Ce travail porte sur les processus psychiques qui sous-tendent l'acte de violence sexuelle à l'âge adulte, avec la perspective de les comprendre à la lumière du processus de subjectivation adolescent, du travail d'historisation et des enjeux identificatoires, en termes de bisexualité psychique, qui y sont liés. Les observations cliniques s'appuient sur plusieurs entretiens non directifs ainsi que sur la passation du Rorschach et du TAT avec 8 hommes auteurs de violence sexuelle étant sous soins pénalement ordonnés après la fin de leur incarcération. Ils ont été rencontrés dans une institution spécialisée en participant de manière libre à cette recherche. Ces hommes ont tous agi leur première violence sexuelle à l'âge adulte et non lors de l'adolescence comme c'est le cas pour la majorité de ces individus. La passation du TAT comporte aussi les planches habituellement destinées aux femmes et la planche 17BM afin d'appréhender davantage les subtilités identificatoires de ces personnes. Ces observations m'ont permis de développer ensuite des hypothèses et réflexions métapsychologiques concernant ces personnes afin de tenter de proposer une interprétation de la manière dont l'acte de violence sexuelle peut s'inscrire dans leur parcours de vie. Enfin, ce travail s'ancre dans des perspectives de soins. En effet, il vise à souligner d'une part l'importance de l'adolescent dans l'adulte au sein de la psychothérapie et, d'autre part, la richesse de la méthode de recherche utilisée ici comme potentiel levier thérapeutique pour les personnes sous soins pénalement ordonnés. / The aim of this study is to examine the psychic processes underlying the act of sexual violence within a population of adult offenders in light of the adolescent process of subjectivation, the work of historicization and the bisexual dispositions of identifications. Clinical data was collected from non-directive interviews and projective assessments of 8 male sexual offenders who had committed their first sexual offense later at adulthood, an uncommon criterion in this population. The sample was selected from a specialized institution for penal imposed treatments after incarceration. Along with the Rorschach, the TAT was administered with the exceptional inclusion of the 17BM and feminine cards in order to gain a better understanding of identification dynamics. The findings highlighted metapsychological basis for the interpretation of the function of sexual violence in the patient's life course, thus yielding important therapeutic implications. Essentially, this research emphasizes the importance of considering the adolescent process in adult psychotherapy. It also underlines the potentially therapeutic benefits yielded by this specific research method amongst the population of sexual offenders undergoing court-ordered treatments.

Le système Tat (pour Twin Arginine Translocation) exporte des protéines repliées depuis le cytoplasme vers le périplasme des bactéries. L’adressage des protéines à exporter au système Tat repose sur une séquence signal spécifique amino terminale clivée après exportation. Chez le phytopathogène Dickeya dadantii, l’homologue de pectine lyase PnlH possède une séquence signal Tat qui assure son adressage au système Tat mais qui n’est pas clivée après exportation et ancre la protéine dans la membrane externe. Chez les protéobactéries, la majorité des protéines de membrane externe sont soit des lipoprotéines soit des protéines intégrales de membrane en tonneau β. L’adressage de ces protéines à la membrane externe repose sur des voies spécifiques du type de protéine : la voie Lol pour les lipoprotéines et la combinaison des chaperons périplasmiques SurA, Skp et DegP et du complexe de membrane externe Bam (β barrel assembly machinery) pour les protéines en tonneau β. Au cours de ce travail, l’étude de l’adressage de PnlH à la membrane externe a montré que SurA se liait à la séquence signal hydrophobe de PnlH pour la protéger de l’environnement hydrophile au cours de son transit dans le périplasme. La séquence signal de PnlH (41 acides aminés) porte l’intégralité de l’information nécessaire à son adressage à la membrane externe. La nature de l’information adressant les protéines au système Tat est bien connue et dans ce travail nous nous sommes efforcés d’identifier les informations requises pour les deux dernières étapes de l’adressage de PnlH à la membrane externe : la traversée du périplasme et l’insertion dans la membrane externe. La délétion d’une région conservée comprise entre les résidus 28 et 41 de la séquence signal de PnlH affecte l’adressage de cette dernière à la membrane externe. Des substitutions des acides aminés conservés de cette région ne semblent pas affecter l’adressage de PnlH, indiquant que l’information nécessaire à l’adressage de PnlH à la membrane externe après exportation ne réside pas dans la séquence en acides aminés de la séquence signal de PnlH. En revanche, nos données suggèrent que la présence d’une hélice α hydrophobe dans la séquence signal de PnlH est importante pour son adressage à la membrane externe. Cette observation est particulièrement intéressante puisqu’une telle structure est généralement considérée comme une caractéristique des protéines de membrane interne. / The Twin Arginine Translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of β-barrel proteins and lipoproteins. Targeting of these proteins to the outer membrane relies on two pathways: the periplasmic chaperones SurA, Skp and DegP work together with the β-Barrel-Assembly Machinery (Bam) to target and insert β-barrel proteins into the outer membrane while the Lol pathway targets and insert lipoproteins. In this work, we showed that SurA binds to the hydrophobic PnlH signal sequence during the course of its periplasmic transit. The PnlH signal sequence (41 residues) carries all the information necessary to the targeting of PnlH to the outer membrane. The nature of the information that targets proteins to the Tat system is well charcterized. Thus, we focused on the nature of the information carried by the PnlH signal sequence and that allows its crossing of the periplasm and its insertion in the outer membrane. The deletion of a conserved region of the PnlH signal sequence between residues 28 and 41 strongly affects the targeting of PnlH to the outer membrane. None of the single amino acid substitutions constructed in this region obviously affected the targeting of PnlH, indicating that the information may not reside in the amino acid sequence of the PnlH signal sequence. Consistently, our data suggest that the presence in the PnlH signal sequence of an α helix with a hydrophobic cluster is important for the targeting of PnlH to the outer membrane. This observation is striking since such a structure is considered as an inner membrane protein property.

