La protéine cellulaire BST2/Tetherin est un médiateur de l’immunité innée qui exerce son activité antivirale contre la dissémination de nombreux virus enveloppés. Ce facteur de restriction retient physiquement les virus néoformés à la surface cellulaire de la cellule infectée, empêchant ainsi le relâchement des virions du VIH-1. La protéine Vpu du VIH-1 est l’un des acteurs viraux capable de contrer cette restriction : elle diminue le niveau d’expression de BST2/Tetherin présent au site de bourgeonnement viral et restaure ainsi une production virale efficace. A ce jour, les mécanismes exacts par lesquels Vpu contre cette barrière cellulaire ne sont pas bien caractérisés. Des études récentes ont montré que HRS et Rab7A, deux protéines contrôlant la voie endo/lysosomale tardive sont impliquées dans le mécanisme déployé par Vpu pour contrer la restriction imposée par BST2. De manière intéressante, ces protéines sont également requises pour l’achèvement des étapes finales de l’autophagie. L’autophagie (macroautophagie) est un mécanisme cellulaire hautement conservé, qui permet la dégradation de composants cytoplasmiques via la formation d’une vésicule à double membrane, appelé autophagosome. L’autophagie est contrôlée par une trentaine de protéines ATG (AuTophaGy-related genes) qui sont recrutés au site de formation du phagophore qui conduira, suite à son élongation, à la formation de l’autophagosome. Il a récemment été décrit que certaine protéines ATG sont également impliquées dans d’autres fonctions cellulaires indépendante de l’autophagie, comme la résistance contre certains pathogènes. C’est le cas notamment de la phagocytose médiée par LC3 (LC3-associated phagocytosis, LAP), un mécanisme indépendant du complexe de pré-initiation de l’autophagie au cours duquel certaines protéines ATG modifient la membrane du phagosome et accélèrent la dégradation des éléments phagocytés. L’objectif de ma thèse était donc de définir si l’autophagie ou certaines protéines de l’autophagie sont impliquées dans le mécanisme moléculaire déployée par Vpu pour contrer la restriction imposée par BST2/Tetherin sur la production virale. Au cours de ma thèse, nous avons démontré l’implication de la protéine LC3C dans le mécanisme par lequel Vpu contrecarre la restriction imposée par BST2 sur la libération des particules virales du VIH-1 dans le milieu extracellulaire. Plus précisément, nos résultats montrent que les protéines ATG5 et Bécline 1, et non tous les composants de l’autophagie, agissent avec la protéine LC3C pour faciliter l’action de la protéine virale Vpu sur la restriction imposée par BST2. L’expression de la protéine LC3C favorise l’élimination par Vpu des molécules de BST2 présentes au site du bourgeonnement, permettant ainsi une libération plus efficace de particules virales VIH-1. Nos expériences d’immunofluorescence montrent que les protéines BST2 et Vpu sont présentes dans des compartiments marqués par LC3, protéine décorant les phagophores, autophagosomes et phagosomes impliqués dans la LAP. Enfin, nos données biochimiques révèlent une interaction spécifique et directe entre la protéine Vpu et la protéine LC3C via un motif LIR non-canonique. L’intégrité de ce motif présent dans le domaine cytoplasmique de Vpu est nécessaire pour la levée par Vpu de la restriction imposée par BST2. En conclusion, mes travaux de thèse montrent que la protéine Vpu du VIH-1 via son interaction avec la protéine d’autophagie LC3C utilise un mécanisme de LAP pour contrer la restriction induite par BST2 sur la production de virus VIH-1. / BST2/Tetherin is a key mediator of the innate immune system that restricts the dissemination of enveloped viruses. This restriction factor impedes the release of de novo formed HIV particles by physically retaining them at the surface of infected cells. The HIV-1 protein Vpu promotes the release of virus by counteracting this restriction. Vpu removes BST2 present at the budding site and downregulates BST2. The mechanisms by which Vpu counteracts BST2 are still not well understood. Recently, we showed that HRS and Rab7A, two regulators of the endocytic and autophagic pathway participates to the mechanism by which Vpu counteracts BST2-mediated restriction on HIV-1 release. Interestingly, these two proteins are also required in the autophagy pathway. Autophagy (macroautophagy) is a highly conserved degradative mechanism that leads to degradation of cytosolic components through the formation of double-membrane vacuoles called autophagosomes that sequester cytosolic material. This process is tightly regulated by the ATG proteins that are hierarchically recruited at the phagophore assembly site to form the autophagosome. Some ATG proteins are additionally involved in non autophagic cell functions involved in maintenance of cell homeostasis and resistance of pathogens. Notably, they participate in microbe clearance through LC3-associated phagocytosis, a process independent of autophagic preinitiation complex in which some ATG proteins directly modify the phagosomal membrane to enhance degradation of phagocytosed elements. The aim of my thesis was to explore if the autophagy pathway or some ATG proteins could be involved in the molecular mechanism by which Vpu counteracts BST2/Tetherin on HIV-1 release. Here, we reveal that the protein LC3C is required in the Vpu-induced antagonism of BST2 restriction. Our results show that only ATG5 and Beclin-1, and not all the components of the autophagy pathway, act with LC3C to favor the counteraction of Vpu on BST2 restriction, and thus enhance HIV-1 release. We report that BST2 and Vpu are present in LC3-positive compartments. We found that Vpu selectively interacts with the ATG8 ortholog, LC3C, through a non-canonical LIR motif by immunoprecipitation and GST pulldown assays. This motif is required for Vpu to antagonize BST2 restriction. LC3C expression favors the removal of BST2 from HIV-1 budding site, and thus HIV-1 release in BST2 expressing cells. Altogether, our data support the view that Vpu uses a non-canonical autophagy pathway reminiscent of LC3-associated phagocytosis to counteract BST2 restriction.

