Human immunodeficiency virus type 1 (HIV-1) infection causes general loss of immune response in humans. Presently, an estimated 34 million (31.4-35.9 million) people worldwide are HIV-1 positive and many more are being newly infected. In the absence of a definitive cure, anti-HIV-1 drug therapy helps to manage the infection by suppressing virus replication. However, extensive drug resistance against most of existing drugs demands alternative anti-HIV-1 strategies. The proper knowledge about HIV-1 replication is essential to guide the development of new anti-HIV-1 strategies. The research presented in this thesis aims to understand the role of HIV-1 Integrase (IN) and cellular co-factors interactions in the early stage virus replication. In the cytoplasm, HIV-1 cDNA exists as a high molecular weight nucleoprotein complex called pre-integration complex (PIC). The cDNA enters the nucleus as a part of PIC by active nuclear import and integrates into the host genome. HIV-1 Integrase (IN) protein has been recognized as a primary viral factor for HIV-1 nuclear import, but the key contributing cellular factor(s) is unknown. We have examined the requirement of different Importinα (Impα) isoforms for HIV-1 replication and identified the requirement of Impα3 for HIV-1 replication in HeLa cells, C8166T cells, and human macrophages. Further investigations showed the specific requirement of Impα3 for HIV-1 nuclear import. By analyzing the Impα3 interaction with HIV-1 proteins, we detected the IN interaction with Impα3 and C-terminal domain (CTD) of IN was essential for Impα3 interaction. These data led to the conclusion that Impα3 is required for HIV-1 nuclear import and interacts with IN. The IN-CTD consists of conserved basic amino acid rich motifs (211KELQKQITK, 236KGPAKLLWK, and 262RRKAK) that closely resemble the consensus classical nuclear localization signal (NLS) for Impα interaction. By substitution mutation and interaction analysis, 211KELQKQITK and 262RRKAK motifs in IN were identified as required for Impα3 interaction, IN nuclear localization, and HIV-1 nuclear import. Together, these data were useful in explaining the molecular mechanism of IN and Impα3 interaction and its requirement for HIV-1 nuclear import. Retrograde transportation of macromolecules in the cytoplasm is one of the prerequisites for their nuclear import. Although an earlier study implicated the dynein complex in retrograde transport of HIV-1, cellular and viral factors that are involved in this process are unknown. In this study, we have elucidated the HIV-1 IN interaction with the dynein light chain 1 (DYNLL1) in 293T cells, in vitro, and in HIV-1 infected cells. DYNLL1 is one of the adapter proteins that mediate the cargo recruitment to dynein complex. However, our data suggested that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not for nuclear import. Surprisingly, DYNLL1 interaction of IN was dispensable for HIV-1 recruitment to dynein complex. These data led to the conclusion that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not required for HIV-1 recruitment to the dynein complex or for retrograde transport. In summary, this study advances our knowledge on the role of IN and cellular factors interactions in different early steps of HIV-1 replication and offers potential contributions in the development of future anti-HIV-1 strategies.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 16, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 112-127).

The aim of this thesis was to optimize the method of detection of phytoplasmas using DNA-microarray. It consisted of testing an appropriate method of genetic material isolation, development and optimization of PCR to amplify different groups of phytoplasmas, optimization of detection of DNA at a microarray, and sequence analysis of phytoplasma in order to design more suitable probes. PCR was first optimized for collection isolates, then also for natural samples. All 16Sr groups from the collection were sequenced and phytoplasmas were detected in them by hybridization. Phytoplasmas were detected also in natural samples: oilseed rape (species Brassica napus), red clover (Trifolium pretense), purple coneflower (Echinacea purpurea), and apple tree (Malus domestica). Using the DNA from insect vectors, only sample 202/6 from the group 16Sr-XII was positive. The sequence of red clover and oilseed rape correspond with the database samples in the group 16Sr-I "Aster yellows".