Ce travail de thèse propose d’approfondir la compréhension de l’anorexie de l’adolescente à partir d’une méthodologie projective (Rorschach et T.A.T.).Il révèle notamment la présence d’une instabilité des frontières du Moi et un dépassement insuffisant des conflits prégénitaux venant influer sur la construction du féminin et favoriser le recours à des défenses de type phalliques narcissiques permettant d’éviter tout autant le vécu de séparation que celui de dépendance.Ce travail met par ailleurs en évidence un traitement singulier des fantasmes de séduction marqué par un renversement de la construction hystérique, l’insuffisance du refoulement et l’impossibilité à investir une position passive venant entraîner un vécu spécifique marqué par une exacerbation des angoisses persécutives qui paraît liée au caractère insuffisamment sécurisé des frontières propres et à la difficulté de régulation de l’excitation pulsionnelle.Dans son approche différentielle, ce travail révèle également chez les patientes présentant un profil restrictif une plus grande efficience des défenses narcissiques en comparaison des patientes présentant un tableau d’anorexie-boulimie où la problématique de porosité des enveloppes et les difficultés de maniement pulsionnel se dévoilent davantage. Les problématiques mobilisées paraissent néanmoins semblables. L’apport de cette recherche se situe davantage dans la mise en évidence de mouvements de clivage au Moi pouvant conduire à deux types d’aménagements distincts venant s’exercer soit sur un mode persécutif, soit sur un mode adhésif, avec une influence sur le type de structuration psychique de ces adolescentes se révélant indépendante des formes symptomatiques tout en pouvant correspondre à deux temps distincts de la maladie. / This paper attempts to go further into female teen anorexia from the perspective of a projective methodology. It brings particularly to light the presence of a blurred frontier of the Ego and an insufficient expansion of pregenital conflicts which affects the construction of the feminine and facilitates resorting to a narcissistic phallic type of defense which helps avoid the experience of separation as much as that of dependency.This paper brings further to light a particular handling of seduction fantasies marked by a reversal of hysterical construction, insufficient repression and the impossibility to invest a passive position. This leads to a specific real life experience marked by an exacerbation of persecution anxiety which seem to be linked to both the insufficiently secured nature of instinctive excitation and the difficulty to regulate it. From its differential approach, this paper also highlights a better efficiency of narcissist defense in those female patients presenting a restrictive profile compared with those presenting anorexia-bulimia stigmas where porosity of envelopes and difficulty of handling drive are more apparent. However, the issues raised appear to be similar.This research paper brings brings further highlight to the cleavage process phenomenon in the Ego leading of anorectic patients to two distinctive types of psychic adjustments which expresses itself either on a persecution mode or adhesive one. This in turn has an influence on the type of psychic structures in female teens which turn out to be independent from symptomatic forms while they may at the same time correspond to two different stages of the illness.

