O neuroblastoma é o tumor sólido mais comum e com maior índice de letalidade em crianças. Diversas famílias de retrovírus endógenos humanos (HERV) estão presentes no genoma humano, em diferentes níveis de integridade, e são reativadas sob diferentes circunstâncias. A atividade de HERV tem sido cada vez mais sido associada a doenças como câncer, doenças autoimunes e ainda com a infecção por vírus exógenos. O principal objetivo deste projeto foi avaliar a expressão de retrovírus endógenos das famílias H (HH), W (HW) e K (HK) em pacientes diagnosticados com neuroblastoma. Amostras tumorais e amostras controle foram submetidas a extração de RNA total, síntese de cDNA e PCR em fase única. Os produtos de HERV foram submetidos ao sequenciamento em larga escala. No total, 43 loci de HH e 14 loci de HW foram diferencialmente expressos entre os grupos e 202 loci de HK foram detectados. As análises de expressão somadas ao contexto genético e epigenético de neuroblastoma, permitiram com que várias hipóteses fossem levantadas acerca da regulação da expressão de HERV neste tumor. A hipometilação geral do tecido tumoral pode ter um papel importante na expressão gênica e na reativação de retrotransposons, podendo ser a principal razão para a expressão de HERV neste contexto. / Neuroblastoma represents the most common solid tumor as well as the most lethal form of tumor in children. Several families of human endogenous retroviruses (HERV) are present in human genome in different integrity levels, and they are reactivated under different circumstances. HERV activity has been linked to diseases such as cancer, autoimmune diseases and even with the infection by exogenous viruses. The main goal of this project was to evaluate the expression of endogenous retroviruses families H (HH), W (HW) and K (HK) in patients diagnosed with neuroblastoma. Tumor samples and control samples were subjected to RNA extraction and a single round in-house RT-PCR. HERVs amplicons were next generation sequenced to access the specific origin of transcripts. Overall, 43 HH loci and 14 HW loci were differentially expressed between groups and, 202 HK loci was detected. Taken together, HERV expression analysis and genetic and epigenetic context of neuroblastoma provided several hypotheses about regulation of HERV expression in this type of tumor. The global hypomethylation of tumoral tissue may have a role in genes expression and retrotransposons reactivation, which may be the main reason for HERV expression in this context.

Les rétrovirus endogènes (ERV) sont constitutifs du génome des eucaryotes et représentent environ 400000 loci dans le génome humain divisés en différentes familles. Ces HERV (Human ERV) sont pour la majorité silencieux en contexte physiologique excepté dans le placenta mais présentent une activité transcriptionnelle en contexte pathologique comme par exemple dans les cancers. Il est difficile de comprendre de façon systématique les mécanismes de régulation/dérégulation des HERV et leur implication en contexte physiopathologique car il n’existe à ce jour aucun critère permettant de distinguer qu’elles sont les longues terminaisons répétées (LTR) transcriptionnellement actives dans l’ensemble de ces éléments de régulation. Nous avons développé deux générations de puces à ADN haute densité afin d’appréhender quelles étaient les LTR réactivées dans les cancers et de comprendre les mécanismes sous-jacents à la transcription des HERV. Avec la première version de la puce HERV, nous avons notamment identifié six loci de la famille HERV-W différentiellement exprimés dans le cancer testiculaire dont le locus ERVWE1 qui code pour la syncytine-1 impliquée dans la morphogénèse placentaire. L’analyse de l’ADN des tumeurs et des tissus sains adjacents démontre que l’hypométhylation des régions U3 promotrices est un pré-requis à l’activation des HERV. La deuxième version de la puce HERV a été utilisée pour une recherche de biomarqueurs pronostiques dans le cancer du poumon non à petites cellules. Ceci a permis de mettre en évidence des réactivations de HERV dans certains échantillons cancéreux et illustre la difficulté d’une telle approche au regard des disparités inter-individus / Endogenous Retroviruses (ERVs) are inherited part of the Eukaryotic genomes, and represent about 400,000 loci in the Human genome divided in distinct families. The majority of HERVs (Human ERV) are mainly silent in most physiological contexts excepted in placenta, whereas a significant expression is observed in pathological contexts such as cancers. It is difficult to understand HERV (de)regulation mechanisms and their implication in physio-pathological