aux protéines G (RCPG) se propage suite à des interactions transitoires et aléatoires entre les RCPGs, les protéines G et leurs effecteurs. Par les techniques de transfert d’énergie de résonance de bioluminescence (BRET), de complémentation bimoléculaire de protéines fluorescentes (BiFC) et de co-immunoprécipitation, nous avons observé que les récepteurs, les protéines G et les effecteurs forment un complexe stable, avant et après l’activation des récepteurs. L’interaction entre l’effecteur Kir3 et le dimère Gbetagamma se produit initialement au réticulum endoplasmique et est sensible à un agoniste liposoluble des récepteurs beta2-adrénergiques. Bien que peu de spécificité pour les nombreux isoformes des sous-unités Gbetagamma ait été observée pour l’activation du canal Kir3, les interactions précoces au RE sont plus sensibles aux différentes combinaisons de Gbetagamma présentes. En plus de son rôle dans la régulation des effecteurs, le dimère Gbetagamma peut interagir avec de nombreuses protéines possédant des localisations cellulaires autres que la membrane plasmique. Nous avons identifié une nouvelle classe de protéines interagissant avec la sous-unité Gbeta, autant en système de surexpression que dans des extraits de cerveaux de rats, soit les protéines FosB et cFos, qui forment le complexe de transcription AP-1, suite à leur dimérisation avec les protéines de la famille des Jun. La coexpression du dimère Gbetagamma réduit l’activité transcriptionnelle du complexe AP-1 induit par le phorbol 12-,myristate 13-acetate (PMA), sans toutefois interférer avec la formation du complexe Fos/Jun ou son interaction avec l’ADN. Toutefois, le dimère Gbetagamma colocalise au noyau avec le complexe AP-1 et recrute les protéines histones déacétylases (HDAC) afin d’inhiber l’activité transcriptionnelle du complexe AP-1. / Based on the classical model of G protein activation, signal transduction occurs by transient and random interactions between the receptor, the G protein and the effectors. Bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation assay (BiFC) and co-immunoprecipitation experiments revealed that receptor, heterotrimeric G proteins and effectors were found in stable complexes that persisted during signal transduction. Kir3 channel and Gbetagamma dimer interacts first in the endoplasmic reticulum (ER) and this interaction can be modulated by the membrane-permeable beta2-adrenergic agonist cimaterol. Little specificity has been reported for several isoforms of the Gbetagamma dimer in the activation of the Kir3 channel. However, we found that the “precocious” interaction in the ER is sensitive to the presence of different combination of Gbeta and Ggamma subunits. Recently, a number of new proteins, which are not classical effectors at the plasma membrane have been shown to interact with GbetagammaThese include histone deacetylases 4 and 5 (HDAC)[1, 2] and the glucocorticoid receptor. We identified a novel interaction between Gbetagamma subunit and the Fos proteins, which form the transcription factor AP-1 following their dimerization with Jun proteins. Gbetagamma and Fos interactions can be detected in HEK 293 cells overexpressing the two proteins as well as in brains from rats pre-treated with amphetamine. Gbetagamma/Fos interaction favours the nuclear translocation of Gbetagamma dimer and inhibits AP-1 transcriptional activity. Gbetagamma did not block Fos/Jun dimerization or the interaction of AP-1 with DNA but recruited HDACs to the AP-1 complex.

Itch est la seule ligase de l'ubiquitine de type C2-WW-HECT capable d'interagir avec les protéines à domaine SH3. Ce domaine est particulièrement représenté parmi les protéines régulatrices de l'endocytose. Les travaux présentés ici visaient à examiner la capacité d'Itch à interagir avec plusieurs protéines endocytiques. Nous avons utilisé la technique du BRET (Bioluminescence Resonance Energy Transfer) pour examiner quelques protéines candidates. Nous avons ensuite confirmé les résultats obtenus par BRET avec des tests d'interaction in vitro, puis déterminé la capacité d'Itch à ubiquityler les protéines liées via leurs domaines SH3. Nous avons ainsi découvert deux nouveaux partenaires d'interaction et substrats d'Itch parmi les protéines endocytiques, amphyphisine et pacsine. De plus, Itch interagit avec les domaines SH3 isolés d'intersectine, mais pas avec la protéine complète, suggérant que cette dernière n'est pas un substrat d'Itch. Itch est donc bien positionnée pour exercer un rôle régulateur de l'endocytose en ubiquitylant ses substrats. / Itch is the only C2-WW-HECT type ubiquitin ligase that can bind SH3 domain proteins. This domain is particularly frequent in accessory endocytic proteins. We have used Bioluminescent Resonance Energy Transfer to examine a few candidate endocytic proteins, in addition to the already known substrate of Itch, endophilin. We then used standard in vitro techniques to confirm these interactions, and tested Itch capacity to ubiquitylate these putative substrate proteins. We thus discovered two new substrates of Itch, amphiphysin and pacsin. We also determined that although Itch interacts with the isolated SH3 domains of intersectin, it does not recognize the full length protein, thus rulling out Intersectin as a substrate of Itch. Itch is thus a putatively important regulator of endocytosis, through its capacity to recognize and ubiquitylate several SH3-domain proteins.