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A raiva é uma enfermidade infectocontagiosa causada por um Lyssavirus, que acomete os mamíferos, inclusive o homem, está presente em todos os continentes com exceção da Antártida. Os cães ainda são considerados os principais responsáveis pela manutenção e transmissão da raiva para o homem. Porém, nos últimos anos os morcegos hematófagos e não hematófagos têm ganhado destaque como potenciais transmissores de raiva para animais e humanos nas Américas. Em 2010, o Brasil registrou três casos de raiva humana, sendo um causado por agressão de morcego. Recentemente, várias epidemias de raiva humana transmitida por morcegos hematófagos foram relatados na região Amazônica. No estado de Roraima até a presente data não há registro de casos de raiva humana. O presente estudo teve o objetivo de detectar a presença e circulação do vírus rábico em quirópteros no estado de Roraima, bem como identificar as espécies de morcegos envolvidas na pesquisa. A técnica Transcriptase Reversa seguida da reação em Cadeia pela Polimerase foi utilizada para a detecção do vírus rábico, utilizando tecido cerebral de morcegos que foram coletados pelas equipes de vigilância epidemiológica e ambiental, da Secretaria de Saúde e Agência de Defesa Sanitária de Roraima. Os morcegos foram identificados utilizando chaves dicotômicas disponíveis para morcegos do Brasil e de outros países sul americanos. No total foram analisadas 94 amostras de morcegos, as quais apresentaram resultados negativos para raiva pela técnica da RT-PCR, no entanto, não é possível afirmar que o vírus rábico não circule em Roraima. Por outro lado, o presente estudo identificou 19 espécies de morcegos distribuídas em seis famílias, sendo uma família (Vespertilionidae) e cinco espécies de quirópteros (Diaemus youngi, Noctilio albiventris, Myotis nigricans, Eptesicus diminutus e Cynomops planirostris) ainda sem relato de ocorrência para Roraima. Morcegos hematófagos foram identificados em cinco municípios. Ressaltando que este trabalho foi um passo inicial e que novos estudos precisam ser desenvolvidos, aprimorando as estratégias de coletas a fim de monitorar a presença do vírus da raiva no Estado. / Rabies is an infectious disease that affects mammals, including human beings. Present on all continents except Antarctica. It is caused by a Lyssavirus. Dogs are considered responsible for the maintenance and transmission of rabies to humans. But in recent years the bats have become a potential source of transmitting rabies to animals and human beings in the Americas. In 2010, Brazil recorded three cases of human rabies. One of them was caused by an attack of bat. Recently, several outbreaks of human rabies transmitted by vampire bats were reported in the Amazon region, so far, in the state of Roraima there is no record of cases of human rabies. This study is aimed to detect the presence and circulation of rabies virus in bats in the state of Roraima, as well as to identify the species involved, it includes, also, the necessity of strengthen the network of epidemiological and environmental surveillance of rabies. The technique followed by reverse transcriptase polymerase chain reaction (RT-PCR) was used for virus research involving brain tissue of bats that were collected by teams of environmental and epidemiological surveillance, belonging to the Department of Health and the health protection agency of Roraima. Species of Bats were identified using dichotomous keys available for bats in Brazil and other Latin American countries. In total of 94 bat samples were analysed. The samples tested were negative for rabies. It can not be said, however, that the rabies virus does not circulate in Roraima. This research identified 19 species of bats distributed in six family. On the other hand, the research points to a richness and abundance of species of bats. This study identified one family (Vespertilionidae) and five species of bats (Diaemus youngi, Noctilio albiventris, Myotis nigricans, Eptesicus diminutus e Cynomops planirostris) not yet reported to the State. Vampire bats were identified in five municipalities. Considering the epidemiological and environmental importance of bats for ecosystems, this study is contributing to the increase of knowledge about both environmental surveillance of rabies and diversity of bats.

Le VIH-1 est un rétrovirus dont le génome est constitué d'ARN (ARNg). La conversion de cet ARNg en ADN est réalisée lors de la rétrotranscription (RTion). Elle permet de convertir l'ARNg simple brin en une molécule d'ADN double brin, qui sera ensuite intégrée dans le génome de la cellule hôte pour assurer la réplication du virus. La RTion est réalisée dans les premières heures de l'infection, dès l'entrée du virus. La protéine de nucléocapside (NC) participe activement à cette conversion. La NC est une petite protéine avec deux motifs en doigt à zinc (ZF1 et ZF2) qui avec sa partie basique N-term sont responsables de son activité chaperonne des acides nucléiques. L'équipe a récemment découvert un nouveau rôle de la NC en observant que la mutation des doigts de zinc ou de la région basique N-term déclenche prématurément la RTion avant le relargage des nouveaux virus, c'est à dire pendant les phases tardives de réplication du VIH. Nous parlons alors de "RTion tardive". Celle-ci génère des virus à forte teneur en ADN viral. Ces résultats originaux indiquent que la NC joue un rôle dans le contrôle spatio-temporel de la RTion au cours de la réplication du VIH. Mon projet de thèse a consisté à identifier les partenaires potentiels de la NC dans cette RT tardive et à élucider les mécanismes moléculaires mis en jeu. L'approche expérimentale principalement utilisée au cours de ma thèse, consiste à la production de particules VIH-1 (pNL4-3) et à l'analyse de leur contenu en acides nucléiques par PCR quantitative en temps réel. En mesurant l'effet de la présence et de l'absence de la protéine virale Vif sur le déclenchement de la RTion tardive dans des cellules produisant le VIH-1, nous avons démontré l'implication de Vif dans la régulation de la RTion tardive. Nous avons également montré que l'ARNg, et plus particulièrement sa région 5'UTR, est un partenaire essentiel de la NC et de Vif dans le contrôle du timing de la RTion tardive. Notre étude comparative avec un rétrovirus plus ancien et plus simple (pas de Vif) comme le Murine Leukemia Virus (MuLV), montre que cette propriété de la NC du VIH-1 à contrôler la RTion tardive n'est pas commune à tous les rétrovirus. Pour mieux comprendre le rôle de la NC au cours des phases tardives de la réplication du VIH-1, nous avons utilisé la technique de microscopie TIRF (Total Internal Reflection Fluorescence microscopy) en cellules vivantes. Cette technique de microscopie permet de visualiser les étapes d'assemblage, de bourgeonnement et de relargage des particules virales à la membrane plasmique de la cellule hôte. Bien que la NC n'influe pas sur la cinétique d'assemblage du virus, elle est en revanche fortement impliquée dans le contrôle du bourgeonnement et du relargage des particules VIH-1. Des expériences de trans-complémentation du VIH-1 mutant (délétion du ZF2) montrent que la NC contribue au recrutement des protéines cellulaires ESCRT (Endosomal Sorting Complex Required for Transport) aux sites d'assemblage viral, notamment via la voie Tsg101. / HIV-1 is a retrovirus whose genome consists of RNA (gRNA). Conversion of this gRNA into DNA is made during reverse transcription (RTion). It can convert single-stranded gRNA in a double-stranded DNA molecule, which is then integrated into the genome of the host cell to ensure the virus replication. The RTion is carried out in the early hours of infection, soon after virus entry. The nucleocapsid protein (NC) is actively involved in this conversion. The NC protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function the NC needs its two conserved zinc fingers and flanking basic residues. The team recently discovered a new role for the NC. When the NC has a mutation of its zinc fingers or its N-terminal basic region, the RTion is made prematurely before the release of new viruses, i.e. during the later stages of HIV replication. We call it «late RTion". This RTion generates virus with a high level of viral DNA. These results indicate that the NC plays a role in the spatio-temporal control of the RTion during HIV replication. My thesis project was to identify potential partners of the NC in the late RT process and to elucidate the molecular mechanisms involved.The experimental approach mainly used in my project is the production of HIV-1 particles (pNL4-3) and the analysis of their nucleic acid content by Q-PCR in real-time. By measuring the effect of the presence and absence of the viral protein Vif on the initiation of late RTion in cells producing HIV-1, we demonstrated the role of Vif in late RTion regulation. We also showed that the gRNA, and particularly its 5'UTR, is a key partner of the NC and Vif in the control of the late RTion. Our comparative study between HIV-1 and an old and simple (no Vif) retrovirus such as the Murine Leukemia Virus (MuLV) showed that the ability of the HIV-1 NC to control late RTion is not common to all retroviruses.To better understand the role of NC during the late stages of the HIV-1 replication, we used the TIRF microscopy (Total Internal Reflection Fluorescence Microscopy) in live cells. The TIRF allows the visualization of the viral assembly, budding and release of virus particles at the plasma membrane of the host cell. Although the NC does not contribute to the kinetic of the virus assembly, it is however strongly involved in the control of the budding and the release of the HIV-1 particles. Experiences of trans-complementation of a HIV-1 mutant (deletion of ZF2) showed that NC contributes to the recruitment of the cellular ESCRT machinery (Endosomal Sorting Complex Required for Transport) at the assembly sites, particularly through the Tsg101 pathway.

Les rétrotransposons L1 sont les seuls éléments transposables autonomes et actifs chez l'Homme et constituent 20% de notre ADN. Ils prolifèrent via un intermédiaire ARN et un processus couplé de réverse transcription et d'intégration, appelé rétrotransposition, et médié par une particule ribonucléoprotéique (RNP). Les L1s sautent de façon active dans les cellules germinales, les cellules souches embryonnaires et l'embryon précoce, ce qui provoque parfois de nouvelles maladies génétiques. Cependant ils sont considérés comme éteints dans la plupart des tissus somatiques. Dans le but d'explorer l'importance et les conséquences de la rétrotransposition des L1s chez l'Homme, nous avons développé une approche de cartographie des L1s actifs dans le génome humain, en combinant amplification sélective des sites d'insertion et séquençage à haut-débit. Nous avons utilisé cette stratégie afin d'obtenir la cartographie différentielle des L1s dans deux lignées cellulaires humaines apparentées. Ainsi, nous avons découvert plusieurs insertions de L1 présentes uniquement dans la lignée fille mais absente dans la lignée parentale, démontrant pour la première fois que les éléments L1 endogènes humains sont capables de mobilité dans des lignées de cellules somatiques en culture. D'autre part, afin d'éclaircir les déterminants qui dictent l'intégration des L1s, nous avons développé un test direct de réverse transcription in vitro à partir de RNP L1 natives partiellement purifiées de cellules humaines. Ceci nous a permis de montrer que la réverse transcriptase du L1 participe à la sélection du site d'insertion, ajoutant une couche additionnelle de spécificité après l'endonucléase L1. En conclusion, notre travail met en lumière la flexibilité de la machinerie des L1s, une propriété qui a certainement participé à l'efficacité de l'invasion des génomes de mammifères par ces éléments génétiques mobiles. / L1 retrotransposons are the only autonomous and active transposable elements in humans and comprise as much as 20% of our DNA. They proliferate via an RNA intermediate and a coupled reverse transcription and integration process, called retrotransposition and mediated by an L1-encoded ribonucleoprotein particle (RNP). L1s are actively jumping in germ cells, embryonic stem cells and in the early embryo, occasionally leading to de novo genetic diseases, but are considered silent in most somatic tissues. To comprehensively map active L1 elements in the human genome and to further explore the importance and consequences of L1 retrotransposition in humans, we combined selective amplification of L1 insertion sites and high-throughput sequencing. We applied this strategy to obtain a differential map of L1 insertions in two related human cultured cell lines and to question the possibility that endogenous L1 elements could be jumping in somatic cultured cells. We discovered several L1 insertions only present in the daughter cell line but absent in the parental cell line, demonstrating for the first time that retrotransposition of endogenous L1s takes place in a human somatic cell line. To get insights into the determinants of L1 integration, we have also developed a novel reverse transcription assay using partially purified native L1 RNPs. This enabled us to show that the L1 reverse transcriptase participates to insertion site selection, adding a second layer of specificity beyond the L1 endonuclease. Finally our work highlights the flexibility of the L1 machinery, which certainly participates to the efficient spreading of L1 elements within mammalian genomes.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21996号 / 医博第4510号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 朝長 啓造, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2 . We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2 . In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

No description available.