Le virus T lymphotropique humain HTLV-1 est l’agent étiologique de la leucémie-lymphome T de l’adulte (ATLL) et de nombreuses maladies inflammatoires. HTLV-1 est associée à de nombreuses modifications quantitatives de l’expression des gènes cellulaires. À ce jour, ces modifications ont été décrites essentiellement à l’échelle transcriptionnelle à travers notamment les effets de l’oncoprotéine virale Tax, et plus récemment HBZ. Outre leurs impacts sur les niveaux d’activité des promoteurs, certains facteurs apparaissent jouer également un rôle dans la régulation de l’épissage alternatif. Ce mécanisme essentiel à la diversité du transcriptome et du protéome cellulaire, apparait étroitement couplé à la transcription et ses dérégulations sont de plus en plus décrites dans les phénomènes cytotoxiques et pathogènes tels que les infections et les cancers. Dans ce contexte, mon travail s’est intéressé à caractériser les profils d’expression des exons des cellules T CD4+ infectées ou non, et transformée ou non par HTLV-1 in vivo. Dans une seconde étude, j’ai abordé les aspects mécanistiques des modifications d’épissage alternatif par HTLV-1. Mes données montrent que, outre ses effets sur la régulation quantitative de l’expression des gènes cellulaires, l’activation de la voie NF-kB par l’oncogène Tax est impliquée dans la reprogrammation de l’épissage alternatif de nombreux gènes. Ces données révèlent un nouveau degré de complexité dans les mécanismes de dérégulation de l’expression des gènes cellulaires par HTLV-1 et ouvre de nouvelles perspectives d’investigations dans la compréhension des processus leucémogènes associés à l’infection par le virus HTLV-1 / Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects. HTLV-1 quantitatively modifies the pattern of cellular gene expression. Exon expression analysis shows that patients’ untransformed and malignant HTLV-1+ CD4+ T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. A total of 486 exon modifications occurred in untransformed infected CD4+ cells were detected in ATLL arguing for a role of AEUs in HTLV-1 leukemogenesis. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patters of 3977 exons that discriminate uninfected, infected, and transformed CD4+ T-cells. Exposing cells to splicing inhibitors revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provides news targets for treating ATLL. Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets

O vírus linfotrópico de células T humanas do tipo 1 (HTLV-1) é o agente etiológico de duas principais manifestações clínicas, a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP) e a leucemia/linfoma de células T do adulto (ATLL). O desenvolvimento de HAM/TSP, uma doença neuroinflamatória crônica é relacionada a uma complexa interação entre vírus e resposta imunológica do hospedeiro. A expressão do gene viral tax desempenha um papel importante na patogênese da HAM/TSP pela ativação de diversos genes celulares, incluindo genes das citocinas inflamatórias IFN-? e TNF- Recentemente, os exossomos surgiram como um importante fator na comunicação célula-célula contribuindo em diversos processos celulares, inclusive na modulação do sistema imunológico. Considerando o potencial papel dos exossomos na imunomodulação e no processo inflamatório, o objetivo deste trabalho foi avaliar se os exossomos produzidos por células infectadas pelo HTLV-1 carreiam moléculas virais e se poderiam participar dos processos de imunomodulação e inflamação crônica observados na HAM/TSP. Os exossomos foram isolados de células infectadas pelo HTLV-1 da linhagem celular C8166, avaliados quanto à presença de RNAm viral tax e testados quanto à capacidade de ativar células mononucleares do sangue periférico (PBMC) à indução de resposta inflamatória. Neste trabalho, foi observado que, após a exposição de PBMC aos exossomos contendo RNAm viral tax, a expressão da citocina inflamatória IFN-? foi aumentada em linfócitos T CD4+ e CD8+ quando comparado ao grupo não exposto aos exossomos. Foi observado um pequeno aumento de TNF-? no grupo exposto aos exossomos. Não houve diferença na expressão das citocinas IL-4, perforina e granzima B. Tomados em conjunto, estes resultados sugerem que exossomos contendo tax isolados de células infectadas pelo HTLV-1 induzem a produção de citocinas inflamatórias ativam a resposta imunológica de padrão Th1, e não Th2. Estes achados podem contribuir na elucidação do papel dos exossomos na infecção pelo HTLV-1 e nos estudos relacionados à patogênese da HAM/TSP e à resposta inflamatória envolvendo a apresentação clínica desta doença. / Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of HTLV-1 associated myelopathy/tropical paraparesis spastic (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). The development of HAM/TSP, a chronic neuroinflammatory disease, is correlated to a complex host-virus dynamic immune response interaction. Tax expression plays an important role in HAM/TSP pathogenesis by activating various cellular genes, including the cytokines IFN-? and TNF-?. Exosomes have emerged as an important factor of cell-to-cell communication contributing to diverse cellular processes, including immune modulation. Considering the potential role of exosomes in modulating the immune response and inflammation, the main objective of this study was to examine if HTLV-1-infected cells produce exosomes can carry viral tax mRNA, and if they can can participate in the chronic inflammation observed in patients with HAM/TSP. Exosomes were isolated from HTLV-1-infected cell line culture, evaluated for the tax mRNA presence and tested for the ability to activate peripheral mononuclear cells (PBMC) in inducing an inflammatory immune response. We observed that the proinflammatory cytokine IFN-? was upregulated in CD4 and CD8 T-cells after treatment of the PBMC with Tax carrying exosomes compared to the negative control. The expression of TNF-? was slightly upregulated. IL-4, Granzyme B and Perforin did not show alterations. Taken together, these results suggest that exosomes carrying Tax isolated from HTLV-1-infected cells might induce the production of proinflammatory cytokines and activate T helper (Th)1, and not Th2, immune response. This study may have important impact on the studies involving the pathogenesis of HAM-TSP and the inflammatory response involved in the clinical presentation of the disease.

