Murine leukemia viruses (MuLV) are retroviruses that play important roles in the study of oncogenes, integration, transcriptional regulation and gene therapy. Mink cell focus-inducing (MCF) viruses are polytropic MuLVs that by definition infect cells from a wide variety of species. Their ability to infect human cells and their utility as gene therapy vectors were not well characterized. To address this issue, primary and immortalized human cells were tested for their ability to be infected by MCF packaged defective vectors as well as replication competent MCF virus. A new packaging cell line, called MPAC, was created to package defective retroviral vectors in virus particles with envelope proteins derived from a Moloney mink cell focus-inducing (Mo-MCF) virus. The cellular tropism of MPAC-packaged retroviral vectors was the same as replication competent MCF viruses. Testing various established cell lines showed some human cell lines could be infected with MPAC-packaged vectors while others cannot. In addition, I show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication. This indicates that some human cells express a protein on their surface that acts as a receptor for MCF viruses and allows MCF viral entry. In addition, the human cells that express a receptor for MCF viral entry did not show any further block to viral replication. An important determinant in the pathogenic phenotype of MCF 247 has been mapped to the enhancer region of the retroviral long terminal repeat (LTR). Recombination of endogenous genetic elements with the 3' portion of envoccurs and incorporates unique LTR sequences. Most strongly pathogenic MCF viruses have a duplication of the enhancer element found in the LTR. AKR mice are an inbred strain of mice that develop spontaneous T-cell lymphomas between 6 and 12 months of age. 12-25 % of MCF induced early lymphomas of AKR mice show MCF viral integration's near c-myc in an opposite transcriptional orientation. A replication competent MCF virus containing a bacterial amber suppressor tRNA gene (supF) was used to investigate the changes in the enhancer region following injection of MCF containing one enhancer in the LTR. Newborn AKR mice were injected with the supF tagged replication competent virus and observed for signs of leukemia development (ruffled fur, lethargy, and tumor development). When these signs were detected, the animals were sacrificed and DNA was prepared from the isolated tumors. Thirty-one tumors DNA were analyzed for the presence of supF tagged virus and rearrangement of the c-myc locus. Nine supF tagged proviral LTRs integrated near c-myc from four animals were PCR amplified, sequenced, and/or cloned. All of the enhancer elements analyzed were derived from proviruses that integrated in a reverse orientation with respect to c-myc locus. Two of the isolated enhancer elements contained only a few base changes whereas the majority contained duplications of different sizes that encompassed different transcription factor binding sites. The duplicated enhancer regions contained duplications from 82-134 bp in length. One tumor contained a proviral enhancer with only 5 bp changes relative to the injected virus. This suggests that the enhancers need only a few specific base changes relative to the injected virus to accelerate leukemogenesis. The other three tumors contained proviral enhancers with various size duplications and additional transcription factor binding sites. These data suggest that the injected virus is not pathogenic unless the enhancer region is altered. One proviral integration site encompassing a duplicated enhancer region and 139 bp of the c-myc gene locus was PCR amplified, cloned and sequenced. A search of the current transcription factor database (Transfac 3.3) showed no known transcription factor binding site sequences were created at the junction of the enhancer duplications. The common motif of LVb, core NF-1, and GRE transcription factor binding sites, described by Golemis at al (57), was conserved throughout the isolated enhancers. Most of the enhancer elements contained additional NF-кB and/or GRE sites in close proximity to the conserved LVb-core region. These results support the hypothesis that additional NF-кB and/or GRE binding sites cooperatively interact with the conserved GRE-NF-1-LVb-core motif in c-myc induced leukemogenesis. In addition, two unique families of enhancer duplications were identified. The two families contained enhancers isolated from different tumors that displayed sequence homology and transcription factor binding site organization unique to each group.

