Einleitung: Die im Genom der Schweine integrierten porzinen endogenen Retroviren (PERV) gehören zu den potentiellen humanpathogenen Erregern, die eines der Risiken bei der Xenotransplantation darstellen. Für die Abschätzung des Infektionsrisikos von PERV sind drei Verfahrensweisen von Bedeutung: Erstens die Evaluierung der PERV-Freisetzung aus porzinen Zellen und Geweben; Zweitens die Etablierung eines In vivo-Infektionsmodells und Drittens ein retrospektives Screenen von Patienten. Methoden: Die PERV-Expression in Inselzellen von Schweinen der Deutschen Landrasse wurde in vitro und in vivo evaluiert. Anschließend wurde PERV auf nicht-humanen Primatenzellen passagiert. In einem zweiten Modellversuch wurde murinen Zellen in vitro und SCID-Mäusen in vivo zellfreies PERV appliziert. Schließlich wurden Seren von Patienten analysiert. Ergebnisse: Die untersuchten Inselzellen setzten keine Viruspartikel frei und konnten somit weder humane Zellen noch BALB/c-Mäuse infizieren. Die verwendeten Affenzellen produzierten infektiöses PERV mit geringer Replikation. Weder in den murinen Modellversuchen noch in den untersuchten Patienten wurde eine Übertragung von PERV beobachtet. Schlussfolgerung: Schweine der Deutschen Landrasse könnten als Ausgangsbasis für die Zucht sicherer Schweine für die Xenotransplantation dienen. Da keine Infektion verschiedener muriner Zellen mit PERV beobachtet wurde, muss angenommen werden, dass Publikationen anderer Arbeitsgruppen, die eine PERV-Infektion in SCID-Mäusen diagnostizierten, ein falsch-positives Ergebnis aufgrund von Mikrochimärismus oder aufgrund von Pseudotypisierungen mit murinen endogenen Retroviren wiedergeben. Unsere Befunde werden dadurch erhärtet, dass der Rezeptor für PERV-A auf murinen Zellen nicht exprimiert wird und diese auch in vitro nicht infiziert werden konnten. In Übereinstimmung mit den weltweit etwa 200 behandelten Patienten konnte auch in den beiden neuen Studien keine Übertragung von PERV festgestellt werden. / Objective: Porcine endogenous retroviruses (PERVs) are integrated in the porcine genome and are able to infect human cells in vitro. Therefore, PERVs are one of the possible pathogens which poses a risk for xenotransplantations. In this study three significant methods were used to evaluate the infectious risk of PERV: The release of PERV particles from porcine cells and tissues, the formation of an in vivo infection model and a retrospective screening of patients. Methods: Islet cells from german landrace pigs were co-cultivated with human cells in vitro and were transplanted in BALB/c mice in vivo. Serial passaging experiments were performed with nonhuman primate cells. Murine cells were incubated in vitro and SCID mice in vivo with PERV. Sera of patients who were treated ex vivo with porcine liver cells and who had received islet cells were investigated for antibodies against PERV. Results: No virus release were observed in german landrace islet cells, thus they were neither able to infect human cells nor BALB/c mice. The used nonhuman primate cells released low replicating PERVs. None of the murine cells could be infected by PERV and no provirus integration was observed in different SCID mice organs. PERV-specific antibodies were found in none of the investigated patients. Conclusion: German landrace pigs could be used as a source for breeding safe genetically modified pigs suitable for xenotransplantation. Since there were no detectable PERV infection of different murine cells and SCID mice, it have to be supposed, that previously reported PERV transmissions to SCID mice might be due to microchimerism or to pseudotyping of murine endogenous retroviruses. Our results were confirmed by the fact, that the receptor for PERV-A is not expressed on murine cells and that these cells could not be infected in vitro. The absence of a PERV transmission in the investigated patients, correspond to the results obtained from approximately twohundred treated patients worldwide.

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