La protéine Tat du VIH-1 est essentielle à la multiplication virale. Elle permet la transactivation de la transcription et, par ses propriétés apoptotiques, elle participe à la pathologie SIDA. D'où l'importance d'étudier les mécanismes régulant sa production. L'épissage alternatif de l'ARN du VIH-1, en particulier, l'utilisation des sites accepteurs d'épissage A3 et A7 est nécessaire pour la production des ARNm tat. L'utilisation du site A3 est fortement régulée par des éléments agissant en cis contenus dans une structure tige-boucle SLS3A3 située en aval du site A3. En purifiant les complexes RNP formés en extrait nucléaire sur un segment de l'ARN viral renfermant le site A3, et en analysant par spectrométrie de masse les protéines contenues dans ces complexes, nous avons pu mettre en évidence la fixation d'une protéine inhibitrice de l'utilisation du site A3, la protéine DAZAP1. Sur la base d'un ensemble de données antérieures du laboratoire et de nouvelles données que j'ai obtenues, nous avons montré que la protéine SRSF7, sans doute en synergie avec SRSF1, limite la fixation de DAZAP1 et active l'épissage au site A3. Nous avons aussi montré que la protéine virale Tat exerce un rétro-contrôle négatif au niveau de la production de l?ARNm tat, ceci en limitant l'activation du site A3 par SRSF7. La partie apicale de la structure tige-boucle SLS3A3 (motif B) est très conservée dans les souches de VIH-1. L'équipe d'E Guittet a déterminé sa structure 3D par RMN. La conformation de sa boucle terminale est caractéristique des structures tige-boucle reconnues par les protéines à domaines dsRBD (double stranded RNA Binding Domain). J'ai pu confirmer cette hypothèse en purifiant les complexes formés par le motif B en extrait nucléaire. Nous avons ainsi pu montrer que la protéine kinase PKR, qui joue chez l'Homme un rôle majeur dans la réponse à une infection virale, est un partenaire du motif B. Par utilisation de sondes chimiques de la structure 2D de l'ARN, j'ai pu montrer que la structure tige-boucle SLS3A3, contenant le codon d'initiation de l'ORF Tat, est présente dans l'ARNm tat1. Nous avons alors développé un système visant à étudier les mécanismes de régulation de l'initiation de la traduction de la protéine Tat. Par l'emploi d'une construction bicistronique, j'ai pu confirmer l'existence d'une activité IRES dans la région 5'UTRtat1 de l'ARNm tat1 et définir deux segments ayant cette activité. Des résultats préliminaires obtenus avec une construction bicistronique, nous ont permis de commencer à tester l'effet de différentes protéines SR et hnRNP sur l'activité de ces IRES / HIV-1 Tat protein is essential for viral replication. It allows the transcription of full-length viral RNAs, and due to its apoptotic properties it contributes to the AIDS disease. Hence, it is important to study the mechanisms regulating its production. Alternative splicing of the HIV-1 RNA, in particular, the use of acceptor sites A3 and A7 is required for tat mRNA production. Splicing at site A3 is highly regulated by cis-acting elements contained in a stem-loop structure SLS3A3, located downstream from site A3. By purifying RNP complexes formed in nuclear extract on a segment of the viral RNA containing site A3 followed by mass spectrometry analysis, we were able to highlight the binding of a new inhibitory protein, DAZAP1. Based on a set of ancient laboratory data and new results that I obtained, we have shown that SRSF7 protein, probably in synergy with SRSF1, limits the binding of DAZAP1 and splicing activation at site A3. We also showed that the viral protein Tat exerts a negative feedback control on tat mRNA production by restricting splicing activation of site A3 by SRSF7. The apical part of the stem-loop structure SLS3A3 (B motif) is highly conserved among HIV-1 strains. E Guittet team determined its 3D structure by NMR. The conformation of this apical loop is characteristic of stem-loop structures recognized by dsRBD proteins (double-stranded RNA binding domain). I was able to confirm this hypothesis by purifying RNP complexes formed by the B motif in nuclear extract. Thus, we have shown that the RNA dependent protein kinase (PKR), which plays in humans a major role in response to viral infection, is a partner of the B motif. By using chemical probes specific of the 2D structure of RNAs, I showed that the stem-loop structure SLS3A3 that contains the initiation codon of Tat is present in tat1 mRNA. We then developed a system to study the mechanisms regulating the initiation of translation of Tat protein. By using a bicistronic construct, I was able to confirm the existence of IRES activity in the 5?UTRtat1 region of tat1 mRNA, and define two segments that contain this activity. Preliminary results obtained with a bicistronic construct allowed us to begin testing the effect of different SR and hnRNP proteins on the activity of the IRES

Os vírus são eficazes na transferência de genes em células devido aos seus mecanismos especializados. No entanto, vírus como veículos de entrega de genes podem acarretar em problemas, particularmente quando proposto para reprogramar células somáticas em células-tronco pluripotentes induzidas (iPS) visando utilização terapêutica. No presente estudo, procurou-se desenvolver um sistema alternativo para entregar diretamente proteínas nucleares (Oct4, Sox2, KLF4, e c-Myc) fusionadas com o domínio de transdução de proteína TAT, para promover a reprogramação de fibroblastos embrionários de camundongos (MEF) ou células mesenquimais derivadas de tecido adiposo humano (hASC) em células iPS. Primeiramente o PTD TAT ou TAT- foi fundido a proteína verde fluorescente (GFP) como modelo para prova de princípio e padronização detalhada. Inesperadamente, TAT-GFP produzido e secretado pelas células NIH-3T3 produtora não foi capaz de ser detectado no meio de cultura por verificação quantitativa fluorimétrica, nem foi capaz de ser detectada em células-alvo, por citometria de fluxo, depois de co-cultura em