contexts, as there is no criteria defining transcriptional active promoters HERV long terminal repeats (LTRs) among all these regulatory élements. We developed two versions of highdensity DNA microarray to specifically detect LTR reactivated in cancers and try to understand transcription mechanism of HERV. With the first version of HERV-microarray, we identified six HERV-W loci over-expressed in testicular cancer, including the domesticated ERVWE1 locus which produces an envelope protein dubbed Syncytin-1 associated with placenta development. The analysis of DNA from tumoral versus normal tissue reveals that hypomethylation of U3 promoters in tumors is a prerequisite of HERV activation. The second version of HERV-microarray was used to identify prognosis biomarkers in non small cell lung cancer. This study identified HERV reactivation in some samples and highlighted difficulties of such approach due to inter-individuals disparities

Recently, the pace of growth in the amount of data sources within Life Sciences has increased exponentially until pose a difficult problem to efficiently manage their integration. The data avalanche we are experiencing may be significant for a turning point in science, with a change of orientation from proprietary to publicly available data and a concomitant acceptance of studies based on the latter. To investigate these issues, a Network of Excellence (EMBRACE) was launched with the aim to integrate the major databases and the most popular bioinformatics software tools. The focus of this thesis is therefore to approach the problem of seamlessly integrating varied data sources and/or distributed research tools. In paper I, we have developed a web service to facilitate allergenicity risk assessment, based on allergen descriptors, in order to characterize proteins with the potential for sensitization and cross-reactivity. In paper II, a web service was developed which uses a lightweight protocol to integrate human endogenous retrovirus (ERV) data within a public genome browser. This new data catalogue and many other publicly available sources were integrated and tested in a bioinformatics-rich client application. In paper III, GeneFinder, a distributed tool for genome-wide association studies, was developed and tested. Useful information based on a particular genomic region can be easily retrieved and assessed. Finally, in paper IV, we developed a prototype pipeline to mine the dog genome for endogenous retroviruses and displaying the transcriptional landscape of these retroviral integrations. Moreover, we further characterized a group that until this point was believed to be primate-specific. Our results also revealed that the dog has been very effective in protecting itself from such integrations. This work integrates different applications in the fields of protein allergology, biotechnology, genome association studies and endogenous retroviruses. / EMBRACE NoE EU FP6

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains xi, 185 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains viii, 146 p. : ill. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Orientador: Mary Marcondes / Banca: Wagner Luís Ferreira / Banca: Raimundo Souza Lopes / Resumo: O objetivo do presente estudo foi determinar em uma população de 302 gatos provenientes de área endêmica para leishmaniose visceral, a prevalência da infecção por Leishmania spp. e a presença de coinfecção pelo Toxoplasma gondii, vírus da imunodeficiência felina (FIV) e vírus da leucemia felina (FeLV). Foram evidenciadas formas amastigotas de Leishmania spp. em 9,93% (30/302) dos animais. A prevalência da leishmaniose observada por meio dos métodos de ELISA-proteína A, ELISA-IgG ou exame parasitológico direto foi de 21,85% (66/302), sendo 13,64% (9/66) positivos no exame parasitológico direto e sororeagentes nas técnicas de ELISA indireto. Doze animais (70,59%) foram sororeagentes para o FIV e a Leishmania spp., enquanto 17 (25,76%) apresentaram anticorpos anti-Toxoplasma gondii e anti-Leishmania spp. e cinco (71,43%) apresentavam infecção pelos três agentes. Não foi observada coinfecção entre Leishmania spp. e o FeLV. Houve associação estatisticamente significante entre a coinfecção por Leishmania spp. e pelo vírus da imunodeficiência felina, bem como entre a presença de Leishmania spp., do vírus da imunodeficiência felina e do Toxoplasma gondii. A sensibilidade e a especificidade dos métodos de ELISA-proteína A, ELISA-IgG e reação de imunofluorescência indireta para o diagnóstico de leishmaniose felina foram de 56,6% e 89,47%, 55,55% e 90,96% e 54,55% e 96,80%, respectivamente. As concordâncias entre a