Le virus T lymphotrope humain de type 1 (HTLV-1, Human T-cell Leukemia Virus type 1) est le premier rétrovirus oncogène humain à avoir été découvert. Sa protéine régulatrice Tax est la principale responsable du processus d’immortalisation et de transformation des lymphocytes T, cibles préférentielles d’HTLV-1 in vivo, qui est associé au développement de leucémies à cellules T de l’adulte. Ce mécanisme oncogénique est dû à la capacité de Tax à interagir avec de nombreux acteurs cellulaires, détournant ainsi différentes voies qui contrôlent notamment la prolifération et la survie des cellules. L’un des événements majeurs de ce mécanisme est l’activation constitutive de la voie NF-κB induite par Tax. Notre laboratoire ainsi que d’autres équipes ont montré que l’ubiquitinylation de Tax, notamment avec des chaînes liées en K63, est nécessaire pour que Tax recrute et active le complexe IKK (IκB Kinase), en se liant à sa sous-unité régulatrice NEMO dans le cytoplasme. Par ailleurs, il a également été décrit que Tax est SUMOylée ce qui lui permet de recruter les dimères NF-κB dans des corps nucléaires pour activer la transcription de leurs gènes cibles. Toutefois, de nombreuses interrogations persistent autour de l’implication et du mode d’action de chacune de ces modifications post-traductionnelles de Tax dans l’activation de la voie NF-κB. L’importance de la SUMOylation de Tax a en effet été remise en cause par notre laboratoire, suscitant une controverse qui nous décida à réexaminer complètement son rôle dans l’activation de la voie NF-κB. Pour cela, nous avons élaboré une nouvelle stratégie basée sur l’inhibition de la machinerie de SUMOylation endogène, grâce au blocage de l’unique enzyme E2 identifiée dans ce processus, l’enzyme Ubc9. Nous avons prouvé qu’une protéine Tax non SUMOylée, mais toujours ubiquitinylée, active la transcription à partir de promoteurs dépendants de NF-κB, qu’ils soient transfectés, intégrés stablement dans la chromatine cellulaire ou endogènes, démontrant ainsi que la SUMOylation de Tax n’est pas nécessaire à l’activation de la voie NF-κB, contrairement à son ubiquitinylation. Un autre point de questionnement concerne l’association de Tax au complexe NEMO/IKKα/IKKβ, pour laquelle l’ubiquitinylation de Tax est essentielle. Nous avons étudié l’implication de l’enzyme TRAF5 dans ces mécanismes, qui semblait être une potentielle E3 ligase de Tax, celle-ci restant encore inconnue. Grâce au blocage de TRAF5, nous avons montré que cette protéine interagit avec Tax et qu’elle est nécessaire à son ubiquitinylation, notamment avec des chaînes K63, sans toutefois que son activité ligase n’entre en jeu, excluant donc son rôle en tant qu’E3 ligase de Tax. Nous avons également découvert que TRAF5 induit la formation du complexe Tax/IKKα/IKKβ, mais n’est pas impliquée dans l’association Tax/NEMO, dessinant alors un nouveau modèle pour le recrutement et l’activation du complexe IKK par Tax. Ce travail permet donc d’apporter un nouvel éclairage sur l’impact de la SUMOylation et de l’ubiquitinylation de Tax dans l’activation de la voie NF-κB et de décrypter les étapes de cet événement crucial dans le mécanisme oncogénique déclenché par la protéine Tax du virus HTLV-1. / The Human T-cell Leukemia Virus type I is the first human oncoretrovirus discovered. The regulatory Tax protein is the main responsible of the immortalization process of primary CD4+ T lymphocytes, the preferential target cells of HTLV-1 in vivo, which is associated with adult T-cell leukemia/lymphoma (ATLL), a highly aggressive malignant proliferation of CD4+ T lymphocytes. The oncogenic property of Tax is mainly due to its capacity to interact with many different cellular proteins belong to several pathways which control cell proliferation and survival. Constitutive activation of the NF-κB pathway induced by Tax plays a crucial role in this oncogenic mechanism. Our laboratory, and others, demonstrated that Tax ubiquitination, notably with K63-linked ubiquitin (Ub) chains, is required for the activation of the cytoplasmic IκB kinase (IKK) complex by directly interacting with the regulatory subunit NEMO. Moreover, in previous studies, we and others describe that Tax is also conjugated to either SUMO-1 or SUMO-2/3 molecules which facilitates its interaction with the NF-κB dimers in particular structures named Tax nuclear bodies, inducing promoter activation of target genes in the nucleus. However, many questions remain concerning the exact role of each Tax post-translational modification in the NF-κB pathway activation process. In the laboratory, we recently reconsidered the importance of Tax SUMOylation, provoking a controversy in our field. So, we decided to reexamine the role of this modification on NF-κB activation. To do this, we designed a novel strategy based on the inhibition of the endogenous SUMOylation machinery by blocking or silencing Ubc9, the unique E2-conjugating enzyme involves in this process. We found that an ubiquitinated but not SUMOylated Tax protein is still able to activate a transfected, an integrated or an endogenous NF-κB promoters, demonstrating that Tax SUMOylation is not required for Tax induced NF-κB pathway activation contrary to its ubiquitination. Another interrogation concerning the formation of the Tax/NEMO/IKKα/IKKβ complex, in which Tax ubiquitination is critical. We studied the role of TRAF5 in these mechanisms because this enzyme could be a potential Ub E3-ligase of Tax, which remains unknown. Thanks to the blockage of TRAF5, we showed that this protein interacts with Tax and that TRAF5 is necessary for Tax ubiquitination, notably with K63-linked Ub chains. However, TRAF5 is not the direct E3-ligase of Tax since we demonstrated that its ligase activity is not involved in Tax conjugation to ubiquitin. We also discovered that TRAF5 induces the formation of the Tax/ IKKα/IKKβ complex but not the association between Tax and NEMO, showing a new model for the recruitment and the activation of the IKK complex by Tax. In conclusion, our results led us to propose a new light on the impact of Tax SUMOylation and ubiquitination in the NF-κB pathway activation and to figure out the different steps of this process, which is crucial for the oncogenic mechanism induced by the HTLV-1 Tax protein.