transwells. Essa observação pode ser explicada por: (1) ineficiência desse tipo de célula em secretar proteínas e (2) falta de resistência à clivagem por endoproteases furinas. Para contornar esses fatores limitantes usou-se citometria de fluxo para avaliar as melhores condições para a transfecção por seis diferentes tipos de células (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t e COS-7) com TAT (modificada para ser resistente à furinas) fundido a GFP. Células 293t-TAT-GFP exibiram a maior eficiência de transfecção e também de secreção. O mesmo pôde ser observado para as seis linhagens celulares expressando fatores de transcrição nucleares TAT, determinados por ELISA. Em seguida, diferentes estratégias de entrega foram testadas. A primeira foi baseada na co-cultura de uma mistura de células produtoras com MEF ou hASC. No entanto, não foi possível observar a reprogramação devido à morte celular. A segunda foi baseada na concentração de meio condicionado de cultura de células por centrifugação usando colunas Amicon, trocando o meio a cada 24h, em quatro ciclos. No entanto, apesar da presença de algumas colônias após 20-30 dias, nenhuma colônia verdadeira iPS foi obtida. Na sequência, as células foram tratadas com cada proteína de forma independente, e as demais foram substituídas pelo retrovírus correspondente, trocando meio a cada 72h, em quatro ciclos. Essa estratégia, apesar de permitir verificar a função de cada proteína, também não resultou em reprogramação. Este achado pode ser explicado pela diferenciação celular induzida por BCS, que também é concentrado no processo. Assim, passou-se a adaptação de \"células produtoras\" em condições de cultura livre de soro, para enriquecer a produção dos fatores nucleares individuais, necessários para a reprogramação. A otimização sistematizada deste processo está sendo realizada em parceria com o IPT e deve resultar em quantidades de proteína de fusão suficientes para o teste final da hipótese proposta. Em conjunto, são apresentados os dados da geração de linhagens celulares expressando estavelmente os vários fatores de transcrição e estratégias para melhorar a eficiência necessária para a produção iPS. Esta nova estratégia garante uma produção eficiente de TAT fundida a fatores nucleares de reprogramação e sua eficácia para promover a reprogramação de células somáticas de maneira livre de vírus merece ser investigado futuramente / Viruses are effective at transferring genes into cells by its specialized mechanisms. However, viruses as gene delivery vehicles entail problems, particularly when proposed to reprogram somatic cells into induced pluripotent stem cells (iPS) for therapeutic uses. In the present study, we aimed to develop an alternative system for directly delivering nuclear proteins (Oct4, Sox2, Klf4, and c-Myc) fused with TAT protein transduction domain to promote reprogramming of mouse embryonic fibroblasts (MEF) or human adipose tissue derived mesenchymal cells (hASC) into iPS cells. First TAT- or TAT- PTD was fused to green fluorescent protein (GFP) as a proof of principle model and for detailed standardization. Unexpectedly, TAT-GFP produced and secreted by NIH-3T3 producer cells was not detected in the culture medium by quantitative fluorimetric verification, nor detected on target cells, by flow cytometry, after being co-cultured using transwells. This observation maybe explained by: (1) inefficiency of this cell type to be transfected and to secrete proteins and (2) lack of resistance to furin endoproteases cleavage on Golgi of TAT sequence. To circumvent these limiting factors we used flow cytometer to assess the best conditions for transfection in six different cell types (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t and COS-7) with TAT- (a modified PTD to be resistant to furin endoproteases) fused to GFP. 293t-TAT-GFP cells displayed the highest transfection efficiency and secretion levels. The same could be observed for the six cell lineages expressing TAT- nuclear transcription factors, determined by ELISA.Next, different delivery strategies were tested for TAT- nuclear transcription factor system. Co-culturing a mix of producer cells with MEF or hASC resulted in not reprogramming and this was associated with cell death. The second was based on the use of microconcentrated conditioned cell culture medium, changed every 24h, in four cycles. However, despite the presence of some emerging colonies after 20-30 days, no true iPS colonies were obtained. Then, cells were treated with each protein independently, and the others were replaced by the corresponding retrovirus, changing cell medium every 72h, in four cycles. We verified the reprogramming potential of each protein, but no true colonies were obtained.One possibility for this finding is that BCS is also concentrated by centrifugation and may induce cell differentiation. To circumvent these problems, we have started the adaptation of producer cells in a serum-free culture condition to enrich the production of the individual factors required for reprogramming. This optimization process is taking place in collaboration with the IPT and shall result in large amounts of the fusion protein to finally test the proposed hypothesis. Altogether, we presented the generation of several cell lines stably expressing the transcription factors and strategies to improve the efficiency required for iPS production. This novel strategy guarantees efficient production of TAT-fused reprogramming nuclear factors and its efficacy to promote somatic cells reprogramming in a virus-free manner deserves to be further investigated