RIFI e as técnicas de ELISA-proteína A e ELISA-IgG foram fracas. No entanto, houve boa concordância entre as duas últimas técnicas. O presente estudo verificou que gatos residentes em área endêmica para leishmaniose visceral são predispostos à coinfecção por Leishmania spp. e vírus da imunodeficiência felina, e que parte deles desenvolvem sintomas inespecíficos e devem ser investigados em um diagnóstico diferencial / Abstract:The aim of this study was to determine, in a population of 302 cats from an endemic area for visceral leishmaniasis, the prevalence of the infection by Leishmania spp. and the presence of co-infection by Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Amastigote forms of Leishmania spp were evidenced in 9.93% (30/302) of the animals. Prevalence of leishmaniasis by ELISA-prot A, ELISA-IgG or direct parasitological examination was 21.85% (66/302), being 13.64% (9/66) positive in both direct parasitological examination and ELISA. Twelve animals (70.59%) were seroreagent for FIV and Leishmania spp., while 17 (25.76%) showed antibodies against Toxoplasma gondii and Leishmania spp. and five (71.43%) showed antibodies against those three agents. Co-infection was not observed between Leishmania spp. and FeLV. There was statistically significant correlation between the co-infection by Leishmania spp. and by the immunodeficiency virus, as well as among the present of Leishmania spp, feline immunodeficiency virus and Toxoplasma gondii. The susceptibility and the specificities of (the methods) ELISA-prot A, ELISA-IgG and reaction of indirect immunofluorescence for the diagnosis of feline leishmaniasis were 56.6% and 89.47%, 55.55% and 90.96% and 54.55% and 96.80%, respectively. The agreements between RIFI and ELISA-prot A and ELISA-IgG techniques were weak. However, there was a good agreement between the last two techniques. This study verified that cats from endemic areas for visceral leishmaniasis are predisposed to co-infection by Leishmania spp. and feline immunodeficiency virus, and that part of them developed nonspecific symptoms and should be investigated in a differential diagnosis / Mestre

O neuroblastoma é o tumor sólido mais comum e com maior índice de letalidade em crianças. Diversas famílias de retrovírus endógenos humanos (HERV) estão presentes no genoma humano, em diferentes níveis de integridade, e são reativadas sob diferentes circunstâncias. A atividade de HERV tem sido cada vez mais sido associada a doenças como câncer, doenças autoimunes e ainda com a infecção por vírus exógenos. O principal objetivo deste projeto foi avaliar a expressão de retrovírus endógenos das famílias H (HH), W (HW) e K (HK) em pacientes diagnosticados com neuroblastoma. Amostras tumorais e amostras controle foram submetidas a extração de RNA total, síntese de cDNA e PCR em fase única. Os produtos de HERV foram submetidos ao sequenciamento em larga escala. No total, 43 loci de HH e 14 loci de HW foram diferencialmente expressos entre os grupos e 202 loci de HK foram detectados. As análises de expressão somadas ao contexto genético e epigenético de neuroblastoma, permitiram com que várias hipóteses fossem levantadas acerca da regulação da expressão de HERV neste tumor. A hipometilação geral do tecido tumoral pode ter um papel importante na expressão gênica e na reativação de retrotransposons, podendo ser a principal razão para a expressão de HERV neste contexto. / Neuroblastoma represents the most common solid tumor as well as the most lethal form of tumor in children. Several families of human endogenous retroviruses (HERV) are present in human genome in different integrity levels, and they are reactivated under different circumstances. HERV activity has been linked to diseases such as cancer, autoimmune diseases and even with the infection by exogenous viruses. The main goal of this project was to evaluate the expression of endogenous retroviruses families H (HH), W (HW) and K (HK) in patients diagnosed with neuroblastoma. Tumor samples and control samples were subjected to RNA extraction and a single round in-house RT-PCR. HERVs amplicons were next generation sequenced to access the specific origin of transcripts. Overall, 43 HH loci and 14 HW loci were differentially expressed between groups and, 202 HK loci was detected. Taken together, HERV expression analysis and genetic and epigenetic context of neuroblastoma provided several hypotheses about regulation of HERV expression in this type of tumor. The global hypomethylation of tumoral tissue may have a role in genes expression and retrotransposons reactivation, which may be the main reason for HERV expression in this context.

