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The pathogenic subgroup C feline leukemia virus (C virus) arises in infected cats by mutations in the envelope gene (env) of subgroup A FeLV (A virus) that switches the host receptor used for infection. To better understand C virus emergence and potential FeLV variants that may arise, I characterized FeLV Env sequence isolated from the primary FY981 FeLV isolate derived from an anemic cat. I show that the FY981 pseudotype virus is capable of using both the A virus receptor THTR1, and the C virus receptor FLVCR1 for infection, consistent with the FY981 Env being a hybrid A virus/C virus Env. Furthermore, I propose that pathogenic C virus arise in infected cats through FeLV variants or intermediates that are multi-tropic in their receptor use.

The pathogenic subgroup C feline leukemia virus (C virus) arises in infected cats by mutations in the envelope gene (env) of subgroup A FeLV (A virus) that switches the host receptor used for infection. To better understand C virus emergence and potential FeLV variants that may arise, I characterized FeLV Env sequence isolated from the primary FY981 FeLV isolate derived from an anemic cat. I show that the FY981 pseudotype virus is capable of using both the A virus receptor THTR1, and the C virus receptor FLVCR1 for infection, consistent with the FY981 Env being a hybrid A virus/C virus Env. Furthermore, I propose that pathogenic C virus arise in infected cats through FeLV variants or intermediates that are multi-tropic in their receptor use.

Gene therapy is the intracellular delivery of genetic material for a therapeutic effect and is currently being used in clinical trials for the treatment of cancer, AIDS and vascular diseases. Recombinant retroviral vectors are one of the most commonly used gene delivery vectors in clinical trials because they can permanently integrate the therapeutic gene into the genome of the target cell resulting in persistent gene expression. However, recombinant retroviral vectors suffer from several limitations. The gene transfer efficiency is not high enough to produce a desired therapeutic effect and the vectors lack the ability to genetically modify target tissue without producing unpredictable side- effects on healthy bystander tissue. The focus of this thesis is to determine target cell factors that affect efficiency and specificity of gene transfer of recombinant retroviruses. Successful gene transfer by recombinant retroviruses is a multi-step process and we have focused our efforts on those target cell factors that affect virus entry into the target cell. We have developed an experimental system to study the effect of pathway of virus entry and the intracellular trafficking itinerary of the targeted receptor, on the efficiency of gene transfer of targeted retroviruses. Our results indicate that interaction with a targeted receptor affects the efficiency of gene transfer of a targeted retrovirus by altering the residence time of the virus on the cell surface, by changing the region of the cell surface that the virus is exposed to, with respect to its natural receptor or by changing the pH that the virus is exposed to during intracellular transport. We have examined if recombinant retroviruses are capable of inducing signaling events in target cells to overcome barriers to efficient gene transfer. We have found that retroviruses are capable of activating actin-regulating-GTPase Rac1 while entering target cells. We have found that retroviruses use non-envelope and non-receptor molecules to induce Rac1 activation. Rac1 activity is important for efficient fusion and intracellular trafficking of the virus and blocking mediators of Rac1 activity on target cells affects the efficiency of gene transfer of recombinant retroviruses. The implications of our findings on efficient retrovirus-cell interactions are discussed.

Endogenous retroviruses (ERVs) account for a substantial portion of the genetic pool of every animal species (e.g. ~ 8% of the human genome). Despite their overwhelming abundance in nature, many questions on the basic biology of ERVs are unanswered. Sheep harbor approximately 20 copies of endogenous betaretroviruses (enJSRVs), which are related to an exogenous oncogenic virus, Jaagsiekte sheep retrovirus (JSRV). Therefore, they are an attractive model for investigation of the potential beneficial roles of ERVs in reproductive biology. Studies were conducted to determine: 1) expression of enJSRVs envelope (env) and HYAL2 mRNAs in the ovine uterus and conceptus (embryo/fetus and extraembryonic membranes) throughout gestation; 2) regulation of enJSRVs expression by progesterone; and 3) the role of enJSRVs in regulating peri-implantation placental growth and differentiation. Study One determined the localization of enJSRVs env and HYAL2 mRNAs throughout gestation. Results demonstrate that alterations in expression of enJSRVs and HYAL2 in the sheep uterus and placenta suggest the probability of a variety of physiological roles in implantation and placentation. Partial sequencing of the transcriptionally active enJSRVs from ovine uteroplacental tissues revealed expression of multiple enJSRV loci. Study Two assessed the influence of progesterone, interferon tau, and pregnancy stage on enJSRVs expression, as an effort to understand factors that may regulate enJSRVs. Results of this study support the hypothesis that expression of enJSRVs is modulated by progesterone, but not IFNτ in vivo. Study Three provides for enJSRVs regulating trophectoderm growth and differentiation in the peri-implantation conceptus. Blocking conceptus enJSRVs Env expression compromised pregnancy by retarding trophoblast outgrowth and differentiation. Inhibition of enJSRVs Env in vitro also reduced proliferation of mononuclear trophectoderm cells. Consequently, these results demonstrate that enJSRVs Env regulates trophectoderm growth and differentiation in the ovine conceptus, strongly supporting the biological significance of ERVs in placental evolution and animal reproduction Collectively, these studies illustrate that enJSRVs play an integral role in success of pregnancy. While the definitive roles of the enJSRVs have not yet been elucidated, it is evident that enJSRVs are an important component of the ovine genome and that they influence recognition and maintenance of pregnancy and placental formation.