Objetivo: Retrovirus Endógeno Humano (HERV) compreende ao redor de 8% do genoma humano. Apesar do fato de que em sua maioria são genes não-funcionais devido a processos de mutação ou perda de material genético no processo de retrotransposição, existem evidências do aumento da expressão de HERVs em tecido de câncer de próstata. Nós estudamos e comparamos a imunogenicidade de peptídeos da família HERV em 2 coortes de pacientes com câncer de próstata. Posteriormente examinamos o estado de ativação e senescência linfocitária nestas coortes. Desenho Experimental: Células Mononucleares de Sangue Periférico (CMSP) de 65 pacientes com câncer localizado da próstata em situação de hormônio-sensibilidade e de 24 pacientes com câncer de próstata metastático e em situação de resistência à castração, comparados a um grupo controle de 12 indivíduos normais foram avaliados em relação ao seu estado de resposta imune pela técnica de ELISPOT contra um conjunto de peptídeos derivados dos exons gag e env do gene da família HERV-K HML-2. Como parte de nosso estudo, foi realizado de forma preliminar uma análise genômica in silico de 500 pacientes com câncer de próstata sequenciados e disponíveis para análise pública do banco de dados TCGA, com o objetivo de reforçar o racional de nossa interrogação científica. Além disso , como estudo de correlação, fizemos uma análise por citometria de fluxo da ativação celular de linfócitos T de nossas coortes para determinarmos a imunofenotipagem e ontogenia linfocitária em nossos indivíduos investigados, no momento de nossa pesquisa de sua resposta imune. Resultados: Nossa análise da resposta imune contra peptídeos de HERV-K HML-2 por ELISPOT-Interferon Gama não mostrou nenhum resultado significativo. Nenhum paciente apresentou dados significativos de resposta de acordo com nossos critérios, apesar de nossos dados preliminares de expressão gênica terem mostrado expressão gênica em torno de 17% em pacientes com doença localizada. Nossos dados de ativação linfocitária mostraram maior ativação e senescência nos pacientes com doença disseminada e resistente à castração. Conclusões: Este parece ser o primeiro estudo a interrogar a presença de resposta celular imune contra peptídeos de HERV-K em pacientes com câncer de próstata. Nosso achados não mostraram resposta imune relevante em doença localizada ou disseminada e em diferentes estados de ativação linfocitária ou status de integridade hormonal. Apesar destes resultados, pesquisa posterior poderia utilizar diferente metodologia, como por exemplo a utilização de citometria de fluxo bem como a busca de diferentes citoquinas envolvidas, tais como as relacionadas a resposta Th2, ao invés de Th1 / Purpose: Human Endogenous Retrovirus (HERV) comprises 8% of human genome. Despite the fact that most of it is non-functional due to mutations or loss of genetic material in the process of retrotransposition, there are some evidence of increased expression of HERV in prostate cancer tissue. We studied the cellular immunogenicity of peptides from HERV-K family in 2 cohorts of prostate cancer patients. Experimental Design: PBMCs from 65 patients with hormone-intact localized prostate cancer and 24 patients with castrate-metastatic disease, matched with 12 normal controls were evaluated for cellular immune response by ELLISPOT against a pool of gag and env peptides from HERV-K family of HML-2 type. As an independent supportive study we did in silico genomic analysis of 500 prostate cancer patients from TCGA database to give another evidence of the prevalence of HERV-K gene expression in prostate cancer genome, reinforcing the rational of our questions. Results: Our analysis of cellular immune response against HERV-K HML-2 peptides by Interferon-gama ELISPOT did not show any meaningful results. No patient showed any minimal criteria of response, despite the fact that in our preliminary genomic analysis we obtained HERV expression in about 17% of a cohort of 500 patients with localized prostate cancer. In regards to the flow cytometry data of the lymphocytes we showed stronger activation and senescence status in the cohort of patients with castration sensitive and resistant disseminated disease, compared to the localized disease cohort. Conclusions: To the authors\'s knowledge this is the first study to look for cellular immune response against peptides derived from coding HERV-K transcripts in prostate cancer patients. Our findings did not show relevant immune response in neither localized nor metastatic castrate prostate cancer patients. Despite those results, further research could continue using different methodology, like flow cytometry as well as looking for different cytokines involved, such as those related to a Th2 response, instead of Th1