Les cancers sont un groupe de maladies diverses et complexes responsables de millions de décès chaque année à travers le monde. Ces maladies sont multicausales et peuvent être engendrées par de nombreux facteurs génétiques ou environnementaux. Parmi les facteurs susceptibles de déclencher l’oncogénèse, les agents infectieux (virus, bactéries et parasites) sont à l’origine de plus de 16 % des cancers. Parmi les virus, l’étude de la famille des Retroviridae a permis de comprendre les mécanismes de l’oncogénèse virale. Certains rétrovirus portent des oncogènes d’origine cellulaire, d’autres activent des oncogènes cellulaires lors de leur insertion dans le génome de l’hôte et d’autres enfin portent des protéines virales oncogènes. Parmi ces derniers, le rétrovirus JSRV (Jaagsiekte Sheep Retrovirus) est responsable de l’adénocarcinome pulmonaire ovin chez les petits ruminants. JSRV transforme des cellules épithéliales alvéolaires et bronchiolaires via sa protéine d’enveloppe (Env) qui dérégule des voies de signalisation cellulaire contrôlant la prolifération, dont la voie Akt/mTOR (Phosphatidylinositol 3-kinase Alpha serine-Threonine-protein Kinase/ mammalian Target Of Rapamycin). Nous avons identifié la protéine cellulaire RALBP1 (RalA Binding Protein 1) comme un partenaire de Env et analysé les effets de cette interaction sur la transformation induite par JSRV. Nous avons confirmé la formation de complexes protéiques RALBP1/ Env dans les cellules de mammifères. Par inhibition de l'expression de RALBP1 avec des siRNA spécifiques, nous avons montré que la protéine cellulaire est impliquée dans le processus de transformation cellulaire induite par l’enveloppe et dans la modulation de la voie mTOR /p70S6K. Nous avons mis en évidence la sous-expression de RALBP1 dans les cellules exprimant Env in vitro, mais aussi ex vivo dans les cellules primaires tumorales et in vivo dans les tissus tumoraux. Nous avons déterminé que CDC42, un activateur de p70S6K dont l’activité est négativement régulée par RALBP1, interagit avec l’enveloppe de JSRV. Nous avons posé l’hypothèse que la diminution de RALBP1 provoquée par l’Env activerait CDC42 ce qui conduirait à l’activation p70S6K. CDC42 étant impliqué dans l’organisation du cytosquelette d’actine, nous nous sommes intéressés à l’effet de l’enveloppe sur le cytosquelette d’actine. Nous avons mis en évidence une désorganisation du cytosquelette d’actine et une perte de la polarisation des cellules exprimant l’enveloppe de JSRV. Comme de nombreux autres virus, JSRV pourrait moduler le cytosquelette d’actine des cellules épithéliales qu’il infecte afin de désorganiser l’épithélium et ainsi affecter son hôte plus efficacement / Worldwide, cancers are a group of diverse and complex diseases responsible for millions of deaths every year. These diseases are multicausal and associated with various genetic and environmental factors. Infectious agents (viruses, bacteria and parasites) are at the origin of more than 16 % of cancers. Among viruses, the study of the Retroviridae family allowed us to understand the mechanisms of viral oncogenesis. They transform cells by carrying oncogenes, by the activation of cellular oncogenes after their integration into the host genomes or by the oncogenic properties of some of their proteins. Among the later, JSRV (Jaagsiekte Sheep Retrovirus) is responsible for ovine lung adenocarcinoma in small ruminants. It transforms alveolar and bronchiolar epithelial cells via its envelope protein (Env). Env expression deregulates pathways involved in the control of cellular proliferation, such as the Akt/mTOR (Phosphatidylinositol 3-kinase Alpha serine-Threonine-protein Kinase/ mammalian Target Of Rapamycin) pathway. We identified RALBP1 (RalA Binding Protein 1) as a cellular partner of Env and analyzed the effects of these interaction on the JSRV induced transformation. We confirmed the formation of RALBP1/Env complexes in cells. By inhibition of RALBP1 expression with specific siRNA, we showed that RALBP1 is involved in Env mediated transformation and in the modulation of the mTOR/p70S6K pathway. We demonstrated the down expression of RALBP1 in vitro in cell lines expressing Env, and importantly ex vivo in primary cells derived from tumoral lungs and in vivo in tumoral lungs. We determined that CDC42, an activator of p70S6K whose activity is negatively regulated by RALBP1, interacts with the envelope of JSRV. We make the hypothesis that the activation of CDC42, following the Env-mediated decrease of RALBP1, would lead to the activation of p70S6K. As CDC42 is involved in the organization of the actin cytoskeleton, we were interested in the effect of Env on actin cytoskeleton. We showed the disorganization of actin cytoskeleton and the loss of polarization in cells expressing Env JSRV. As many viruses, JSRV could modulate the actin cytoskeleton in epithelial cells via its envelope in order to disrupt the epithelium and affect its host more efficiently