Le récepteur de chimiokine CXCR7 a été récemment identifié comme liant la chimiokine SDF-1, anciennement considérée comme ligand exclusif du récepteur CXCR4. Ces deux récepteurs sont exprimés majoritairement dans les mêmes types cellulaires et, ainsi, la découverte de CXCR7 incite à réévaluer les effets respectifs de SDF-1 sur CXCR4. Étant donné son rôle dans le cancer, CXCR4 est une cible de choix pour le développement de molécules thérapeutiques. Également, CXCR7 semble être impliqué dans la croissance tumorale. AMD3100, un antagoniste «sélectif» pour CXCR4, est maintenant commercialisé. Cet antagoniste a été identifié comme liant lui aussi CXCR7. De plus, sur CXCR7, l’AMD3100 agit comme agoniste puisqu’il induit le recrutement de la β-arrestine, à l’opposé de son effet sur. En revanche, AMD3100 n’induit pas le recrutement de la β-arrestine à CXCR4. Basé sur ces résultats, il est nécessaire de revoir la sélectivité d’autres antagonistes synthétiques de CXCR4. À l’aide de la technique de BRET (Résonance d’un transfert d’énergie par bioluminescence), nos résultats montrent que le Tc14012, un autre antagoniste synthétique de CXCR4, et structurellement distinct de l’AMD3100, interagit avec CXCR7. Contrairement à CXCR4, les deux antagonistes de CXCR4 agissent comme agonistes sur CXCR7 en induisant le recrutement de la β-arrestine. Nos résultats suggèrent que l’organisation spatiale du corps du récepteur serait responsable de cet effet opposé. En conclusion, AMD3100 et Tc14012 ne sont pas sélectifs pour CXCR4, puisqu’ils interagissent avec CXCR7. Lors du développement de nouvelles molécules synthétiques ciblant CXCR4, il serait alors nécessaire d’en évaluer leur sélectivité, et leurs effets en les testant aussi sur CXCR7. / ASBTRACT SDF-1 was at first thought to exclusively bind CXCR4, but it was subsequently found to also bind to the chemokine receptor CXCR7. CXCR4 is a promising target for drug development due to its role in cancer. AMD3100 is newly commercialised synthetic antagonist of CXCR4. This drug leads to massive release of hematopoietic stem cell into the peripheral blood. It was found that AMD3100 also binds to CXCR7 and acts as an agonist of β-arrestin recruitment to CXCR7. An antagonist of CXCR4 acts as an agonist on CXCR7. Prompted by this observation, we tested whether this might hold true for other CXCR4 antagonist. Tc14012, a peptidomimetic of T140, has been extensively described as a potent CXCR4 antagonist. We find that TC14012 also interacts on CXCR7. Like AMD3100, TC14012 alone induces β-arrestin recruitment to CXCR7. Thus, two structurally unrelated CXCR4 antagonists, AMD3100 and TC14012, are agonists of the CXCR7-arrestin pathway. This suggests distinct activation mechanisms of the arrestin pathway by CXCR4 and CXCR7. The results we obtained using a BRET (Bioluminescence Resonance Energy Transfer)-based arrestin recruitment assay, suggest that the CXCR7 receptor core is responsible for the recruitment of beta-arrestin in response to AMD3100 and TC14012. The finding that both AMD3100 and TC14012 do not only bind CXCR4, but also CXCR7, with opposite effects on arrestin recruitment, is important for the use of the compounds as tools to dissect SDF-1-mediated effects. This may be a general feature of synthetic ligands of the two receptors, with potential consequences for drug development. Key words: Chemokine receptor, CXCR4 and CXCR7, BRET, β-arrestin recruitement, TC14012, AMD3100 and SDF-1.