O HIV-1 é o agente etiológico da síndrome da imunodeficiência adquirida (AIDS) e o HTLV-I da leucemia/linfoma de célula T no adulto (ATL) e da paraparesia espástica tropical ou mielopatia associada ao HTLV (HAM/TSP), principalmente. Ambos são retrovírus com genoma RNA e possuem o gene gag que codifica as proteínas p24 (HIV-1 e HTLV-1) e p19 (HTLV-1) que formam o capsídeo e a matriz do vírus, respectivamente, e o gene env que codifica as proteínas gp41 e gp120 (HIV-1) e gp21 e gp46 (HTLV-1) que compõem o envelope viral. Os primeiros anticorpos produzidos nas infecções por ambos os vírus são destinados a essas proteínas e os diferentes testes diagnósticos disponíveis no mercado usam uma combinação dessas proteínas virais. O diagnóstico precoce é de extrema importância para o controle da epidemia, tratamento dos indivíduos e planejamento dos gastos com saúde pública. Os kits diagnósticos usados em laboratórios clínicos, bancos de sangue e hospitais brasileiros para o diagnóstico destas viroses são na sua maioria de empresas estrangeiras e o Brasil despende milhares de reais importando esses materiais. No Brasil, há a necessidade e incentivo para a produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, os genes das proteínas p24, gp41 e gp120 do HIV-1 e p19 do HTLV-1 foram clonados com sucesso em diferentes vetores e em diferentes linhagens de E. coli, porém essas proteínas não foram expressas. As proteínas gp21, p24 e gp46 do HTLV-1 foram produzidas em bactérias BL21(DE3) com vetor pET28a(+). Essas três proteínas foram solubilizadas dos corpos de inclusão, purificadas por IMAC e identificadas pelas técnicas de Western Blotting e por espectrometria de massas. As proteínas recombinantes gp21, p24 e gp46 foram reconhecidas pelos soros de indivíduos com HTLV-1 e não foram reconhecidas por soros de indivíduos com HIV-1 e saudáveis, o que confere a elas especificidade e grande potencial diagnóstico. Os resultados deste trabalho são os primeiros passos para atingir o objetivo maior de produzir todas as sete proteínas em maior escala e, por fim, chegar a produção de um kit diagnóstico sensível, específico e barato com tecnologia nacional, diminuindo os gastos com a importação destes produtos e fomentando a indústria biotecnológica nacional. / HIV-1 is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and HTLV-I is the cause of adult T-cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). Both are retroviruses with RNA genome and possess the gene gag and env. The gag gene encodes for p24 protein (HTLV-1 and HIV-1) and p19 (HTLV-1) forming the viral capsid and matrix, respectively, and the env gene encodes for proteins gp120 and gp41 (HIV-1) and gp21 and gp46 (HTLV-1) making the viral envelope. The first antibodies produced in infections by both viruses are against these proteins and the various diagnostic tests on the market use a combination of those viral proteins. Early diagnosis is extremely important to control the epidemia, treatment of individuals and planning of public health expenditures. The diagnostic kits used in clinical laboratories, blood banks and in Brazilian hospitals for the diagnosis of these viruses are mostly from foreign companies. Brazil spends thousands of reais importing these materials. In Brazil, there is a need and incentive for the production of diagnostic systems with national technology. In this study, the genes of p24, gp41 and gp120 of HIV-1 and p19 of HTLV-1 have been successfully cloned in different vectors and different strains of E. coli, but these proteins were not expressed. The proteins gp21, gp46 and p24 of HTLV-1 were produced in bacteria BL21 (DE3) with vector pET28a (+). These three proteins were solubilized from inclusion bodies, purified by IMAC and identified by Western blotting techniques and mass spectrometry. The recombinant proteins gp21, p24 and gp46 were recognized by sera from patients with HTLV-1 and were not recognized by sera from individuals with HIV-1 and healthy people, which gives them great specificity and diagnostic potential. These results are the first steps to achieve the ultimate goal of producing all seven proteins on a larger scale and finally get the production of a diagnostic kit sensitive, specific and cheap with national technology, reducing spending on imports of these products and fostering the national biotechnology industry.

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-04-26T13:41:04Z No. of bitstreams: 1 Dinaura Maramaldo Cruz.pdf: 3121635 bytes, checksum: 83c3cab538352aacd4eaa7920d4fa684 (MD5) / Made available in DSpace on 2017-04-26T13:41:04Z (GMT). No. of bitstreams: 1 Dinaura Maramaldo Cruz.pdf: 3121635 bytes, checksum: 83c3cab538352aacd4eaa7920d4fa684 (MD5) Previous issue date: 2011-10-04 / The type 1 and type 2 virus human T-lymphotropic type 1 are human retroviruses, that destroy or transform lymphocytes causing diseases that are externalized years or even decades after primary infection. The virus has a worldwide prevalence varies by geographic region and population, Brazil is assigned the highest absolute number of carriers. Risk factors such as transfusions, sharing needles, sexual contact and breastfeeding are no ways to prevent transmission. The objective of this study was the epidemiological profile of the sociodemographic and blood donors, the Blood Center of Maranhão - HEMOMAR with positive serology for HTLV-1/2, identifying the virus types. We analyzed the records of 924 blood donors of the Blood Center of Maranhão - HEMOMAR, which showed positive pattern and indeterminate ELISA test (OD / CO > 0.800) and the Western blot test in the period January 2002 to December 2009. Reactivity was identified for HTLV-1 / 2 by ELISA in the general population of donors is 0.24% (n = 924/398.362) and 0.02% in the Western blot test. Sex, age and color were statistically significant. Considering the group studied, there was a significant number of females, average age 40 years predominantly brown and black. Most had less than eight years. Among the cases 35.6% had co-infection with other serological markers, with higher prevalence for hepatitis B, and Hepatitis C, syphilis and HIV. The Western Blot test revealed the circulation of HTLV type 2 (2,4%) between subjects, but with higher prevalence for HTLV type 1 (70,1%). The presence of HTLV-1/2, the situation of co-infections associated with HTLV indicate the need for adoption of effective public health measures focused on the implementation of prevention strategies, since even in the presence of population studies suggests the existence of risk factors for transmission. Epidemiological studies have been relevant to advances in scientific knowledge about HTLV. The availability of qualified information on the means of transmission, prevention and risk perception, considering cultural and social aspects, are mechanisms for intervention in controlling the virus. / Os vírus linfotrópicos de células T humanas do tipo 1 (HTLV-1) e do tipo 2 (HTLV-2) são retrovírus humanos, que destroem ou transformam os linfócitos causando doenças que se exteriorizam anos ou mesmo décadas após a infecção primária. O vírus tem prevalência mundial que varia de acordo com a região geográfica e população estudada; ao Brasil é atribuído maior número absoluto de portadores. Fatores de risco como transfusões, uso compartilhado de agulhas, aleitamento materno e contato sexual sem prevenção são formas de transmissão. O objetivo deste trabalho foi conhecer o perfil epidemiológico e sociodemográfico dos doadores de sangue, do Hemocentro do Maranhão – HEMOMAR, com sorologia positiva para HTLV-1/2, identificando os tipos virais. Foram analisados os registros de 924 doadores de sangue do Hemocentro do Maranhão – HEMOMAR, que apresentaram padrão positivo e indeterminado no teste ELISA (DO / CO > 0,800) e no teste Western Blot no período janeiro de 2002 a dezembro de 2009. Identificou-se reatividade para o HTLV-1/ 2 pelo teste ELISA na população geral de doadores de 0,24% (n=924/398.362) e 0,02% no teste Western Blot. Sexo, idade e cor foram estatisticamente significantes. Considerando o grupo estudado, houve um número significativo do sexo feminino, média de idade de 40 anos predominando pardos e negros. A maioria tinha escolaridade inferior a oito anos. Entre os casos, 35,6% apresentaram co-infecção com outros marcadores sorológicos, com maior prevalência para Hepatite B, além de Hepatite C, Sífilis e HIV. O teste Western Blot revelou a circulação do HTLV tipo 2 (2,4%) entre os sujeitos, mas com maior prevalência para o HTLV tipo 1 (70,1%). A presença do HTLV-1/2, a situação de coinfecções associadas ao HTLV indica a necessidade de adoção de medidas eficazes de saúde pública focadas na implantação de estratégias de prevenção, uma vez que a presença do mesmo na população estudada sugere a existência de fatores de risco à transmissão. Estudos epidemiológicos têm sido relevantes para avançar no conhecimento científico sobre o HTLV. A disponibilização de informação qualificada sobre os meios de transmissão, prevenção e percepção de risco, considerando aspectos culturais e sociais, constitui mecanismos para intervenção no controle do vírus.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-04T18:19:56Z No. of bitstreams: 1 Filipe Ferreira de Almeida Rego Fatores....pdf: 6473179 bytes, checksum: 008855484cf612885a04f114f51214ba (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-04T18:20:12Z (GMT) No. of bitstreams: 1 Filipe Ferreira de Almeida Rego Fatores....pdf: 6473179 bytes, checksum: 008855484cf612885a04f114f51214ba (MD5) / Made available in DSpace on 2016-04-04T18:20:12Z (GMT). No. of bitstreams: 1 Filipe Ferreira de Almeida Rego Fatores....pdf: 6473179 bytes, checksum: 008855484cf612885a04f114f51214ba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Ba, Brasil / Nesta tese foram realizados três trabalhos distintos sendo que todos envolvem identificar possíveis fatores genéticos ou clínicos relacionados com a infecção pelo HIV-1 ou pelo HTLV-1 ou por ambos. No primeiro trabalho nós objetivamos identificar mutações que poderiam