Objetivo: Retrovirus Endógeno Humano (HERV) compreende ao redor de 8% do genoma humano. Apesar do fato de que em sua maioria são genes não-funcionais devido a processos de mutação ou perda de material genético no processo de retrotransposição, existem evidências do aumento da expressão de HERVs em tecido de câncer de próstata. Nós estudamos e comparamos a imunogenicidade de peptídeos da família HERV em 2 coortes de pacientes com câncer de próstata. Posteriormente examinamos o estado de ativação e senescência linfocitária nestas coortes. Desenho Experimental: Células Mononucleares de Sangue Periférico (CMSP) de 65 pacientes com câncer localizado da próstata em situação de hormônio-sensibilidade e de 24 pacientes com câncer de próstata metastático e em situação de resistência à castração, comparados a um grupo controle de 12 indivíduos normais foram avaliados em relação ao seu estado de resposta imune pela técnica de ELISPOT contra um conjunto de peptídeos derivados dos exons gag e env do gene da família HERV-K HML-2. Como parte de nosso estudo, foi realizado de forma preliminar uma análise genômica in silico de 500 pacientes com câncer de próstata sequenciados e disponíveis para análise pública do banco de dados TCGA, com o objetivo de reforçar o racional de nossa interrogação científica. Além disso , como estudo de correlação, fizemos uma análise por citometria de fluxo da ativação celular de linfócitos T de nossas coortes para determinarmos a imunofenotipagem e ontogenia linfocitária em nossos indivíduos investigados, no momento de nossa pesquisa de sua resposta imune. Resultados: Nossa análise da resposta imune contra peptídeos de HERV-K HML-2 por ELISPOT-Interferon Gama não mostrou nenhum resultado significativo. Nenhum paciente apresentou dados significativos de resposta de acordo com nossos critérios, apesar de nossos dados preliminares de expressão gênica terem mostrado expressão gênica em torno de 17% em pacientes com doença localizada. Nossos dados de ativação linfocitária mostraram maior ativação e senescência nos pacientes com doença disseminada e resistente à castração. Conclusões: Este parece ser o primeiro estudo a interrogar a presença de resposta celular imune contra peptídeos de HERV-K em pacientes com câncer de próstata. Nosso achados não mostraram resposta imune relevante em doença localizada ou disseminada e em diferentes estados de ativação linfocitária ou status de integridade hormonal. Apesar destes resultados, pesquisa posterior poderia utilizar diferente metodologia, como por exemplo a utilização de citometria de fluxo bem como a busca de diferentes citoquinas envolvidas, tais como as relacionadas a resposta Th2, ao invés de Th1 / Purpose: Human Endogenous Retrovirus (HERV) comprises 8% of human genome. Despite the fact that most of it is non-functional due to mutations or loss of genetic material in the process of retrotransposition, there are some evidence of increased expression of HERV in prostate cancer tissue. We studied the cellular immunogenicity of peptides from HERV-K family in 2 cohorts of prostate cancer patients. Experimental Design: PBMCs from 65 patients with hormone-intact localized prostate cancer and 24 patients with castrate-metastatic disease, matched with 12 normal controls were evaluated for cellular immune response by ELLISPOT against a pool of gag and env peptides from HERV-K family of HML-2 type. As an independent supportive study we did in silico genomic analysis of 500 prostate cancer patients from TCGA database to give another evidence of the prevalence of HERV-K gene expression in prostate cancer genome, reinforcing the rational of our questions. Results: Our analysis of cellular immune response against HERV-K HML-2 peptides by Interferon-gama ELISPOT did not show any meaningful results. No patient showed any minimal criteria of response, despite the fact that in our preliminary genomic analysis we obtained HERV expression in about 17% of a cohort of 500 patients with localized prostate cancer. In regards to the flow cytometry data of the lymphocytes we showed stronger activation and senescence status in the cohort of patients with castration sensitive and resistant disseminated disease, compared to the localized disease cohort. Conclusions: To the authors\'s knowledge this is the first study to look for cellular immune response against peptides derived from coding HERV-K transcripts in prostate cancer patients. Our findings did not show relevant immune response in neither localized nor metastatic castrate prostate cancer patients. Despite those results, further research could continue using different methodology, like flow cytometry as well as looking for different cytokines involved, such as those related to a Th2 response, instead of Th1