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

Le récepteur de chimiokine CXCR3 est un récepteur couplé à la protéine G (RCPG) exprimé, entre autre, sur les cellules T activées lors d’une réponse immune. CXCR3 est activé par trois ligands inductibles par l’interféron-γ (CXCL9, 10, 11) et, plus récemment, il a été découvert que CXCL4 liait CXCR3. Nous savons que CXCR3 joue un rôle dans la chimiotaxie des leucocytes, mais peu d’attention a été portée sur la signalisation biaisée induite par ces quatre ligands. Alors que l’homodimérisation entre récepteurs de chimiokine est un concept grandement observé, l’hétéromérisation entre deux récepteurs reste un domaine de recherche active. La signalisation biaisée et l’hétéromérisation ont été testées grâce à la technique de bioluminescene resonance energy transfer (BRET) dans des cellules HEK293E. Nous présentons une caractérisation pharmacologique des quatre ligands de CXCR3 et démontrons l’hétéromérisation de CXCR3 avec CXCR4 et avec CXCR7. Nos résultats suggèrent que les ligands de CXCR3 n’agissent pas de manière redondante. / The chemokine receptor CXCR3 is a G-protein-coupled receptor (GPCR) rapidly induced on naïve T cells upon activation. CXCR3 is activated by three interferon-γ inducible ligands (CXCL9, 10, 11) and, more recently, CXCL4 has been discovered as a functional ligand for CXCR3. It is known that CXCR3 acts as a chemotactic receptor, but limited attention has been directed to the biased signaling induced by all four ligands. Chemokine receptor homodimerization is now a widely accepted concept, but the extent to which heterodimerization is prevalent remain matter of active research. In this work, biased signalling and heterodimerization were assessed with bioluminescence resonance energy transfer (BRET) in HEK293E cells. We present pharmacological characterization of all four ligands of CXCR3 and heterodimerization of CXCR3 with CXCR4 or CXCR7. Our results suggest that CXCR3 ligands are not redundant and that CXCR3 heterodimerizes with CXCR4 and with CXCR7.

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

Itch est la seule ligase de l'ubiquitine de type C2-WW-HECT capable d'interagir avec les protéines à domaine SH3. Ce domaine est particulièrement représenté parmi les protéines régulatrices de l'endocytose. Les travaux présentés ici visaient à examiner la capacité d'Itch à interagir avec plusieurs protéines endocytiques. Nous avons utilisé la technique du BRET (Bioluminescence Resonance Energy Transfer) pour examiner quelques protéines candidates. Nous avons ensuite confirmé les résultats obtenus par BRET avec des tests d'interaction in vitro, puis déterminé la capacité d'Itch à ubiquityler les protéines liées via leurs domaines SH3. Nous avons ainsi découvert deux nouveaux partenaires d'interaction et substrats d'Itch parmi les protéines endocytiques, amphyphisine et pacsine. De plus, Itch interagit avec les domaines SH3 isolés d'intersectine, mais pas avec la protéine complète, suggérant que cette dernière n'est pas un substrat d'Itch. Itch est donc bien positionnée pour exercer un rôle régulateur de l'endocytose en ubiquitylant ses substrats. / Itch is the only C2-WW-HECT type ubiquitin ligase that can bind SH3 domain proteins. This domain is particularly frequent in accessory endocytic proteins. We have used Bioluminescent Resonance Energy Transfer to examine a few candidate endocytic proteins, in addition to the already known substrate of Itch, endophilin. We then used standard in vitro techniques to confirm these interactions, and tested Itch capacity to ubiquitylate these putative substrate proteins. We thus discovered two new substrates of Itch, amphiphysin and pacsin. We also determined that although Itch interacts with the isolated SH3 domains of intersectin, it does not recognize the full length protein, thus rulling out Intersectin as a substrate of Itch. Itch is thus a putatively important regulator of endocytosis, through its capacity to recognize and ubiquitylate several SH3-domain proteins.