estar relacionadas com o desenvolvimento da TSP/HAM ou carga proviral. Para isto sequenciamos a região LTR5’ do HTLV-1 por Ion Torrent para verificar mutações com baixa frequência. Nós encontramos mutações em 52 posições que estavam presentes em mais de um indivíduo, porém apenas 11 destas estavam presentes em TFBS previamente descritos. Três mutações que não estavam presentes em TFBS previamente descritos foram estatisticamente significantes quando comparadas entre os grupos," sendo" que" estes" sítios" podem" ser" importantes" para" a"mediação" da" transcrição" viral."No" segundo" estudo" nós" objetivamos determinar a prevalência do genótipo selvagem em Hlabisa, Kwazulu-Natal na África do Sul além de identificar possíveis fatores associados a presença deste genótipo em 220 pacientes submetidos a ART. O genótipo selvagem foi detectado em 28 amostras (12,7%). Selecionamos 11 pacientes para realizar o sequenciamento pelo Ion Torrent, nove confirmaram não ter mutações de resistência aos antirretrovirais em alta frequência. Foi encontrada uma alta contagem de CD4+ no início da terapia associado a falha terapêutica assim como uma alta carga viral antes da genotipagem e não foi encontrado associação entre aderência a terapia auto-reportada e a presença do genótipo selvagem. Aproximadamente um em cada oito adultos que falham a terapia possuem o genótipo selvagem sendo este dado confirmado através de sequenciamento de nova geração. Devido ao alto número de genótipos selvagem encontrados, o teste de resistência genotípica deve ser solicitado para se obter um melhor desfecho clinico em níveis individuais e populacionais. No terceiro estudo nós analisamos as diferenças na contagem de linfócitos T CD4+ entre indivíduos infectados apenas com o HIV-1 e indivíduos coinfectados HIV-1/HTLV-1 com falha terapêutica, além de analisarmos a soroprevalência do HTLV-1 em indivíduos infectados pelo HIV-1. Foram encontrados oito pacientes coinfectados (2,1%) dos 381 pacientes analisados. Nós não observamos nenhuma diferença estatística quando analisamos transversalmente os dados clínicos dos pacientes, exceto na primeira contagem de linfócitos T CD4+ após o início do tratamento que estava maior nos indivíduos coinfectados (p=0.03). A análise multivariada longitudinal mostrou que a media de linfócitos T CD4+ ao longo do tratamento, foi estatisticamente maior nos pacientes coinfectados levando em consideração características demográficas, carga viral, fatores relacionados a terapia, entre outros. Nos pacientes coinfectados também não foram encontrados marcadores de HLA relacionados com os supressores de elite do HIV-1. Os dados deste trabalho sugerem que os pacientes coinfectados em terapia antirretroviral deveriam ter um acompanhamento clínico diferenciado dos indivíduos apenas infectados pelo HIV-1, pois a coinfecção poderia estar levando ao aumento do número dos linfócitos T CD4+ sem um possível ganho de resposta imune. / We performed three studies to analyze risk factors associated with retroviruses infections. In the first study we attempted to analyze mutations related to TSP/HAM development or proviral load. For that purpose we have sequenced the LTR 5’ region of HTLV-1 by Ion Torrent. We found that mutations in 52 positions were present in more than one individual, but only 11 were present in the previously described TFBS. Three mutations that were not present in the previously described TFBS were statistically significant comparing groups. Despite the absence of previously described TFBS, these sites might be important for the viral transcription. In the second study we analyzed the prevalence of HIV-1 wild type genotype in adults failing first-line ART. A total of 220 adults were included. The wild type genotype was detected by population sequencing in 28 (12.7%). No major drug resistance mutations were detected by deep sequencing for 81.8% (9/11) of those sampled. Higher baseline CD4+ cell count was associated with a greater likelihood of wild type genotype as was a higher viral load prior to resistance testing but there was no evidence of an association between selfreported adherence and the presence of wild type genotype. Approximately one in eight adults failing first-line ART had wild type genotype and this result was confirmed trough deep sequencing in some samples. Access to genotypic resistance testing may be required in this region to achieve optimal individual-level and population-level outcomes. In the third study we proposed to verify the prevalence of HTLV-1 and to statistically assess differences in CD4+ counts between HTLV-1/HIV-1 co-infected and HIV-1 mono-infected patients living in rural KwaZulu-Natal. The HTLV-1 seroprevalence was 2.1% (8 out 381 patients). The patients were grouped by HTLV-1/HIV-1 co-infected and HIV-1 mono-infected status for the statistical analysis. There were no cross-sectional differences between the groups regarding CD4+ count before therapy, CD4+ count at genotype, age, gender, viral load, duration of ART, immunological failure, ART failure and first ART regimen. However, the first CD4+ count after treatment was higher in the co-infected group (p=0.03). A multivariate, longitudinal model showed that the mean CD4+ count over time for the HTLV-1/HIV-1 coinfected group was significantly higher than the HIV mono-infected group (p<0.05) when adjusting for demographic characteristics, viral load and ART treatment factors. This finding was independent of expression of HLA Class 1 genotypes previously associated with HIV-1 infected elite suppressors. This study suggests that the increase of CD4+ count after therapy suggests a differential clinical management for the HTLV-1/HIV-1 co-infected patients should be implemented.

Le virus T lymphotrope humain de type 1 (HTLV-1) est l'agent étiologique de la leucémie à cellules T de l'adulte (ATL) et de la paraparésie spastique tropicale/myélopathie associée à HTLV-1 (HAM/TSP). Chez les patients chroniquement infectés, le provirus d'HTLV-1 est majoritairement retrouvé dans les lymphocytes T CD4+. Ex vivo, on peut aussi retrouver le provirus dans les lymphocytes T CD8+, les lymphocytes B, les monocytes, les cellules dendritiques (DCs) myéloïdes, les DCs plasmacytoϊdes (pDCs) et les macrophages. In vitro, HTLV-1 est capable d'infecter productivement les cellules lymphoïdes et les cellules dendritiques dérivées de monocytes humains (MDDCs). De par leur fonction et leur distribution dans l'organisme, les DCs pourraient être les premières cellules à interagir avec HTLV-1 au cours de la primo-infection. Elles seraient ensuite capables de transmettre HTLV-1 aux lymphocytes T CD4+. Cette hypothèse est soutenue par les travaux de notre équipe qui ont montré que les MDDCs sont plus susceptibles que des lymphocytes T autologues à l'infection par HTLV-1. Ainsi, les DCs constitueraient des relais importants pour l'établissement de l'infection chronique. Dans ce contexte, nous nous sommes demandés si toutes les populations de DCs étaient également susceptibles à l'infection par HTLV-1 et si elles transmettaient similairement HTLV-1 aux lymphocytes T. Pour cela, nous avons différencié trois sous types de MDDCs après l'exposition de monocytes humains à divers cocktails de cytokines : - les IL4-DCs (pour interleukine 4 - DCs) miment les DCs immatures myéloϊdes du sang, - les TGF-β DCs (pour tumor-growth factor β - DCs) miment les DCs mucosales à phénotype tolérogène, - les IFN-α DCs (pour interféron α DC) miment les DCs activées et inflammatoires recrutées au niveau des sites d'inflammation. Nous avons aussi traité au lipopolysaccharide (LPS) des IL-4 DCs afin de générer des DCs qui sur-expriment les marqueurs de maturation CD80 et CD86. Nos résultats montrent que les IFN-α DC et les IL-4 DCs traités au LPS ne supportent pas une infection productive au contraire des TGF-β DCs et des IL4-DCs qui sont productivement infectés par HTLV-1. La restriction virale des IFN-α DC et les IL-4 DCs traitées au LPS n'est pas due à leur production d'IFN. Nous avons montré que la susceptibilité des IL4-DCs à l'infection productive par HTLV-1 est liée à leur phénotype immature. De plus, nos résultats montrent qu'HTLV-1 est internalisé par macropinocytose dans les IL-4 DCs alors qu'il est internalisé par endocytose médiée par la clathrine dans les IFN-α DCs. Enfin, nous avons pu restaurer partiellement la susceptibilité à l'infection productive des IL-4 DCs traités au LPS et celle des IFN-α DCs et nous avons pu restreindre celle des IL-4 DCs immatures en modulant le pH de leurs endosomes. Ces résultats suggèrent que le virus utilise le trafic vésiculaire pour infecter les DCs et que le pH des vésicules conditionne, au moins partiellement, le devenir de l'infection productive. De plus, parmi les IL-4 DCs, les IL-4 DCs traités au LPS et les IFN α DCs, seules les IL-4 DCs qui sont productivement infectées peuvent transmettre HTLV-1 aux lymphocytes T. En conclusion, nos résultats suggèrent que c'est le sous type de DC que rencontre HTLV- 1 lors de la primo-infection ainsi que le trafic viral d'HTLV-1 dans la DC qui conditionnent ou pas l'établissement de l'infection productive de la DC ainsi que la transmission aux lymphocytes T / HTLV-1 (Human T cell leukemia/lymphoma virus type 1) is the etiological agent of Adult T cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). In chronically infected patients, the provirus is mainly detected in the CD4 T-cell population and, to a lesser extent in myeloid dendritic cells (DCs), plasmacytoid DCs (pDCs), macrophages and monocytes. Among the different DCs subsets found in vivo, myeloid DCs from the blood, tolerogenic or inflammatory DCs from mucosa may first encounter HTLV-1 during blood transmission, breast-feeding or sexual transmission, respectively. They would then be able to transmit HTLV-1 to CD4 + T cells. This hypothesis is supported by the recent work of our team that showed that monocyte derived dendritic cells (MDDCs) are more susceptible to HTLV-1 infection in comparison to autologous T cells. We therefore asked whether all these DCs subsets were equally susceptible to HTLV-1 and whether the nature of the DC subset would impact HTLV-1 spread to T-cells. Human monocytes obtained from healthy blood donors were differentiated into IL-4 DCs, TGF-ß DCs or IFN-a DCs. In vitro-derived immature IL-4 DCs, TGF-ß DCs and IFN-a DCs mimic myeloid, tolerogenic and inflammatory DCs, respectively. We also generated LPS-matured IL-4 DCs that exhibited a strong maturation profile with over-expression of maturation markers. We observed HTLV-1 protein expression and provirus accumulation in IL-4 DCs and TGF-ß DCs but not in IFN-a DCs and LPS-matured IL-4 DCs. Despite their increased ability to capture HTLV-1 virion compared to IL-4 DCs and TGF-ß DCs, IFN-a DCs and LPS-matured IL-4 DCs restricted HTLV-1 productive infection. This was not due to the antiviral activity of type–I interferon produced by IFN-a DC or LPS-matured IL-4 DCs. In contrast, we showed that these differences in susceptibility to HTLV-1 infection might be linked to the maturation phenotype of the DCs subsets and to a different trafficking of HTLV-1 in IL-4 DC vs. IFN-a DC. Finally, using IL-4DCs, LPS-matured IL-4 DCs and IFN-a DCs, we demonstrate that productive infection rather than trans-infection is required for HTLV-1 transmission from DCs to CD4 T-cells. Thus, our results demonstrate that the nature of the DCs encountered by HTLV-1 during primo-infection and the trafficking route of the virus through the vesicular pathway of these cells determine the efficiency of viral transmission to T-cells

O HIV-1 é o agente etiológico da síndrome da imunodeficiência adquirida (AIDS) e o HTLV-I da leucemia/linfoma de célula T no adulto (ATL) e da paraparesia espástica tropical ou mielopatia associada ao HTLV (HAM/TSP), principalmente. Ambos são retrovírus com genoma RNA e possuem o gene gag que codifica as proteínas p24 (HIV-1 e HTLV-1) e p19 (HTLV-1) que formam o capsídeo e a matriz do vírus, respectivamente, e o gene env que codifica as proteínas gp41 e gp120 (HIV-1) e gp21 e gp46 (HTLV-1) que compõem o envelope viral. Os primeiros anticorpos produzidos nas infecções por ambos os vírus são destinados a essas proteínas e os diferentes testes diagnósticos disponíveis no mercado usam uma combinação dessas proteínas virais. O diagnóstico precoce é de extrema importância para o controle da epidemia, tratamento dos indivíduos e planejamento dos gastos com saúde pública. Os kits diagnósticos usados em laboratórios clínicos, bancos de sangue e hospitais brasileiros para o diagnóstico destas viroses são na sua maioria de empresas estrangeiras e o Brasil despende milhares de reais importando esses materiais. No Brasil, há a necessidade e incentivo para a produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, os genes das proteínas p24, gp41 e gp120 do HIV-1 e p19 do HTLV-1 foram clonados com sucesso em diferentes vetores e em diferentes linhagens de E. coli, porém essas proteínas não foram expressas. As proteínas gp21, p24 e gp46 do HTLV-1 foram produzidas em bactérias BL21(DE3) com vetor pET28a(+). Essas três proteínas foram solubilizadas dos corpos de inclusão, purificadas por IMAC e identificadas pelas técnicas de Western Blotting e por espectrometria de massas. As proteínas recombinantes gp21, p24 e gp46 foram reconhecidas pelos soros de indivíduos com HTLV-1 e não foram reconhecidas por soros de indivíduos com HIV-1 e saudáveis, o que confere a elas especificidade e grande potencial diagnóstico. Os resultados deste trabalho são os primeiros passos para atingir o objetivo maior de produzir todas as sete proteínas em maior escala e, por fim, chegar a produção de um kit diagnóstico sensível, específico e barato com tecnologia nacional, diminuindo os gastos com a importação destes produtos e fomentando a indústria biotecnológica nacional. / HIV-1 is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and HTLV-I is the cause of adult T-cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). Both are retroviruses with RNA genome and possess the gene gag and env. The gag gene encodes for p24 protein (HTLV-1 and HIV-1) and p19 (HTLV-1) forming the viral capsid and matrix, respectively, and the env gene encodes for proteins gp120 and gp41 (HIV-1) and gp21 and gp46 (HTLV-1) making the viral envelope. The first antibodies produced in infections by both viruses are against these proteins and the various diagnostic tests on the market use a combination of those viral proteins. Early diagnosis is extremely important to control the epidemia, treatment of individuals and planning of public health expenditures. The diagnostic kits used in clinical laboratories, blood banks and in Brazilian hospitals for the diagnosis of these viruses are mostly from foreign companies. Brazil spends thousands of reais importing these materials. In Brazil, there is a need and incentive for the production of diagnostic systems with national technology. In this study, the genes of p24, gp41 and gp120 of HIV-1 and p19 of HTLV-1 have been successfully cloned in different vectors and different strains of E. coli, but these proteins were not expressed. The proteins gp21, gp46 and p24 of HTLV-1 were produced in bacteria BL21 (DE3) with vector pET28a (+). These three proteins were solubilized from inclusion bodies, purified by IMAC and identified by Western blotting techniques and mass spectrometry. The recombinant proteins gp21, p24 and gp46 were recognized by sera from patients with HTLV-1 and were not recognized by sera from individuals with HIV-1 and healthy people, which gives them great specificity and diagnostic potential. These results are the first steps to achieve the ultimate goal of producing all seven proteins on a larger scale and finally get the production of a diagnostic kit sensitive, specific and cheap with national technology, reducing spending on imports of these products and fostering the national biotechnology industry.