No contexto da pesquisa e utilização prática das técnicas de autoexpressão, o Teste de Apercepção Temática (TAT) apresenta reconhecidas vantagens. Apesar de linhas de pesquisa bastante desenvolvidas internacionalmente, não existem estudos atuais sobre as propriedades deste instrumento no Brasil, levantando a necessidade de conhecer seus alcances e limites nesse contexto cultural. Desse modo, o desenvolvimento de normas que descrevam o desempenho típico de uma população não clínica se mostra um importante passo inicial rumo à retomada da pesquisa com o TAT e sua adoção por profissionais. Tendo em vista que a validação das técnicas de autoexpressão se refere aos sistemas de classificação e não aos instrumentos em si, o presente estudo adotou, dentre a miríade de sistemas de categorização das histórias do TAT descritos na literatura, o sistema proposto por Monique Morval, que foi desenvolvido para estudantes e profissionais com pouca experiência com o TAT, utilizando pressupostos da Personologia de Henry Murray, Psicologia Cognitiva e Psicologia do Ego, possibilitando leituras objetivas e qualitativas do material. Considerando o potencial de utilização desse sistema por parte dos psicólogos brasileiros - visto sua semelhança com o atual sistema aprovado para uso pelo Sistema de Avaliação dos Testes Psicológicos (SATEPSI) do Conselho Federal de Psicologia - foram feitos aprimoramentos no assim chamado sistema morvaliano, possibilitando sua adoção no estudo normativo proposto pelo presente projeto. Tais aprimoramentos consistiram na revisão e adição de categorias e subcategorias, a partir de trabalhos brasileiros e internacionais sobre o instrumento. Desta forma, o presente estudo teve por objetivo desenvolver normas para o TAT (Sistema Morvaliano) em adultos, a partir de uma amostra não clínica, aleatória e estratificada por critérios de idade, sexo, escolaridade e nível socioeconômico (NSE) da cidade de Ribeirão Preto (SP). Os participantes foram 100 adultos (25 a 44 anos de idade), selecionados através de visita a domicílios sorteados em bairros de diferentes NSE. Foram realizadas entrevistas de avaliação psicológica individuais, utilizando um roteiro de entrevista semiestruturada, o Teste de Inteligência Geral Não-Verbal (TIG-NV) e 20 cartões do TAT, pré-selecionados de acordo com as recomendações do manual original do instrumento. As histórias do TAT foram categorizadas no sistema morvaliano revisado e seus dados passaram por análises estatísticas e sínteses qualitativas em busca de diferenças de desempenho relacionadas ao sexo, escolaridade e NSE. Os resultados mostram poucas diferenças entre os grupos que justifiquem a elaboração de normas específicas; apesar disso, foi identificada uma tendência (ainda que pequena) de o nível de escolaridade e o NSE favorecerem um melhor desempenho nas variáveis relativas à organização formal das histórias. São discutidas as implicações dos dados obtidos em termos do perfil normativo de desempenho nas variáveis do sistema morvaliano, suas evidências preliminares de validade e futuras direções para estudos sobre o TAT nesse sistema. / Within the context of research and practice with self-expression techniques, the Thematic Apperception Test (TAT) presents several advantages widely acknowledged. However, in spite of well-developed research with this instrument being seen in other countries, no recent Brazilian studies have been carried out up to date. Such situation arise the need of efforts in order to show the properties of the TAT in the current Brazilian cultural context, so that its strengths and weaknesses can be known. In this sense, the development of norms on the typical performance of a non-clinic population is an important first step toward resuming the Brazilian studies on the TAT, at the same time such norms could be used by applied professionals. Considering that the validation of self-expression techniques is related to classification systems (and not the instruments themselves), the present project adopts - among the myriad of categorizing systems for TAT stories - the one proposed by Monique Morval, which was developed for students and professionals with no experience with the TAT. Such system adopts assumptions from Henry Murray\'s Personology, Cognitive Psychology and Ego Psychology, providing both objective and qualitative data on the stories. Due to resemblance of this system with the one currently approved for use by the Evaluation System of Psychological Tests (Sistema de Avaliação dos Testes Psicológicos, SATEPSI) of the Brazilian Federal Council of Psychological, there is a potential for the adoption of such system by Brazilian psychologists. In order to carry out the present project, the now called morvalian system was improved by revising and adding new categories and subcategories, which was based on both Brazilian and international studies regarding. The present project, thus, aimed to develop norms for the TAT (morvalian system) in adults from a randomized, non-clinical sample, which was stratified in terms of age, gender, schooling and socialeconomical levels. The study was carried out in the city of Ribeirão Preto (Brazilian state of São Paulo), with a n = 100 sample (25-44 years of age). Participants were invited to take part of the study during visits to randomly selected houses, located in regions of different socialeconomical levels. Participants were individually assessed, using a semi-structured interview script, a measure of intelligence (the TIG-NV - General Non-Verbal Intelligence Test) and 20 TAT cards, selected according to the original manual of this instrument. Data was compiled with the morvalian system\'s categories and went through both statistically and qualitative analyzes in search for performance differences related to gender, schooling and socialeconomical levels. The results showed few differences across the groups, which do not support for the elaboration of specific norms. However, results also showed a significant (although small) trend for better performance of groups with better schooling and socialeconomical levels on the variables related to stories\' formal organization. Such results are discussed in terms of their implication for describing the normative performance on the morvalian system, preliminary validity evidence and future directions for the TAT within this system.