No description available.

No description available.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Le Doux, Joseph; Committee Member: Bellamkonda, Ravi; Committee Member: Lyon, Andrew; Committee Member: Prausnitz, Mark; Committee Member: Spencer, Trent. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-16T20:46:25Z No. of bitstreams: 1 Domingos Ramon Moreau da Cunha Lasp-HIV1-Restool Desenvolvimento de uma ferramenta de bionformática para análise de resistencia do HIV-1 aos antirretrovirais.pdf: 2043486 bytes, checksum: 1cd7e640420b0a4675e14d21f7f04c9d (MD5) / Made available in DSpace on 2012-07-16T20:46:25Z (GMT). No. of bitstreams: 1 Domingos Ramon Moreau da Cunha Lasp-HIV1-Restool Desenvolvimento de uma ferramenta de bionformática para análise de resistencia do HIV-1 aos antirretrovirais.pdf: 2043486 bytes, checksum: 1cd7e640420b0a4675e14d21f7f04c9d (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / As terapias antirretrovirais (TARV) trouxeram um grande benefício para os pacientes infectados pelo HIV, porém não conseguem impedir totalmente o surgimento de formas virais resistentes, causadas principalmente pela elevada taxa mutacional do vírus. O desenvolvimento de resistência do HIV aos antirretrovirais é um fator limitante para o sucesso da TARV. Os portadores de vírus resistentes, além de não responderem adequadamente ao tratamento, podem também transmitir estes vírus mutantes, representando grave problema de saúde pública. O acúmulo de mutações de resistência aos antirretrovirais representa um desafio importante na melhoria do tratamento de pacientes com vírus multirresistentes. Atualmente, dois tipos de métodos estão estabelecidos para avaliação da resistência ou sensibilidade do HIV aos antirretrovirais: os testes fenotípicos e genotípicos de resistência. As análises de resistência do HIV aos antirretrovirais, avançaram muito nos últimos anos com o desenvolvimento dos algoritmos para avaliação de resistência. Atualmente, uma série de sistemas de interpretação de resistência fenotípica e genotípica do HIV aos antirretrovirais estão disponíveis na Internet. Estes sistemas identificam respectivamente os níveis de sensibilidade in vitro aos antirretrovirais e mutações associadas à resistência em sequêcias virais, e retornam o perfil de resistência do HIV, funcionando portanto, como importantes ferramentas para utilização em análises de resistência. No presente trabalho, foi desenvolvida uma ferramenta de bioinformática para análise de resistência do HIV-1 aos antirretrovirais, chamada de LASP-HIV1-ResTool. A ferramenta LASP-HIV1-ResTool é capaz de otimizar a análise de dados genômicos do HIV-1, é de fácil manuseio e está disponível no site da unidade de bioinformática do LASP/CPqGM/FIOCRUZ, Bioafrica e IOC/FIOCRUZ para domínio público. Esta ferramenta pode acessar um banco de dados com seqüências gênicas previamente armazenadas, extrair informações relativas à resistência aos antirretrovirais, comparar sequências de HIV-1 dos bancos de dados públicos, identificando mutações nos genes que codificam a transcriptase reversa e a protease e associar esses dados a resistência a cada antirretroviral utilizado nas terapias anti-HIV/AIDS. Adicionalmente, a ferramenta possibilita localizar sítios pós-traducionais e traçar um perfil de distribuição das sequências armazenadas no banco de dados local, tanto por resistência, quanto por sítios pós-traducionais, para servirem de parâmetro de comparação com as sequências iv submetidas pelos usuários da ferramenta. A ferramenta LASP-HIV1-ResTool apresenta uma tabela de quantidade e percentual de sequências brasileiras do HIV-1, dispostas por subtipo e outra tabela apresentando a quantidade e o percentual de seqüências das regiões Protease e Trasncriptase Reversa do HIV-1 armazenadas no Banco de Dados. Os usuários da ferramenta podem analisar sequências do banco de dados que compõe a ferramenta ou submeter suas próprias sequências de HIV-1 no formato FASTA. Durante o processamento das sequências, o sistema identifica a sequência através do número de acesso e localiza a posição inicial da sequência dentro da região gênica escolhida. Em seguida identifica a fase de leitura correta e realiza a conversão da sequência de nucleotídeos em sequência de aminoácidos. Inicia-se então