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-11T16:43:01Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoPolimorfismosGenes.pdf: 2189382 bytes, checksum: c2c98617b43c03be9d1706ad6571449a (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-03T15:40:43Z (GMT) No. of bitstreams: 2 Dissertacao_InvestigacaoPolimorfismosGenes.pdf: 2189382 bytes, checksum: c2c98617b43c03be9d1706ad6571449a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-03T15:40:43Z (GMT). No. of bitstreams: 2 Dissertacao_InvestigacaoPolimorfismosGenes.pdf: 2189382 bytes, checksum: c2c98617b43c03be9d1706ad6571449a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2009 / No presente estudo foram investigadas as freqüências dos polimorfismos nos genes FAS e FASL em um grupo de 198 indivíduos soropositivos para o HIV-1 e 191 indivíduos controles soronegativos, com o objetivo de avaliar a ocorrência de uma possível associação entre os polimorfismos nestes genes e a infecção pelo HIV-1. A identificação dos alelos A e G do polimorfismo -670 FAS foi realizada por meio da técnica de PCR, utilizando seqüências de iniciadores específicos e posterior digestão enzimática (RFLP) com a enzima MvaI. A identificação dos alelos A e G do polimorfismo -124 FASL, bem como T e delT do polimorfismo -169 FASL foi realizada através da técnica de ACRS, seguido de RFLP com as endonucleases de restrição FokI e HincII, respectivamente. As análises das freqüências alélicas e genotípicas dos polimorfismos analisados não mostraram qualquer diferença significativa entre soropositivos e soronegativos. A análise da quantificação dos linfócitos T CD4⁺ entre os portadores dos diferentes genótipos do polimorfismo -670 FAS revelou uma associação significativa, sugerindo que o estado de portador do alelo G, em homo ou heterozigose, nos indivíduos infectados pelo HIV-1 pode ser um fator de proteção à depleção destas células no curso da infecção pelo HIV-1. As associações entre o número de linfócitos TCD8⁺, a carga viral plasmática e os polimorfismos analisados não foram estatisticamente significantes. Desse modo, pode-se sugerir, que o polimorfismo -670 do gene FAS, influencie na apoptose dos linfócitos T CD4⁺ no curso da infecção pelo HIV-1, assim, faz-se necessário estudos adicionais visando confirmar ou não esta associação, uma vez que a identificação desse polimorfismo pode ser, no futuro, uma importante ferramenta a ser utilizada no acompanhamento da infecção. / The present study investigated the frequency of the polymorphisms in the FAS and FASL gene in a sample of 198 HIV-1 seropositives individuals and 191 healthy control individuals, in order to evaluate the occurrence of a possible association between the polymorphisms and HIV-1 infection. The A and G alleles identification of the -670 FAS polymorphism was performed through a PCR followed by restriction endonucleases analyses (RFLP), with the enzyme MvaI. The identification of A and G alleles of -124 FASL polymorphism, T and delT of the -169 FASL polymorphism was performed using ACRS assay, followed by RFLP with the restriction endonucleases FokI and HincII, respectively. The analysis of allele and genotype frequencies of polymorphisms examined did not show any differences between seropositives and seronegatives individuals. The analysis of the quantification of CD4⁺ T lymphocytes from individuals with different genotypes of -670 FAS polymorphism showed a significant association, suggesting that the state of carrier of the G allele in homo or heterozygosity in HIV-1 individuals infected may be a protection factor from depletion of these cells in the course of HIV-1 infection. Associations between the FAS and FASL genes polymorphisms and the number of CD8⁺ T-cells and plasma viral load were not statistically significant. These findings suggest that the -670 FAS gene polymorphism, influences the apoptosis of CD4⁺ T lymphocytes in the course of HIV-1 infection, thus it is necessary further studies to confirm or not this association since that the identification of this polymorphism may be in the future, an important tool to be used in monitoring the infection.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-143).

Reproduction de : Thèse d'exercice : Médecine : Nancy 1 : 2004. / Titre provenant de l'écran-titre.