La membrane bactérienne a fait l'objet de nombreuses études de localisation de protéines et de phospholipides. Par fusion d’une protéine fluorescente (GFP) aux gènes d’intérêt, il est alors possible d‘observer la localisation des protéines associées par microscopie. La plupart de ces observations ont été réalisées à l’aide de microscopes dits à épifluorescence. Afin d’obtenir une qualité d’image suffisante, il était nécessaire de surexprimer la protéine observée, insérée à un locus ectopique non naturel. Ce travail de thèse a permis d’utiliser une nouvelle technologie acquise dans notre laboratoire, le microscope à fluorescence par réflexion totale interne (TIRFM), plus puissant que le microscope à épifluorescence utilisé précédemment. Cette technologie a permis une caractérisation plus détaillée de la localisation de protéines d’intérêt, placées sous contrôle de leur promoteur naturel. Il a également été possible de caractériser la dynamique des foci observés. Nous avons concentré notre étude sur 3 protéines: (i) SecA pour l’étude de la translocation des protéines du cytoplasme vers la membrane, (ii) YidC pour l’insertion des protéines dans la membrane, (iii) PgsA pour la synthèse des phospholipids. Les foci se déplacent dynamiquement et s’associent de manière transitoire dans la membrane. L’observation sur la durée de ces foci, et l’analyse de leur intensité moyenne au cours des observations, montre que SecA se déplace sur l’ensemble de la membrane de manière uniforme. L’analyse du déplacement des foci montre une relation quadratique entre la distance moyenne parcourue par les foci en fonction du temps. Ce résultat est en accord avec l’hypothèse d’un mouvement brownien des foci. Les foci sont observés dans les différentes phases de croissance des cellules, et le nombre de foci présents dans une cellule de la longueur de celle-ci. SecA-GFP a été testés dans un certain nombre de contextes génétiques. La localisation a été perturbée lors de la déplétion de pgsA. Cependant, comme PgsA est une protéine essentielle, il ne peut être exclu que ce changement de localisation apparaît des cellules qui sont mortes ou mourantes. Dans une souche mutante ΔclsA, on n’observe aucune différence dans la localisation de SecA en phase exponentielle, mais on aperçoit une relocalisation aux poles en phase stationnaire de croissance. La voie Tat est responsable du transport des protéines devant être exportées dans un état structuré, par exemple dans le cas de l’incorporation d’un co-facteur. À ce jour, la régulation du système Tat est peu connues, de même que les interactions entre les différentes sous-unités du système Tat et d'autres protéines dans le cytoplasme, dans la membrane ou dans la paroi cellulaire. Des fusions de les gènes de la voie Tat ont été co-exprimées deux à deux dans des cellules de levure, et leur capacité à interagir in vivo a été testée par la méthode dite du double hybride chez la levure. Nous avons généré un réseau d’interaction autour des cinq composants de système Tat. Pour déterminer les implications fonctionnelles des composants du réseau, nous avons travaillé en collaboration avec le laboratoire de Jan-Maarten van Dijl. Nous avons utilisé une collection de souches mutantes pour lesquels certains composants individuels du réseau ont été retires. Trois a été observe d’etre nécessaires pour la sécrétion Tat-dépendante. Nous avons étudié la localisation des fusions GFP avec ces proteins. On a observé une localisation double de HemAT selon l’état physiologique de la cellule. En phase exponentielle, les cellules de B. subtilis sont généralement présentes sous forme de chaînes dans lesquelles le septum de division a déjà été formé, mais la séparation cellulaire n'a pas encore eu lieu. Une fusion de la GFP à CsbC apparaît de façon homogène dans la membrane. / In the years since the cloning of GFP, the field of bacterial cell biology has characterized a variety of specific protein localization patterns in the bacterial membrane. The vast majority of early subcellular localization studies made use of inducible GFP fusions, which generally required the presence of high concentrations of inducer, and can therefore be considered to be overexpressed. An outstanding question remains over the organization of natively expressed proteins in the membrane. Here, we have investigated the localization of functional GFP fusions to proteins catalyzing important membrane processes; the secretion motor protein SecA, the membrane insertase YidC1, and the essential phospholipid synthase PgsA using total internal reflection fluorescence microscopy (TIRFM). This allowed natively expressed proteins to be localized with temporal resolution that can capture their dynamics. We characterized dynamic complexes dispersed throughout the membrane displaying diffusive movement with no preferred trajectories. Further characterization focused upon identifying conditions in which the localization pattern was disturbed. A polar mislocalization was identified in a cardiolipin mutant strain. The yeast two-hybrid (Y2H) approach is a robust approach to detect binary interactions on a proteome-scale. We performed genome-wide Y2H screens as well as targeted Y2H analyses for specific interactions involving components of the Sec and Tat secretion machineries of B. subtilis, revealing an intricate protein-protein interaction network involving 71 proteins. Furthermore, three proteins identified in the Tat network, WprA, CsbC and HemAT, were shown to be important for effective protein secretion via the B. subtilis Tat system, indicating that our yeast two hybrid assays reveal biologically significant interactions involving membrane proteins. The studies provide a novel proteomic view on the interaction network of the secretion systems of B. subtilis.