O reconhecimento da flagelina é compartilhado pelo receptor transmembrânico TLR5 e citosólico NAIP5/NLRC4. Entretanto, pouco se sabe sobre os mecanismos efetores individuais induzidos a partir do reconhecimento extra e intracelular da flagelina. Aqui, nós demonstramos que macrófagos estimulados com a flagelina citosólica (FLA-BSDot) induziu a expressão de iNOS, enzima responsável pela produção do óxido nítrico (NO). A expressão de iNOS foi dependente do eixo NAIP5/NLRC4/caspase-1 e independente de IL-1β, IL-18 e MyD88, descartando a via de ativação dos TLRs. Ainda, esta via não requer a ativação do fator de transcrição IRF-1, mas envolve a ativação do NF-kB, assim como a clivagem da enzima PARP-1 (poly(ADP-ribose)polymerase-1). Por fim, avaliamos a relevância biológica desta via no controle das infecções por L. pneumophila e S. Typhimurium, dados que definem um mecanismo efetor adicional no controle de patógenos. / Recognition of flagellin is shared by transmembranic TLR5 and cytosolic NAIP5/NLRC4. However, little is known about the individual effector mechanisms induced by extra and intracellular flagellin. Here, we have demonstrated that cytosolic flagellin-stimulated macrophages (FLA-BSDot) induced iNOS expression, an enzyme responsible for the production of nitric oxide (NO). iNOS expression was dependent of the NAIP5/NLRC4/caspase-1 axis and independent of IL-1β, IL-18 and MyD88, discarding TLRs signaling pathway. Still, this pathway do not require the activation of IRF-1 transcriptional factor, but involves NF-kB activation as well as the cleavage of the enzyme, PARP-1 (poly(ADP-ribose)polymerase-1). Finally, we have evaluated the biological relevance of this pathway in the control of the infections by L. pneumophila e S. Typhimurium, which define an additional effector mechanism to the control of pathogens.

Introdução: O desenvolvimento de antirretrovirais (AVRs) para o vírus HIV em pacientes multiexperimentados é prioridade em saúde pública e, além de supressão virológica máxima, recuperação e estabilização imunológica, os ensaios clínicos randomizados (ECRs) que envolvem esquemas de antirretrovirais utilizados em terapias de resgate devem ser capazes de demonstrar segurança na sua utilização, a curto e longo prazos. Objetivo: Realizar revisão sistemática e caracterizar os achados de segurança da terapia antirretroviral (TARV) para a infecção pelo vírus HIV-1 nos ECRs que utilizaram esquemas otimizados de base (EOB) em pacientes multiexperimentados através, principalmente, da avaliação da qualidade da informação sobre riscos baseada na extensão do CONSORT sobre riscos e da avaliação da apresentação de achados laboratoriais que são proxy de desfechos conhecidamente relevantes. Metodologia: Foram revisadas as bases de dados MEDLINE, EMBASE, LILACS, Colaboração Cochrane, SCOPUS e ISI Web of Science visando identificar publicações entre janeiro/2003 e agosto/2014. Também foram revisadas as bases de dados dos seguintes congressos científicos internacionais: International AIDS Conference; Conference on Retroviruses and Opportunistic Infections (CROI); Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); e International Congress on Drug Therapy in HIV Infection. Não foram feitas restrições em relação à língua da publicação dos estudos. Os critérios para inclusão dos estudos foram: ECRs com pelo menos 16 semanas de duração e com pelo menos 100 indivíduos que avaliaram segurança da terapia antirretroviral (TARV) em pacientes com infecção pelo HIV- 1 caracterizados como multiexperimentados. Resultados: Vinte e quatro ECRs foram incluídos, sendo 16 estudos originais e 8 extensões. Vinte e três (95,8%) estudos foram exclusivamente financiados pela indústria farmacêutica. Apenas 15 (62,5%) estudos apresentaram eventos adversos (EAs) clínicos graves. Ocorrência de limiar para a apresentação de resultados foi observada em 13 (86,7%) estudos que apresentaram EAs graves, 22 (95,6 %) estudos que apresentaram EAs considerados comuns e em 8 (42,1%) estudos que apresentaram informações sobre desfechos laboratoriais. Seis (25%) ECRs não apresentaram informações sobre os desfechos laboratoriais investigados. Oito (33,3%) estudos não apresentaram informações sobre triglicerídeos, colesterol total, LDL (low density