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.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-12T13:44:18Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PerfilNutricionalPortadoresHiv.pdf: 656593 bytes, checksum: c3122ef5d4a4f56f507c6403371aa19a (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-01T13:58:30Z (GMT) No. of bitstreams: 2 Dissertacao_PerfilNutricionalPortadoresHiv.pdf: 656593 bytes, checksum: c3122ef5d4a4f56f507c6403371aa19a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-01T13:58:30Z (GMT). No. of bitstreams: 2 Dissertacao_PerfilNutricionalPortadoresHiv.pdf: 656593 bytes, checksum: c3122ef5d4a4f56f507c6403371aa19a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2009 / A estimativa de pessoas vivendo com HIV-1 no Brasil, até junho de 2008, é de 432.890 casos, sendo que 3% destes residem na região Norte. O presente trabalho teve como objetivo descrever o perfil nutricional de portadores do HIV-1 no Estado do Pará e sua correlação com fatores da dieta usual, aspectos demográficos, sociais e laboratoriais que possam estar influenciando a sua qualidade de vida. O grupo populacional constou de 58 indivíduos, atendidos na Unidade de Referência Especializada em Doenças Infecciosas e Parasitárias Especiais (URE-DIPE) e avaliados longitudinalmente (19 meses). Houve predominância do sexo masculino, na faixa etária entre 20 e 50 anos e com escolaridade menor que 8 anos. O perfil antropométrico da maioria, com relação ao peso corporal (% peso atual, % peso usual e IMC) mostrou eutrofia, porém quando avaliadas as reservas de gordura (prega cutânea triciptal, PCT) e proteínas (circunferência braquial, CB e circunferência muscular braquial, CMB), a maioria mostrou desnutrição. A contagem de linfócitos T CD4+ foi maior do que 350 células/mm³ e a carga viral, menor do que 10.000 cópias/mL. Níveis de colesterol total, LDL-colesterol, triglicerídeos, glicemia, ferro sérico e hemograma encontravam-se dentro dos padrões de referência, mas não o de HDL-colesterol. A correlaçãodos resultados laboratoriais com o estado nutricional mostrou significância estatística em pelo menos uma variável antropométrica (CMB), a exceção da contagem de linfócitos T CD4+ e triglicerídeos. O uso da TARV foi observado em 83% do grupo, no entanto, não houve correlação com o perfil nutricional. A dieta usual mostrou equilíbrio qualitativo (normoproteica, normoglicídica e normolipídica), porém insuficiente quantitativamente (hipocalórica). A correlação entre a alimentação utilizada e o perfil nutricional mostrou diferença estatística apenas quando associada ao consumo de proteínas e carboidratos. / There are approximately 432,890 persons in Brazil (reported until June 2008) infectedwith HIV-1 and 3% of them are located in the North region of the country. The present work intended to describe the nutritional profile of HIV-1 infected persons in the State of Para and its correlation with dietary habits, demographic, social and laboratory variables influencing their life quality. The group included 58 individuals, attending the State Reference Unit for Infectious Diseases (URE-DIPE), and was followed for 19 months. There was a predominance of males, aged 20 to 50 years old, with less than 8 school years. The anthropometrical profile of the majority in relation to body weight (% of the current weight, % of usual weight and body mass index) showed a majority of eutrophic individuals, but lipid reserves (triceps skinfold thickness, TST) and protein reserves (arm circumference, AC, and arm muscle circumference, AMC) showed undernourishment. Counts of T CD4+ lymphocytes were greater than 350 cells/mL and plasma viral loads were lower than 10,000 copies/mL. Blood measurements of lipids (cholesterol, LDL, HDL and triglycerides), glucose, iron, red blood cell counts, white bood cell counts, hemoglobin and hematocrit were within reference values, except for HDL-cholesterol. Nutritional status correlated most of the time, with laboratory results when considering arm muscle circumference (AMC), except for T CD4+ lymphocyte counts and triglycerides. Most of the persons (83%) were currently undergoing ARVT, but no statistical correlation was associated to their nutritional profile. The nutritional assessment indicated that the group was placed as normal in relation to protein, carbohydrate and lipid intake, but quantitatively insufficient (hypocaloric). The correlation between food intake and nutritional profile did not show statistical significance, except when associated to the consumption of proteins and carbohydrates.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves : 325-355). Also available on the Internet.