Le lymphome de Burkitt (BL) est due dans 80% des cas à une translocation chromosomique t(8;14)(q24;q32). Cette translocation marque l’évènement initial de la transformation maligne d’une cellule B normale, par délocalisation de l’oncogène CMYC à proximité du locus du gène codant pour la chaîne lourde d’immunoglobuline IGH par le mécanisme de réparation de l’ADN NHEJ durant l’hypermutation somatique (SMH). La probabilité de cette translocation est inversement proportionnelle à la distance qui sépare les loci portés par les deux chromosomes. La translocation (8;14) (q24;q32) qui apparaît durant les étapes de différentiation des lymphocytes B est encore plus importante chez les patients infectés par le virus Epstein-Barr (EBV) et le virus de l’immunodéficience humaine (VIH-1). L’objectif de notre étude est de déterminer les origines possibles de la translocation t(8;14) (q24;q32)dans les lymphocytes B normaux humains. Nous nous sommes intéressés tout d’abord à la dynamique de la localisation nucléaire des loci IGH et CMYC dans les lymphocytes B activés. Nous avons particulièrement étudié l’impact des virus EBV et VIH-1 sur l’organisation des gènes IGH et CMYC.Nous avons utilisé la technique d’hybridation in situ à fluorescence FISH pour la détection de CMYC (8q24) et IGH (14q32). Dans les lymphocytes B naïfs, CMYC est localisé du côté de la périphérie nucléaire, en revanche IGH est central, les deux loci sont complétement distants dans le noyau.L’activation des lymphocytes B induisait une augmentation de la colocalisation IGH-CMYC. La proximité physique entre les deux loci augmente la probabilité de leur translocation durant la SHM et favorise la t(8;14) (q24;q32) dans les lymphocytes B. Nous avons montré que les virus EBV et VIH-1ont un effet important sur la délocalisation IGH-CMYC dans les lymphocytes B. Nous avons aussi déterminé une molécule virale VIH-1 qui intervenait aussi dans la dérégulation de la localisation nucléaire des gènes IGH et CMYC. Nous avons déterminé deux mécanismes différents et indépendants impliqués dans la dynamique des loci IGH et CMYC : le premier mécanisme intervient dans le processus de développement normal des lymphocytes B, et le deuxième mécanisme dépend des virus ainsi que des molécules virales (particulièrement la Tat-HIV-1). / Eighty percent of Burkitt's lymphomas (BL) cases bear translocation t(8;14)(q24;q32). Thistranslocation is the initial event in malignant transformation of normal B-cell and derives from nonhomologousend joining of the oncogene CMYC to the immunoglobulin heavy chain locus IGH duringSomatic Hypermutation (SHM) of IGH. The probability of this translocation is inversely proportionalto the distance between the loci of involved chromosomes. The translocation t(8;14)(q24;q32) occursduring normal development of B-lymphocytes and more probable in patients infected with Epstein-Barr virus (EBV) and the human immunodeficiency virus (HIV-1).The subject of this study was to determine the possible origin of the translocation t(8;14)(q24;q32) inhuman normal B-lymphocytes. We followed the dynamics of the nuclear localization of IGH andCMYC genes in activated B-lymphocytes. We payed particular attention to the impact of EBV andHIV-1 viruses on dynamics of both IGH and CMYC. We applied Fluorescence in situ hybridization(FISH) for detection of CMYC (8q24) and IGH (14q32). In naïve B-cells CMYC is mainly localized inthe periphery of nucleus, whereas IGH is preferentially localized in the nuclear centre, i.e. these lociare distanced by a radius of cell nucleus. Activated B-lymphocytes displayed dramatic increase ofnumber of cells with colocalized IGH and CMYC. Close physical proximity of CMYC to IGH duringSHM amplifies the probability of occurance of translocation t(8;14)(q24;q32) in human Blymphocytes.Interestingly, we observed even more pronounced impact of EBVand HIV-1onproximity of IGH and CMYC. Finaly, among the molecules of HIV-1 we revealed those which possessthe most regulative role on dynamics of both IGH and CMYC. Our results suggest about twoindependent mechanisms of IGH and CMYC dynamics: the first is appropriate for normal developmentof B-lymphocytes and the second depends on virus and viral molecules, such as transactivator of viraltranscription HIV Tat.

Este Doutorado teve como objetivo o entendimento sobre a construção da conjugalidade homossexual feminina e masculina, a partir da eleição de casais longevos, com mais de vinte anos de relacionamento. A partir de coleta de dados por meio de entrevista semidirigida com o casal, acrescida da aplicação de pranchas previamente selecionadas do teste projetivo TAT (Teste de Apercepção Temática), individualmente, sob o método clínico-qualitativo, foram analisados quatro casais, dois masculinos e dois femininos, com e sem filhos vindos de relação heterossexual anterior, sob a perspectiva da psicanálise vincular. Os resultados apontaram para casamentos românticos, com vínculos protetivos e de afeto, porém com influências pós-modernas no que diz respeito ao fato de estarem juntos somente pelo desejo, com flexibilização das performances de gênero e democratização das relações. Os quatro casais demonstraram empenho na construção e manutenção do vínculo conjugal e harmonia no convívio, ressaltando a singularidade de cada um. Esses casais concretizaram suas relações amorosas em uma época não permissiva e patologizante da homossexualidade, fazendo uso do disfarce e da vida dupla como estratégia de sobrevivência, atitude presente ainda na vida de dois dos quatro pares. Essa característica em particular pode fazer com que casais gays necessitem de maiores espaços de fusionamento do que os heterossexuais, para que possam se fortalecer ante os desconfortos sociais pelos quais passam - principalmente os que têm filhos de uniões heterossexuais anteriores. Neste caso, além da própria separação, há também o confronto com a orientação sexual do ex-parceiro. Somente um casal de homens havia se casado legalmente, por temor aos bens conquistados frente aos filhos de um deles; o de mulheres com filho também havia legitimado a relação com União Estável e testamento. Os dois casais sem filhos não haviam providenciado nenhuma legitimação e nem estavam preocupados com isso. A herança heteronormativa que receberam foi adaptada aos seus estilos de vida, embora não tenha havido um espaço familiar e social a ser ocupado a homossexualidade e a homoconjugalidade, embora vistas, são ainda muitas vezes não-nomeadas ou reconhecidas. Não houve referências a traições, sendo que os filhos foram os deflagradores de crises em dois dos casais, ambas superadas. Os resultados alcançados confirmam a teoria psicanalítica vincular no sentido de enfatizar o conceito de alteridade, entre um e outro, como base para a compreensão desse tipo de conjugalidade / This Doctoral thesis aimed to understand the construction of female and male homosexual conjugality by selecting long-lived couples, with more than twenty years of relationship. Based on data collection through semi-structured interviews with the couples and the use of previously selected cards of the projective measure TAT (Thematic Apperception Test), under the clinical-qualitative method, four couples - two male and two female, with and without children from previous heterosexual relationships - were analyzed under the perspective of linkage psychoanalysis. Results point to romantic marriages, with protective and emotional bonds, but under post-modern influence based on the fact they are together only by their desire, with flexibility of the gender performances and democratization of the relations. The four couples demonstrated a commitment to building and sustaining the conjugal bond as well as coexisting in harmony, highlighting the individuality of each one. These couples materialized their love relationships at a nonpermissive and pathologizing time for homosexuality, using disguises and double lives as a strategy for survival, an attitude still present in the lives of two out of the four couples. This characteristic, in particular, may lead a gay couple to need bigger spaces of fusioning when compared to heterosexual couples, so that they can strengthen themselves in the face of the social discomfort which they go through particularly the ones who have kids from previous heterosexual relationships. In this case, in addition to the divorce itself, there is also the confrontation related to the sexual orientation of the ex-partner. Only one couple of men had legally married due to the fear of losing the assets acquired to the children of one of them; the couple of women with a child had also legitimated their relationship through common-law marriage and testament. Both couples without children hadnt sought any type of legal legitimacy nor were worried about it. The heteronormative heritage they received was adapted to their lifestyles, although there hadnt been a family and social place to be occupied homosexuality and homo-conjugality, while seen, are often not named nor recognized. There were no references to betrayals, although, among two of the couples, the children had been the triggers of already overcome crises. The findings confirm the linkage psychoanalysis theory, which emphasizes the concept of otherness, between one and another, as a basis to comprehend this type of conjugality

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