Chez les mammifères, les lymphocytes T CD4 sont essentiels à la défense de l’organisme contre des infections par des pathogènes ou le développement de tumeurs. Après activation, les lymphocytes T CD4 naïfs ont la capacité de se différencier en divers lymphocytes T helper (Th1, Th2, Th17…) en fonction des signaux reçus. Le choix du lignage permet d’adapter le phénotype et la fonction des cellules au type de danger détecté. Le processus de différenciation des lymphocytes T helper implique l’établissement de programmes d’expression des gènes distincts. La dynamique et la stabilité de ces programmes sont notamment régulées par l’activité d’éléments cis-régulateurs. Le but de ma thèse était de comprendre les mécanismes épigénétiques qui contrôlent la programmation des lymphocytes T CD4. Dans cet objectif, nous avons étudié le rôle de la H3K9 méthyl-transférase SETDB1 dans la différenciation des lymphocytes T CD4 en Th1 et Th2, deux lignages T helper fortement antagonistes. Nous avons découvert que SETDB1 réprime de manière critique le programme d’expression des gènes Th1. En effet, en l’absence d’expression de Setdb1, la différenciation Th1 est exacerbée. De plus, lorsqu’elles sont exposées à un signal pro-Th1, les cellules Th2 franchissent les barrières de lignage et se transdifférencient en Th1. De manière surprenante, SETDB1 ne cible pas directement les enhancers Th1. Au contraire, l’enzyme dépose de manière type cellulaire spécifique la marque répressive H3K9me3 au niveau d’un set restreint de rétrovirus endogènes (ERVs). Des analyses bio-informatiques ont indiqué que les rétrotransposons ciblés sont fortement associés à des gènes impliqués dans les processus immunitaires. La suite de ces analyses a indiqué que ces ERVs flanquent et répriment l’activité d’éléments cis-régulateurs des gènes Th1, ou agissent eux même comme des enhancers du lignage. En conclusion, la déposition de H3K9me3 par SETDB1 garantit l’intégrité des lymphocytes T helper en réprimant un panel d’ERVs qui ont été exaptés en modules cis-régulateurs pour façonner et contrôler le réseau de gènes Th1. / CD4 T lymphocytes play a central role in the defense of mammal organisms against infections by pathogens and the development of tumors. Upon activation, naïve CD4 T cells differentiate into distinct helper cell subsets depending on environmental cues. T helper cells are key players of the immune system as they finely orchestrate immune responses in a danger-adapted manner. The process of T helper differentiation relies on the establishment of complex and lineage-specific gene expression programs. The dynamics and stability of these programs are regulated at the chromatin level through epigenetic control of cis-regulatory elements. My thesis objective was to investigate the epigenetic pathways involved in the regulation of enhancer activity in CD4 T cells. In this purpose, we studied the role of the H3K9 specific methyltransferase SETDB1 in the differentiation of Th1 and Th2 cells, which are strongly antagonistic. We report that SETDB1 critically represses the Th1 gene expression program. Indeed, Setdb1-deficient naïve T cells show exacerbated Th1 priming. Moreover, when exposed to a Th1-instructive signal, SETDB1-deficient Th2 cells cross lineage boundaries and transdifferentiate into Th1 cells. Surprisingly, SETDB1 does not directly target Th1 enhancers to heterochromatin. Instead, SETDB1 deposits the repressive H3K9me3 mark at a restricted and cell type specific set of endogenous retroviruses, strongly associated with genes involved in immune processes. Further bioinformatic analyses indicated that these retrotransposons flank and repress Th1 gene cis-regulatory elements or behave themselves as Th1 gene enhancers. Thus, H3K9me3 deposition by SETDB1 ensures T cell lineage integrity by repressing a repertoire of ERVs that have been exapted into cis-regulatory modules to shape and control the Th1 gene network.