lipoprotein) e hiperglicemia, resultando em ausência de informação sobre os referidos desfechos para os medicamentos maraviroque (MVC) e vincriviroc (VIC). Nove estudos não apresentaram informações sobre marcadores hepáticos, resultando em ausência de informação destes desfechos para o medicamento enfuvirtida (ENF). Dezoito (75%) estudos não apresentaram informações sobre creatinina, resultando em ausência de informação sobre este desfecho para os medicamentos ENF, tipranavir (TPV) e darunavir (DRV). Conclusões: A informação sobre riscos em ECRs envolvendo TARV para pacientes multiexperimentados é altamente seletiva e insuficiente. Além disso, desfechos conhecidamente relevantes não tem sido incluídos a priori nestes estudos. Os ECRs que avaliam novos ARVs ainda apresentam as características dos estudos do início da era TARV, quando, diante de um cenário com grande mortalidade associada ao HIV, o principal objetivo era preservar a vida dos pacientes, em detrimento das questões de segurança da terapia. / Introduction: The development of antiretroviral drugs (ARVs) for treating the HIV virus in treatment-experienced patients is a priority in public health. In addition to virological suppression, recovery and stabilization of the immune system, randomized clinical trials (RCTs) involving antiretroviral regimens used in recovery therapies must be able to demonstrate short and long term safety in their use. Objective: To perform a systematic review and to characterize the findings regarding the safety of cART for HIV-1 virus infections in RCTs that used optimized background regimen in treatmentexperienced patients primarily through assessing the quality of information based on the extent of the CONSORT on risks and evaluating the overall presentation of laboratory findings that are known to proxy relevant outcomes. Methodology: The following databases were researched: MEDLINE, EMBASE, LILACS, The Cochrane Collaboration, SCOPUS and ISI Web of Science. We searched for works published between January/2003 and August/2014. The databases of the following international scientific conferences were also researched: International AIDS Conference; Conference on Retroviruses and Opportunistic Infections (CROI); Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC); and International Congress on Drug Therapy in HIV Infection. There were no restrictions regarding the language of the studies. The criteria for inclusion were the following: RCTs that lasted at least 16 weeks and with at least 100 participants who evaluated the safety of antiretroviral therapies (cART) in treatment-experienced patients with HIV-1. Results: Twenty-four RCTs were included, sixteen original studies and eight extensions. Twenty-three (95.8%) of the studies were funded exclusively by the pharmaceutical industry. Only fifteen (62.5%) of the studies showed serious clinical adverse events (AEs). The occurrence threshold for the presentation of results was observed in thirteen (86.7%) of the studies that showed serious AEs, twenty-two (95.6%) of the studies that showed AEs considered to be common, and eight (42.1%) of the studies which presented information on laboratory outcomes. Six (25%) RCTs have not provided information on laboratory outcomes investigated. Eight (33.3%) studies did not provide information on triglycerides, total cholesterol, LDL (low density lipoprotein) and hyperglycemia, resulting in lack of information on these outcomes for the drugs maraviroc (MVC) and vicriviroc (VIC). Nine studies did not provide information on hepatic markers, resulting in lack of information on these outcomes for the drug enfuvirtide (ENF). Eighteen (75%) studies did not provide information on creatinine, resulting in lack of information on this outcome for the drugs ENF, tipranavir (TPV) and darunavir (DRV). Conclusions: Information on the risks of RCTs involving cART for treatment-experienced patients is highly selective and insufficient. Moreover, known relevant outcomes have not been included a priori in these studies. RCTs evaluating new ARVs have the same characteristics of studies of early ARVs, when, faced with a scenario of high mortality associated with HIV, the main goal was to preserve the lives of patients, at the expense of the safety in therapy.

L’autophagie est un mécanisme vacuolaire de dégradation de matériel cytoplasmique permettant le maintien de l’homéostasie cellulaire, mais elle peut être également activée par de nombreux stress, comme l’infection virale. Le virus de l’Herpès Simplex de type 1 (HSV 1) est capable de contrecarrer ce mécanisme de défense antivirale. HSV-1 possède une protéine ICP34.5 capable d’inhiber l’autophagie en se liant à Beclin 1, une protéine de la machinerie autophagique. Nous avons mis en évidence une deuxième protéine d’HSV-1 capable d’inhiber l’autophagie, la protéine tardive Us11, qui pourrait avoir un rôle complémentaire à celui d’ICP34.5 dans le contrôle de l’autophagie par le virus.Nous montrons que l’expression ectopique d’Us11 permet de bloquer l’autophagie induite par différents stimuli, et ce de manière similaire à ICP34.5. De plus, dans un contexte viral, l’expression précoce d’Us11 dans des cellules infectées par un virusICP34.5 permet un contrôle de l’autophagie comparable à celui d’un virus sauvage. Nous avons ensuite recherché le mécanisme d’action d’Us11. La protéine Us11 a été décrite comme pouvant interagir avec la kinase dépendante de l’ARN double brin PKR, empêchant ainsi la phosphorylation de son substrat eIF2, un facteur d’initiation de la traduction. Nous avons observé qu’en l’absence de PKR, Us11 n’est plus capable d’inhiber l’autophagie. Nous avons pu confirmer qu’Us11 a besoin de se lier à PKR pour exercer son activité inhibitrice par la construction de formes tronquées d’Us11, permettant de montrer l’importance de son domaine d’interaction avec PKR dans l’inhibition de l’autophagie. L’étude des formes tronquées d’Us11 a soulevé le fait que le domaine N-terminal était également nécessaire. Aucune interaction de ce domaine avec une protéine cellulaire n’a été identifiée à ce jour, mais il pourrait permettre l’interaction d’Us11 avec une autre protéine de la machinerie autophagique. Cependant, nous avons montré qu’Us11 n’interagissait pas avec Beclin 1 et n’avait pas d’effet sur la kinase mTOR, une autre voie importante de l’autophagie. Enfin, nous avons étudié la modulation de la voie PKR/eIF2 lors de la stimulation de l’autophagie par la carence, et nos résultats suggèrent que cette voie joue un rôle sous-estimé dans la réponse à la carence.Le mécanisme d’action de la protéine Us11, qui consiste en un blocage de l’autophagie en inhibant PKR, n’avait jamais été décrit auparavant. Ce travail ouvre de nombreuses perspectives dans l’étude de la voie PKR/eIF2 vis à vis de la régulation de l’autophagie, ainsi que dans la compréhension de l’implication de l’autophagie dans la neurovirulence d’HSV-1. / Autophagy is an evolutionary conserved vacuolar mechanism allowing to degrade cytoplasmic components and to maintaining cellular homeostasis, but it can also be triggered by a variety of stress-related conditions, including viral infection. The herpes simplex virus 1 (HSV-1) is able to counteract this antiviral mechanism. Notably, HSV-1 encodes a protein, IPC34.5, which inhibits autophagy through its interaction with the autophagy machinery protein Beclin 1. In the present work, we uncovered a second anti-autophagic protein from HSV-1, the late protein Us11, which likely plays a complementary role to ICP34.5 regarding the inhibition of autophagy by the virus. We demonstrated that ectopic expression of Us11 inhibited autophagy triggered by different stimuli, as observed for ICP34.5. Moreover, during viral infection, early expression of Us11 was sufficient to block autophagy in cells infected with a ICP34.5 virus, similarly to the wild-type virus. We then explored the mechanism of action of Us11. Us11 has been described as capable of interacting with the dsRNA-dependent kinase PKR, therefore preventing it to phosphorylate its substrate eIF2, a translation initiation factor. We demonstrated that Us11 was no longer able to inhibit autophagy when expressed in PKR-deficient cells. We confirmed that Us11 binding to PKR was necessary for its function by constructing various truncated forms of Us11 that showed that the PKR-binding domain was crucial. We also unveiled the importance of a domain located within the N-terminal part of Us11. This domain has no cellular molecular partner known, but it can allow Us11 to interact with another protein of the autophagy machinery. However, we further showed that Us11 did not interact with Beclin 1 nor affected the kinase activity of mTOR, another important pathway regulating autophagy. In our work, we also gained insights into regulatory mechanisms of starvation-induced autophagy.The inhibition of autophagy through the specific blockade of PKR by Us11 had never been previously described. This work thus paves the way for studying the involvement of PKR/eIF2 pathway in the regulation of autophagy and for exploring the role of autophagy in HSV-1 neurovirulence.

O modelo não linear supersimétrico em l+l dimensões é acoplado à supergravidade. Ao quantizarmos a teoria, os campos de matéria adquirem massa dinamicamente, o que ocasiona a quebra da invariança de Weyl. Este fato implica em que as funções de dois pontos do Gravitino e do Graviton, obtidas a partir da ação efetiva, não sejam triviais. Em particular a função de dois pontos do Gravitino apresenta um polo no infravermelho. Nós conjeturamos que este polo está ligado com o confinamento dos graus de liberdade supersimétricos da teoria. Isto porque, ao restringirmos o domínio de integração de x IND. 1 à uma medida finita L (quebrando as invarianças da teoria), aparece na função de dois pontos do Gravitino um termo de massa, que decresce exponencialmente em função de L. Neste contexto relacionamos este model o como da corda supersimetrica e definimos um critério de estabilidade para a mesma. / The nonlinear o supersymmetric model in l+l dimensions is coupled to supergravity. When we quantize the theory, the matter fields acquire mass dynamically, which leads to the breaking of the Weyl invariance. This fact implies that the two point functions of the Gravitino and the Graviton, obtained from the effective action, become non trivial. Particularly, the two point function of the Gravitino presents a pole in the infrared region. We conjecture that this pole is related to the confinement of all supersymmetric degrees of freedom of the theory. If we restraind the integration domain of x1 to a finite lengh L (breaking all invariances of the theory), there appears a mass term in two point function of the Gravitino, which decreases exponentially with L. In this context we relate this model with that of the supersymmetric string and define a stability criteria for the latter.

Islet transplantations have been successful in restoring glucose homeostasis in patients with diabetes; however, the limited number of donor organs limits the success of this treatment. The lineage reprograming of different cell sources to beta cells potentially provides an unlimited supply of insulin-producing cells for regenerative therapy for patients with diabetes. The aim of this study was to investigate the ability to transdifferentiate two cell lines into an endocrine lineage. Insulin production in pancreatic beta cells can be increased using a small molecule, 3,5-disubstituted isoxazole, N-cyclopropyl-t-(thiophen-2-yl)isoxazole-3-carboxamide (isoxazole) but its effect on other cell types has not been reported. Here, we investigated the lineage reprogramming of PANC-1 pancreatic ductal cells to insulin producing cells by isoxazole treatment. Gene expression was performed using RT-PCR and qPCR for approximately 30 genes critical to beta cell development and function. In addition, quantitative proteomic profiling was performed using LC-MS by monitoring protein abundance in isoxazole-treated PANC-1 cells compared to time-matched controls. Isoxazole treatment stimulated PANC-1 cells to aggregate into islet-like clusters and gene expression analysis revealed induction of important developmental beta cell markers including NGN3, NEUROD1 and INSULIN. In addition, beta cell surface markers were also upregulated such as CD200, GPR50, TROP-2, GLUT2 and SLC30A8. Using LC-MS a catalogue of approximately 2400 identified proteins was generated; 257 proteins were differentially expressed in isoxazole-treated cells compared to DMSO-vehicle controls at p < 0.05. Amongst the proteins upregulated were molecules that regulate metabolic processes and cytoskeletal reorganisation. The expression of the majority of these proteins has not been previously reported or studied in the context of beta cell differentiation. Functional analysis of the relative protein changes was determined using Ingenuity Pathway Analysis, IPA, and gene ontology, GO, software, which revealed the regulation of several cellular canonical pathways including metabolic pathways, cell adhesion, remodelling of epithelial adherens junctions and actin cytoskeleton signalling. The effects of isoxazole were further studied in the A549 lung cancer cell line. Similar effects were observed, such as the induction of pro-endocrine markers NGN3 and NEUROD1 and endocrine-specific hormones INS and GCG. These results indicate that isoxazole has the capacity to transdifferentiate pancreatic and non-pancreatic cell origins into an endocrine lineage. This study reveals the powerful induction capacity of isoxazole in inducing cellular reprogramming events.

Infants born to HIV-1 positive mothers are at risk of acquiring the infection through pro-longed breastfeeding due to the presence of HIV-1 cell-free RNA and cell-associated DNA in breast milk. However, there is limited focus on vaccines to prevent mother-to-child transmission (MTCT) via breastfeeding. Mycobacterium tuberculosis (M. tuberculosis) the causative agent of Tuberculosis (TB) is the most common cause of AIDS-related deaths. Most infants in Africa receive Bacillus Calmette-Guérin (BCG) at birth or soon after birth and it is the only licensed vaccine for TB. The development of a dual platform vaccine against HIV-1 and TB would be a logical effort to combat these two deadly diseases. Thus, rBCG expressing an HIV-1 derived immunogen may induce HIV-1 responses at birth and these responses can be boosted at adolescence, by a heterologous vector such as modified vaccinia Ankara (MVA) or Chimpanzee adenovirus serotype 63 (ChAdV63). In the first study, I assessed BCG-based vaccines derived from BCG Danish SSI-1331 (BCG₁₃₃₁), expressing an HIV-1 immunogen HIVconsv either by an episomal plasmid (BCG.HIVconsv401^epi) or integrated into the BCG chromosome (BCG.HIVconsv401^int) in a prime-boost regimen. BALB/c mice were immunised with the different prime-boost regimens. rBCG alone was unable to induce detectable HIV-1-specific T-cell responses, however, when used in a prime-boost strategy, elevated HIV-1-specific T-cell responses were observed. In the second study, I aimed to construct marker-less mycobacterium-vectored HIV-1 vaccines using the operator-repressor titration (ORTA®) system as an alternative system for antibiotic resistance gene free vaccines. This rBCG vaccine would express the HIVconsv immunogen. I first constructed plasmids carrying the lac operator lacO and tetracycline operator tetO to enable use in ORT Escherichia coli (E.coli) and Mycobacterium strains, respectively. The ORT system was successful in E. coli and not in mycobacterium. I also constructed plasmids carrying mycobacterium essential genes that would allow for genetic manipulation in Mycobacterium and the use of ORT in mycobacterium. Although the plasmid construction was successful, in the end, genetic manipulations in Mycobacterium and the production of an ORT based BCG (ORT-VAC) was not successful. Finally, I evaluated the immunogenicity of conventional DNA plasmid pTH.HIVconsv compared to Semliki Forest virus replicon DREP.HIVconsv in rhesus macaques. Immunisations were done in a prime-boost strategy with heterologous vectors MVA or ChAdV63 delivering the same immunogen, HIVconsv. It was found that DREP.HIVconsv which was at least 20-fold lower dose than pTH.HIVconsv was capable of inducing comparable T-cell responses and in some experiments, the responses were superior to the conventional DNA plasmid pTH.HIVconsv.

Mon travail de thèse a porté sur le développement de méthodes avancées de RMN des solides. Nous avons notamment introduit de nouvelles méthodes de corrélation homonucléaire compatibles avec une rotation rapide de l’échantillon et des champs magnétiques élevés. Nous avons montré que la robustesse de la technique fp-RFDR peut être améliorée par l’utilisation d’un supercyclage (XY8)41. Cette méthode a été employée pour sonder les proximités 13C-13C and 31P-31P dans les solides. Nous avons aussi introduit expériences de corrélation 13C-13C du deuxième ordre avec assistance des protons, appelées SHA+, afin d’observer les proximités 13C-13C à longue distance dans les solides. Au cours de ma thèse, nous avons aussi amélioré les méthodes de corrélation hétéronucléaire pour l’observation indirecte des noyaux 14N via les protons. Nous avons montré que la résolution spectrale dans la dimension indirecte des spectres HMQC peut être accrue par l’utilisation de séquences de découplage homonucléaire pendant le temps t1. Nous avons aussi proposé l’utilisation d’impulsion sélective de la bande centrale pour l’excitation des noyaux 14N dans les expériences HMQC 1H{14N}. L’efficacité de ces impulsions sélectives de la bande centrale est comparable aux méthodes d’excitation large bande, compte tenu des champs radiofréquence produits par les sondes RMN pour l’étude des solides. La dernière partie de mon travail de thèse a porté sur l’amélioration des séquences d’écho quadripolaire pour l’acquisition des spectres 2H de solides. Les distorsions de ces spectres ont été réduites par l’introduction de nouvelles impulsions composites. / My PhD work has focused on the development of advanced solid-state NMR methods. We have notably developed homo-nuclear correlation methods compatible with high MAS frequencies and high magnetic fields. First, we have shown that the robustness of finite pulse RadioFrequency Driven Recoupling (fp-RFDR) technique can be improved by the use of nested (XY8)41 super-cycling. Such method has been employed to probe 13C-13C and 31P-31P proximities in solids. Second, we have also introduced a second-order proton-assisted 13C-13C correlation experiment, denoted “Second-order Hamiltonian among Analogous nuclei plus” (SHA+), to observe long-range 13C-13C proximities in solids at fast MAS and high magnetic field. During my PhD, we have also improved the heteronuclear correlation methods for the indirect observation of 14N nuclei via protons. We have shown that the spectral resolution along the indirect dimension of proton-detected Heteronuclear Multiple Quantum Correlation (HMQC) spectra can be enhanced by applying homonuclear dipolar decoupling schemes during the t1 period. We have also proposed the use of centerband-selective radio-frequency (rf) pulses for the excitation of 14N nuclei in 1H{14N} HMQC experiment. The efficiency of these centerband-selective pulse is comparable to that of broadband excitation given the rf field delivered by common solid-state NMR probes. The last part of my PhD focuses on the improvement of the quadrupolar echo sequence for the acquisition of the 2H spectra of solids. The distortions of such spectra were reduced by the introduction of novel composite pulses.

Introdução: A disfunção erétil (DE) é definida como a incapacidade de alcançar ou manter a ereção do pênis para um desempenho sexual satisfatório, contribuindo significativamente para a baixa qualidade de vida e morbidade psicossocial masculina. A endotelina-1 (ET-1), um potente peptídeo vasoconstritor que promove contração lenta e sustentada em células de músculo liso vascular, possui grande importância na fisiopatologia da DE. Diversos estudos mostram que o aumento da expressão de mediadores inflamatórios está intimamente ligado ao desenvolvimento da DE. O inflamassoma é um complexo multiprotéico do sistema imune inato que atua através da ativação da caspase-1 e resulta na maturação de citocinas pró- inflamatórias, tais como interleucina- IL (IL-l?). O receptor NLRP3 faz parte do inflamassoma e sua ativação leva a clivagem de caspase-1 e consequente secreção de IL-1?. A ET-1, também possui papel importante na inflamação crônica vascular, mediando a liberação de citocinas pró-inflamatórias. No entanto, ainda é desconhecido se a ação pró- inflamatória da ET-1 em células de músculo liso é mediada pela ativação da via do inflamassoma. Hipótese: A ET-1 ativa o NLRP3 em células do músculo liso do corpo cavernoso (CMLCC), promovendo alterações na reatividade do corpo cavernoso (CC). Objetivo: Avaliar o papel da endotelina-1 na ativação do NLRP3 em CMLCC de camundongos. Métodos: CMLCC de camundongos C578BL/6 (WT) e NLRP3-/- foram cultivadas em meio de cultura DMEM acrescido de soro fetal bovino (SFB), 10%, foram pré- incubadas com endotelina-1 nas concentrações de 10-9, 10-8 e 10-7 M, em presença de LPS ou veículo. Avaliamos o efeito da deleção do NLRP3 sobre a reatividade do CC (contratilidade e relaxamento mediante estímulos por campo elétrico e/ou farmacológico). Após, avaliamos o efeito da ET-1 na ativação do NLRP3, nas alterações sobre a reatividade do CC de camundongos WT, e se estas persistiriam nos camundongos NLRP3-/- e caspase1/11-/- . Resultados: As células apresentaram-se fluorescentes para marcação para ?-actina e não para Von Willebrand, caracterizando assim que não houve contaminação com células endoteliais. A incubação com a ET-1 10-7 M por 24 h na presença de LPS ou veículo aumentou a atividade da caspase-1 em CMLCC de camundongos WT e este efeito não ocorreu nas CMLCC de camundongos NLRP3-/-. Não se observou diferença com relação à massa corporal ou massa dos órgãos entre os animais WT e NLRP3-/-. O CC de animais NLRP3-/- apresenta prejuízo para o relaxamento mediado por nitroprussiato de sódio (NPS) quando comparado com as tiras de CC de camundongos WT. A incubação com ET-1 10-7 M por 4 horas promove aumento na contração para fenilefrina (PE) e prejuízo no relaxamento induzido por nitroprussiato de sódio (NPS), e o mesmo efeito não é observado nas tiras de CC de camundongos NLRP3-/- e caspase1/11-/-. Conclusão: O NLRP3 contribui para o aumento na contração e prejuízo no relaxamento produzido pela ET-1 em CC de camundongos, possivelmente através da ativação da caspase-1 / Introduction: Erectile dysfunction (ED) is defined as the inability to achieve or maintain penile erection to perform sexual intercourse, it contributes significantly to the low quality of life and male psychosocial morbidity. Endothelin-1 (ET-1), a potent vasoconstrictor peptide that promotes slow and sustained contraction of vascular smooth muscle cells, has great importance in the pathophysiology of ED. Several studies show that increased expression of inflammatory mediators is closely linked to the development of ED. The inflammasome is a multiproteic complex of the innate immune system that acts through activation of caspase-1, which leads to maturation of pro-inflammatory cytokines such as interleukin-1 beta (IL-l?). The activation of NLRP3 receptor, part of the inflammasome, leads to caspase-1 cleavage and subsequent secretion of IL-1?. ET-1 also plays an important role in chronic vascular inflammation by mediating the release of pro-inflammatory cytokines. However, it is still unknown whether pro-inflammatory actions of ET-1 on smooth muscle cells is mediated by the activation of the inflammasome. Hypothesis: ET-1 activates NLRP3 in smooth muscle cells of the corpora cavernosa (SMCCC), promoting changes in corpus cavernosum (CC) reactivity. Objective: To evaluate the role of endothelin-1 in the activation of the NLRP3 in SMCCC of mice. Methods: SMCCC of C57BL/6 (WT) and NLRP3-/- mice were grown in DMEM culture medium supplemented with bovine fetal serum (FBS) 10%, pre-incubated with endothelin-1 at concentrations of 10-9, 10- 8 and 10-7M, in the presence of LPS or vehicle. We evaluated the effect of the NLRP3 deletion on the reactivity of the CC (contractility and relaxation by electric field and/or pharmacological stimulation). After that, we evaluated the ET-1 effect on activation NLRP3, changes on the reactivity of the CC of WT, and if these alterations would persist NLRP3-/- and caspase1/11-/- mice. Results: The cells presented fluorescent labeling to ?-actin, but not for Von Willebrand factor, characterizing absence of endothelial cells contamination. The incubation with 10-7 M ET-1 for 24 h in the presence of LPS or vehicle increased caspase-1 activity in SMCCC from WT, but not from NLRP3-/- mice. No difference was observed in body mass or weight of the organs between WT and NLRP3-/- animals. The CC from NLRP3-/- animals displayed impaired relaxation mediated by sodium nitroprusside (SNP) when compared to WT CC. The incubation with ET-1 10-7 M for 4 hours promoted an increase in the contraction to phenylephrine (PE) and reduced relaxation induced by sodium nitroprusside (SNP). The same effect was not observed in CC strips from NLRP3-/- and caspase1/11-/- mice. Conclusion: NLRP3 contributes to the increase in contraction and impaired relaxation produced by ET-1 in mice CC, possibly by activation of caspase-1

O HIV é dividido em HIV-1 e 2. O HIV-1 é classificado em três grupos, o M (main) responsável pela maioria das infecções, O (outlier) e N(new). Na atualidade, nove subtipos puros e 43 formas recombinantes do grupo M são reconhecidos incluindo sub subtipos. O subtipo C é o mais prevalente, representando aproximadamente 56% das infecções no mundo. Não foram descritas as conseqüências exatas destes subtipos, algumas evidências indicam que alguns subtipos podem ter vantagem na transmissão viral, enquanto outros na replicação, além de influenciar nas vias de mutação, aparecimento de resistência mais rapidamente e fracasso do tratamento. No Brasil os subtipos B e C são os mais freqüentes e acima de 50% dos indivíduos recentemente infectados pertencem aos subtipos não-B. O Rio Grande do Sul (RS) exibe a prevalência mais alta do subtipo C e há uma forma recombinante específica C e B – CRF-31. Entre julho de 2002 e janeiro de 2003 foi realizada a genotipagem e subtipagem do vírus HIV-1 em 178 pacientes, consecutivamente, atendidos no ambulatório de HIV/AIDS do Hospital de Clínicas de Porto Alegre (HCPA) desde 1983, os quais assinaram o termo de consentimento livre e informado. Após revisão dos resultados destes testes restaram 161 pacientes devido a perda de dados relacionados a nome e prontuário (não foi possível relacionar as 17 amostras a um nome e/ou prontuário). Em busca das variáveis demográficas e epidemiológicas realizou-se revisão de prontuário e busca ativa através de telefonema aos pacientes sem seguimento por pelo menos 2 anos. Consultou-se também o obituário de HIV/AIDS. O objetivo principal deste estudo é avaliar os padrões variáveis do HIV-1, prevalência nos indivíduos assistidos no Hospital de Clínicas de Porto Alegre e relacionar estes subtipos com características demográficas e epidemiológicas. Dos 161 pacientes, 90 eram do sexo masculino e 71 do sexo feminino. O subtipo B foi o mais prevalente em nossa amostra sendo encontrado em 53% dos pacientes. Até 1990 o subtipo B era encontrado na grande maioria dos pacientes com HIV, contribuindo com 75% das infecções. A partir de 2000 o subtipo C passou a representar a maioria das infecções, ficando com 33,3% dos casos, porém este aumento não foi estatisticamente significativo. Dos 161 pacientes com subtipo identificado, 19 não se conhecia a categoria de exposição. Revelando entre o subtipo B, a grande maioria dos pacientes nas categorias de exposição sexual, subdivisão homossexual e bissexual, o subtipo C foi relacionado com categorias de exposição heterossexual e uso de drogas injetáveis, quando se analisou separadamente o subtipo C com as categorias de exposição, não se demonstrou relação com categoria específica. Já o subtipo B analisado separadamente mostrou grande associação com a categoria de exposição MSM e bissexual. Em relação a procedência dos pacientes, houve associação estatisticamente significativa entre o subtipo B e a cidade de Porto Alegre, onde o mesmo foi encontrado na maioria dos pacientes. O subtipo C foi encontrado na maioria dos pacientes procedentes de regiões metropolitanas e do interior do estado. O conhecimento da prevalência dos subtipos de acordo com a procedência dos pacientes pode ajudar as autoridades competentes a dirigir esforços na prevenção da infecção pelo HIV.