La leucémie T de l’adulte (ATL) est une pathologie lympho-proliférative aiguë associée à l’infection par le virus HTLV-1 (human T-cell leukemia virus type 1). La réactivation de la télomérase observée lors de la phase tardive du développement de l’ATL est un évènement crucial dans la progression tumorale. Elle est induite au niveau transcriptionnel par la protéine HBZ (HTLV-1 bZIP factor) et est dépendante du facteur de transcription JunD. Ce dernier est normalement associé en complexe avec le produit du gène suppresseur de tumeur MEN-1, la ménine, dont l’interaction avec JunD réprime la transcription JunD-dépendante et convertit JunD en inhibiteur de croissance.Mes résultats démontrent que la protéine virale HBZ inhibe la fonction suppresseur de tumeur de la ménine, induisant l’activité transcriptionnelle de JunD et donc l’activation de la transcription de son gène cible : la transcriptase inverse télomérase humaine (hTERT). J’ai démontré que HBZ, JunD et la ménine peuvent coexister dans un même complexe protéique et que HBZ et la ménine ont des effets opposés sur l’activité transcriptionnelle de JunD. En effet la ménine inhibe l’activation du promoteur proximal d’hTERT par JunD, alors que HBZ est capable de contre balancer cet effet. Finalement, je propose qu’en recrutant l’histone acétyltransférase p300, HBZ réverse la déacétylation des histones induite par le recrutement des HDACs par la ménine et par conséquent active le promoteur d’hTERT. L’ensemble de ces résultats a permis d’identifier les mécanismes moléculaires aboutissant à l’inhibition fonctionnelle de la protéine suppresseur de tumeur ménine, résultant en la dérégulation de la voie AP-1 dans les cellules infectées par HTLV-1. Finalement, ce travail apporte de nouvelles précisions sur le mécanisme de la surexpression transcriptionnelle de la télomérase lors de l’infection par HTLV-1, une étape importante de la mise en place et du développement de la leucémie T de l’adulte vers des stades plus agressifs. / Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Reactivation of telomerase, a critical event in tumor progression observed in late phases of ATL development, has been shown to be caused by HBZ (HTLV-1 bZIP factor), a regulatory protein encoded by the negative strand of the HTLV-1 genome. The HBZ-mediated up-regulation of the telomerase catalytic subunit is dependent on JunD, which in the cellular context occurs in the complex with menin, the product of the MEN-1 tumor suppressor gene. Interaction with menin represses JunD-dependent transcription and converts JunD into a growth suppressor, whereas it acts as a growth promoter in the absence of menin. My results demonstrate that the viral protein HBZ abrogates tumor suppressor function of menin, resulting in the activation of JunD transcriptional activity and finally in the up-regulation of its target gene, the human telomerase reverse transcriptase (hTERT). I showed that HBZ, JunD and menin can coexist in the same protein complex and that HBZ and menin exert opposite effects on JunD transcriptional activity. Moreover menin inhibits the JunD-mediated activation of the hTERT proximal promoter and HBZ is able to counteract this effect. Finally, I proposed that HBZ, by recruiting p300 histone acetyltransferase, reverses the histone deacetylation conducted by menin-recruited HDACs and therefore up-regulates the expression of the hTERT gene. Altogether, my work led to the identification of the molecular mechanism leading to the functional impairment of the menin tumor suppressor, which results in the deregulation of AP-1 signaling in HTLV-1 infected cells. Finally this work gave new insights into the mechanism of the transcriptional up-regulation of the hTERT gene upon HTLV-1 infection, being a key event during the development of Adult T-cell leukemia and a necessary step towards the progression into more aggressive courses.

Na cidade de São Paulo, cerca de 50 mil pessoas são portadoras do HTLV-1. O HTLV-1 é o agente causador da paraparesia espástica tropical/mielopatia associada com o HTLV-1 (TSP/HAM) e os mecanismos desta patogênese são obscuros. A TSP/HAM é considerada uma doença imuno-mediada e algumas citocinas, podem estar associadas, com a desmielinização da membrana de mielina da coluna espinhal, provavelmente, estimuladas pela presença de antígenos virais. A PCR em tempo real é uma técnica utilizada para a detecção de citocinas e apresenta maior sensibilidade na expressão de mRNA e carga proviral do HTLV-1. Objetivos: Detecção de citocinas pelas técnicas sorológicas e de biologia molecular (PCR em tempo real) que auxiliaram no aconselhamento e avaliação de pacientes infectados pelo HTLV-1, além de uma avaliação da carga proviral dos pacientes com infecção pelo HTLV-1. Casuística: Grupo I: indivíduos soronegativos para HCV, HIV-1 e HTLV-1 (Controle); Grupo II: pacientes HTLV-1 assintomáticos; Grupo III: pacientes com TSP/HAM. Observamos que o INF-<font face=\"symbol\">g apresenta-se alterado na infecção HTLV-1, sendo um dos fatores mais importantes da evolução para TSP/HAM, ambos os ensaios apresentaram o mesmo resultado. A carga proviral pode ser um marcador de progressão para a TSP/HAM. A interação complexa existente entre o HTLV-1 e as células responsáveis pela liberação de citocinas e <font face=\"symbol\">b-quimiocinas pró-inflamatórias, assim como elementos de ativação celular, resultam na ativação imunológica. Isto, lentamente, pode levar ao processo inflamatório crônico que atua diretamente no micro ambiente neuronal e/ou membrana de mielina da coluna espinhal, em alguns portadores de HTLV-1. Com a persistente ativação do sistema imunológico, este processo provocará destruição das células gliais e dos neurônios, dando início ao processo de desmielinização. / In São Paulo, about 50 thousand people are HTLV-1 carriers. HTLV-1 is the agent that causes tropical spastic paraparesis and HTLV-1 associated myelopathy (TSP/HAM) despite the mechanisms of this disease are still unclear. TSP/HAM is considered an immune mediated illness and some cytokines might be involved with axonal damage and demyelination, most pronounced in the midthoracic spinal cord, probably stimulated by the presence of viral antigens. Real Time PCR is a used technique to cytokine detection and shows higher sensitivity on mRNA expression and HTLV-1 proviral load. Objectives: cytokine detection through molecular biology (Real time PCR) and serologic techniques that helped on advising and assessment of HTLV-1 infected patients and also on their proviral load. Casuistic: Group I: seronegative individuals for HCV, HIV-1 and HTLV-1 (control); Group II: asymptomatic HTLV-1 infected patients; Group III: TSP/HAM patients. We\'ve been observed that <font face=\"symbol\">g-interferon presents changing on the HTLV-1 infection, being one of the most important factors to TSP/HAM progression, both essays showed the same results. The proviral load may be an important marker for TSP/HAM development.