o processo de localização de todas as mutações ocorridas, tanto na sequência de ácido nucléico como na sequência de aminoácido. O sistema então apresenta uma tabela apenas com as mutações que conferem a resistência do HIV-1 contra o antirretroviral. Além disso, exibe o grau de resistência de cada mutação e indica, ao final da tabela, se a amostra de HIV-1 submetida na análise é susceptível, apresenta resistência intermediária ou se é resistente ao antirretroviral. O sistema exibe dois gráficos que relacionam: a quantidade de sítios pós-traducionais em função do total de sequências submetidas e os padrões de resistência aos antirretrovirais (susceptível, intermediária e resistente) em função do total de sequências. Além de apresentar um gráfico de barras mostrando a quantidade de ocorrências de cada mutação de resistência no conjunto de sequências analisadas. A ferramenta LASP-HIV1-ResTool também gera uma página em formato XML contendo os dados das análises do usuário, que podem ser salvas em um arquivo. Os arquivos XML podem ser importados de outras ferramentas tais como: planilhas eletrônicas, programas de análise estatística e sistemas gerenciadores de bancos de dados. Para validação da aplicabilidade da ferramenta LASP-HIV1-ResTool foram utilizadas 111 amostras correspondentes a região gênica da protease e da transcriptase reversa derivadas da genotipagem da resistência do HIV-1 provenientes de pacientes infectados, que apresentavam falha virológica a TARV. Quando comparados os dados obtidos na ferramenta LASP-HIV1-ResTool com os dados obtidos através das análises realizadas com a ferramenta de Stanford, pode-se observar a similaridade entre os resultado. Por outro lado, pode-se observar diferenças entre os resultados das análises realizadas com a ferramenta LASP-HIV1-ResTool quando comparados com a ferramenta o sistema RENAGENO. / The antiretroviral therapy (HAART) has brought a great benefit to HIV-infected patients, but can not completely prevent the emergence of resistant viral forms, mainly caused by the high mutation rate of the virus. The development of HIV resistance to antiretroviral drugs is a limiting factor for the success of HAART. The patients with resistant virus, that do not respond adequately to treatment, may also transmit this virus mutants, representing a serious public health problem. The accumulation of resistance mutations to antiretroviral drugs represents a major challenge in improving the treatment of multiresistant virus. Currently, two types of methods are established to assess resistance or susceptibility of HIV to antiretroviral drugs: the phenotypic and genotypic resistance. However, the analysis of HIV resistance to antiretrovirals, have advanced greatly in recent years with the development of algorithms for evaluation of resistance. Currently, a number of systems for interpretation of HIV resistance to antiretrovirals, using algorithms, are available on the Internet. These systems identify the mutations associated with resistance, in amino acids sequences and return the viral resistance profile of HIV, acting therefore as, important tools for use in resistance analysis. In this study, we developed a bioinformatics tool for analysis of HIV-1 resistance to antiretrovirals. This tool is easy to use, will be available at the bioinformatics unit of LASP / CPqGM / FIOCRUZ, Bioafrica and IOC / FIOCRUZ in public domain and is able to optimize the analysis of genomic data of HIV-1. This tool can access a database of gene sequences previously stored, extract information, compare HIV-1 sequences from public databases, identifying mutations in genes that encode reverse transcriptase and protease, and associate that data with resistance to the individual anti -retroviral therapies used in anti-HIV/AIDS. Additionally, the tool allows locate post-translational sites and establish a distribution profile of the sequences stored in local databases, both for resistance and by post-translational sites to serve as parameter for comparison with the sequences submitted by users of the tool.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18897号 / 医博第4008号 / 新制||医||1009(附属図書館) / 31848 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松岡 雅雄, 教授 朝長 啓造, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM