No description available.

O HPgV (Pegivirus Humano), conhecido anteriormente por GBV-C, causa infecção assintomática, persistente e com alta carga viral. Sendo o vírus de RNA sem patologia associada mais prevalente no mundo até os dias de hoje, os estudos in vitro ainda não foram capazes de mimetizar a replicação in vivo, permanecendo pouco esclarecidos vários aspectos de sua biologia. Estudos em macacos mostraram que os órgãos responsáveis pela maior replicação viral são o baço e a medula óssea, no entanto as células específicas permissíveis à infecção ainda não foram determinadas. Na década de 90, o HPgV ganhou notoriedade por diminuir os efeitos patológicos do HIV e aumentar a sobrevida de pacientes coinfectados HIV/HPgV, através da diminuição da ativação do sistema imune. Devido a essa característica, diversos estudos tentaram associar a presença de viremia a doenças hematológicas e um deles mostrou que a viremia de HPgV parece estar associada com o desenvolvimento de linfoma não- Hodgkin, porém muitas variáveis de confusão parecem influenciar esse resultado. Em conjunto, os dados mostram que o HPgV pode ter influência sobre a maturação e liberação de células progenitoras da medula óssea e, portanto, tornou-se primordial avaliar o impacto da viremia em pacientes com doenças hematológicas e definir o tropismo viral. Para tanto, o presente trabalho analisou a dinâmica de exposição à viremia de HPgV em pacientes submetidos ao transplante de células-tronco hematopoiéticas (HSCT), em cadáveres autopsiados pelo Sistema de Verificação de Óbitos da Capital de São Paulo (SVOC-SP) e em pacientes submetidos à cirurgia de artroplastia pelo Instituto de Ortopedia e Traumatologia (IOT) do HCSP. A carga viral de HPgV foi mensurada por qRT-PCR em Tempo Real levando em consideração valores adquiridos com um curva padrão desenvolvida para este trabalho. Para os pacientes HSCT, o efeito da presença de viremia antes e após o procedimento foi avaliada quanto aos principais desfechos do transplante: doença enxerto-hospedeiro aguda (aGVHD), recaída da doença hematológica primária e mortalidade. Para amostras SVOC e IOT, células específicas dos tecidos (cérebro, fígado, baço, linfonodo, medula óssea e sangue) foram avaliadas quanto à quantidade de RNA viral de polaridade positiva e negativa, a fim de quantificar a replicação e definir o tropismo do vírus. Os resultados mostraram que a prevalência de HPgV encontrada nos pacientes HSCT foi maior do que em estudos similares: 42% para as amostra pré- HSCT e 31% para as amostras pós-HSCT; porém, para as amostras SVOC, a prevalência foi menor do que o esperado: 1,2%. A presença de alta carga viral de HPgV em pacientes HSCT foi associada com aumento da taxa de incidência de aGVHD (95% CI 1,05-5,37, p=0,038). Dada a alta prevalência de HPgV na população brasileira, é essencial confirmar esse achado em outras coortes, de modo a determinar se o monitoramento do HPgV pode beneficiar o cuidado a esses pacientes. Nas amostras SVOC e IOT, a medula óssea apresentou maior número de populações celulares com replicação viral, com destaque para as células B progenitoras e para as células dendríticas. Porém, devido às condições da coleta e do processamento das amostras, o tipo celular permissível à infecção não pôde ser identificado com clareza, portanto fazendo-se necessária a coleta de um número maior de amostras SVOC / HPgV (Human Pegivirus), previously known as GBV-C, causes asymptomatic, persistent and high viral load infection. To date, it is the most prevalent RNA virus without associated pathologies in the world, yet in vitro studies have not yet been able to mimic the in vivo replication, consequently many aspects of its biology remaining unclear. Studies in non-humam primatas have shown that the organs responsible for the greatest viral replication are spleen and bone marrow, however the specific cells permissible for infection have not yet been determined. In the 1990s, HPgV gained notoriety by decreasing the pathological effects of HIV and increasing the survival of coinfected HIV/HPgV patients, by decreasing the activation of the immune system. Because of this characteristic, several studies have attempted to associate the presence of viremia with haematological diseases, and one has shown that HPgV appears to be associated with the development of non-Hodgkin\'s lymphoma, but many confounding variables still seems to influence this outcome. Together, the data show that HPgV may influence the maturation and release of bone marrow progenitor cells and therefore it has become crucial to evaluate the impact of HPgV viremia in patients with haematological diseases and to define viral tropism. Therefore, the present study analyzed the dynamics of HPgV viremia exposure in patients submitted to hematopoietic stem cell transplantation (HSCT), in cadavers autopsied by the São Paulo City Death Verification System (SVOC-SP) and in patients submitted to arthroplasty surgery by the Orthopedics and Traumatology Institute (IOT) of the Clinics Hospital of Sao Paulo. The HPgV was identified by real-time qRT-PCR and the viral load quantification was estimated by a standard curve developed for this work. For HSCT patients, the effect of viremia before and after the procedure was evaluated for the main transplant outcomes: acute graft vs host disease (aGVHD), relapse of primary haematological disease and mortality. For SVOC and IOT samples, tissue-specific cells (brain, liver, spleen, lymph node, bone marrow and blood) were evaluated for the amount of viral positive and negative RNA strains, in order to quantify replication and define virus tropism. The results showed that the prevalence of HPgV found in HSCT patients was higher than in similar studies: 42% for pre-HSCT samples and 31% for post-HSCT samples; however, for SVOC samples, the prevalence was lower than expected: 1.2%. The presence of high viral load of HPgV in HSCT patients was associated with an increase in the incidence rate of aGVHD (95% CI 1.05-5.37, p = 0.038). Given the high prevalence of HPgV in the Brazilian population, it is essential to confirm this finding in other cohorts, in order to determine if HPgV monitoring can improve the care of these patients. In the SVOC and IOT samples, the bone marrow presented a higher number of cell populations with viral replication, especially the progenitor B cells and the dendritic cells. However, due to the conditions of specimen collection and processing, the major permissible cell could not be clearly identified, thus making it necessary to collect a larger number of SVOC samples

La fertilisation chez les plantes dépend de la livraison des cellules spermatiques contenues dans le pollen à l’ovule. Au contact du stigmate, le grain de pollen s’hydrate et forme une protubérance, le tube pollinique, chargé de livrer les noyaux spermatiques à l’ovule. Le tube pollinique est une cellule à croissance rapide, anisotrope et non autotrophe; ainsi tout au long de sa croissance à travers l’apoplaste du tissu pistillaire, le tube pollinique puise ses sources de carbohydrates et de minéraux du pistil. Ces éléments servent à la synthèse des constituants de la paroi qui seront acheminés par des vésicules de sécrétion jusqu’à l’apex du tube. Ce dernier doit aussi résister à des pressions mécaniques pour maintenir sa forme cylindrique et doit répondre à différents signaux directionnels pour pouvoir atteindre l’ovule. Mon projet de doctorat était de comprendre le rôle du cytosquelette dans la croissance anisotrope du tube pollinique et d’identifier les éléments responsables de sa croissance et de son guidage. Le cytosquelette du tube pollinique est composé des microfilaments d’actine et des microtubules. Pour assurer une bonne croissance des tubes polliniques in vitro, les carbohydrates et les éléments de croissance doivent être ajoutés au milieu à des concentrations bien spécifiques. J’ai donc optimisé les conditions de croissance du pollen d’Arabidopsis thaliana et de Camellia japonica qui ont été utilisés avec le pollen de Lilium longiflorum comme modèles pour mes expériences. J’ai développé une méthode rapide et efficace de fixation et de marquage du tube pollinique basée sur la technologie des microondes. J’ai aussi utilisé des outils pharmacologiques, mécaniques et moléculaires couplés à différentes techniques de microscopie pour comprendre le rôle du cytosquelette d’actine lors de la croissance et le tropisme du tube pollinique. J’ai trouvé que le cytosquelette d’actine et plus précisément l’anneau d’actine localisé dans la partie sub-apicale du tube est fortement impliqué dans la croissance et le maintien de l’architecture du tube à travers le contrôle de la livraison des vésicules de sécrétion. J’ai construit une chambre galvanotropique qui peut être montée sur un microscope inversé et qui sert à envoyer des signaux tropistiques bien précis à des tubes polliniques en croissance. J’ai trouvé que les filaments d’actine sont impliqués dans la capacité du tube pollinique à changer de direction. Ce comportement tropistique dépend de la concentration du calcium dans le milieu de croissance et du flux de calcium à travers des canaux calciques. Le gradient de calcium établi dans le tube pollinique affecte l’activité de certaines protéines qui se lient à l’actine et dont le rôle est la réorganisation des filaments d’actine. Parmi ces protéines, il y a celles de dépolymérisation de l’actine (ADF) dont deux spécifiquement exprimées dans le gamétophyte mâle d’Arabidopsis (ADF7 et ADF10). Par marquage avec des proteins fluorescents, j’ai trouvé que l’ADF7 et l’ADF10 ont des expressions différentielles pendant la microsporogenèse et la germination et croissance du tube pollinique et qu’elles partagent entre elles des rôles importants durant ces différents stades. / Fertilization in plants depends on the delivery of the sperm cells in the pollen grain through the pollen tube to the ovule. The pollen tube is a highly anisotropic, fast growing cellular protuberance. Because the pollen tube is non autotrophic, it requires a steady supply of carbohydrates and minerals supplied by the pistil to sustain its growth. These elements serve for the synthesis of cell wall material, delivered to the site of cell wall assembly in secretory vesicles that are transported along the actin cytoskeleton and deposited at the growing apex of the tube. The tube has to resist external deformation forces in order to maintain its cylindrical shape and to respond to various directional signals in order to reach its target. My objectives were to identify the role of the cytoskeleton in the anisotropic growth of the pollen tube and to determine how the tube responds to directional cues. The cytoskeleton in the pollen tube consists of microfilaments and microtubules, both forming long filamentous elements. For in vitro growing pollen tubes, carbohydrates and growth minerals have to be added to the growth medium in specific amounts order to sustain pollen tube growth. I optimized the growth conditions of Arabidopsis thaliana and Camellia japonica pollen tubes which, in addition to pollen from Lilium longiflorum, were used as model species for my experiments. I developed a microwave based, fast and efficient fixation and labelling protocol for pollen tubes. I used pharmacological, mechanical, molecular and microscopical tools to study the role of the cytoskeleton in pollen tube growth and tropism. I found that the actin cytoskeleton, and more specifically the subapical actin fringe, plays an important role in the regulation of pollen tube growth and architecture through the controlled delivery of secretory vesicles to the growing apex. I constructed a galvanotropic chamber that can be mounted on an inverted microscope to induce controlled tropic triggers. I found that the actin cytoskeleton is also involved in the ability of the pollen tube to change its direction. This tropic behaviour was shown to be dependent on the concentration of calcium ions in the growth medium and calcium influx through calcium channels. The cytosolic calcium gradient in the pollen tube regulates the activity of various actin binding proteins that are responsible for remodelling the actin cytoskeleton. Among these proteins are two Arabidopsis gametophyte-specific actin depolymerizing factors (ADFs) that I tagged with two intrinsically fluorescent proteins. I found that ADF7 and ADF10 are differentially expressed during microsporogenesis and pollen tube germination and growth and that they likely divide important functions between them.

No description available.

L’épidémiologie moléculaire des virus VIH-1 en France est caractérisée par une augmentation constante de la diversité virologique et de la fréquence des virus de sous-types non-B chez les patients en primo-infection. Entre 1997 et 2007, 28.4% des 591 patients suivis étaient infectés par des virus de sous-types non-B. De plus, 49 patients (8.3%) étaient infectés par des souches de sous-types différents sur les gènes pol et env, témoignant d’évènements de recombinaisons entre ces gènes. Ces virus recombinants étaient isolés à la fois chez des patients originaires d’Afrique sub-saharienne (28.3%) et des sujets caucasiens (6.3%). Ces résultats témoignent des échanges de souches virales entre les populations d'origine africaine et caucasienne, contribuant encore à augmenter la diversité virologique dans ces deux populations. Parmi 131 virus de sous-types non-B, 12.2% étaient classés comme ayant un tropisme CXCR4 par méthode génotypique, mais seulement 0.8% par méthode phénotypique, indiquant d’une part la faible proportion de virus non-B de tropisme CXCR4 en primo-infection en France, et d’autre part le manque de spécificité des méthodes génotypiques de détermination du tropisme pour ces sous-types, rendant nécessaire la mise au point d’autres algorithmes spécifiques pour ces virus.L’analyse de 987 virus isolés dans la cohorte entre 1999 et 2010 a mis en évidence que 12.7% d’entre eux étaient regroupés en "clusters" de transmission. Les patients en primo-infection contribuent donc de façon significative à la propagation de l’épidémie de VIH en France, particulièrement les hommes homo/bi-sexuels, avec une fréquence augmentant au cours de la période récente (2006-2010).La comparaison des quasi-espèces virales circulant concomitamment chez 8 patients en primo-infection (« receveurs ») et leurs 8 partenaires sexuels respectifs (« donneurs ») a révélé dans tous les cas la transmission d’un virus unique, présent de façon minoritaire parmi les sous-populations virales du donneur. La transmission virale muqueuse implique donc une sélection génétique drastique. / High genetic diversity is a major characteristics of HIV-1. In France, although subtype B strains are still predominant, the proportion of non-B viruses isolated in patients at the time of primary HIV-1 (PHI) infection increases over time. Between 1997 and 2007, 28.4% of patients were infected with non-B subtypes strains. Forty-nine viruses showed different phylogenies between the pol and env genes, indicating that recombinations have occurred in 8.3% of cases. These recombinants were isolated both in patients from Sub-Saharan Africa (28.3%) and in white subjects (6.3%).The phenotypic analysis of viral tropism of 131 non-B strains showed a very low (0.8%) proportion of CXCR4-tropic strains (X4 strains) at the time of PHI. Compared to phenotypic tests, genotypic predictions can overestimate (12.2% versus 0.8%) the proportion of X4 strains in non-B subtypes.The phylogenetic analysis of 987 strains isolated in 1999-2010 showed that 12.7% of PHI cosegregated into 56 transmission chains. PHIs are a significant source of onward transmission, especially in men having sex with men, with increasing frequency during the recent years (10.2% in 1999-2006 versus 15.2% in 2006-2010, p=0.02).The comparison of the viral quasispecies isolated in plasma and PBMC samples from 8 patients at the time of PHI ("recipients") and their transmitting partners ("donors") suggested that a severe genetic bottleneck occurrs during HIV-1 heterosexual and homosexual transmission. Indeed, we observed in all cases the transmission of a single variant, which was derived from an infrequent variant population within the blood of the donor. The proportion of X4 quasispecies in donors were higher in case of X4 versus CCR5-tropic viral transmission, suggesting that X4 transmission may be associated with a threshold of X4 circulating quasispecies in donors.

The Human Immunodeficiency Virus (HIV) is able to infect CD4+ T cells as well as macrophages. Macrophage-tropism has been linked to determinants in the envelope of HIV. These determinants allow envelopes to exploit low levels of CD4 for infection. Macrophages are an important reservoir of virus, especially during chronic infection, and are likely responsible for the bulk of virus produced after CD4+T cells have declined. Viral factors that may impact the ability to infect macrophages are worth studying because this cell type is so important in infection. It was previously reported that the macrophage-tropic primary isolate AD8 was vpu-independent. The molecular clone YU-2, derived from brain tissue without culture, was also reported to be macrophage-tropic despite having a mutation in the vpu start codon. It was therefore possible that vpu-independent envelopes could evolve in vivo. To examine this possibility, I constructed chimeras containing wild type or defective vpu start codons, and gp160 sequences from AD8, YU-2 or SF162 (a vpu-dependent control). I also used full length AD8 and YU-2 with wild type or defective vpu start codons. I infected macrophages with equal amounts of virus, and measured viral output over two weeks. Viruses with defective vpu start codons were released to lower levels compared to their wild type vpucounterparts. In contrast to previous reports, the AD8 envelope is not vpu-independent for replication in macrophages. The YU-2 envelope is also not vpu-independent. Macrophage-tropic envelopes from late stages of infection can be sensitive to antibodies that bind the CD4 binding site on gp120, implying that macrophage-tropic envelopes have more exposed CD4 binding sites. Neutralizing antibodies may act as modulators of macrophage-tropism over the course of infection. Using chimeras containing gp120 sequences derived from the PBMC of four HIV+patients, I examined the capacity for envelopes to infect macrophages. Three patients (MM1, 4, and 8) had macrophage-tropic envelopes before and after developing autologous neutralizing antibodies. Three patients (MM1, 4, and 23) developed heterologous antibodies against IIIB, an easily neutralized T-cell line adapted strain of HIV-1. This data indicates that macrophage-tropism in these patients is not modulated by the presence of neutralizing antibodies. The macrophage-tropism of envelopes tends to segregate depending on the tissue origin of the virus. Envelopes from two separate tissues from the same patient exhibit very different infectivity characteristics. The B33 envelope, from brain tissue, is very infectious and is macrophage-tropic, while the LN40 envelope, from lymph node tissue, is weakly infectious and is not macrophage-tropic. Replacing the entire gp41 of LN40 with that of B33 restores some infectivity to LN40. The cytoplasmic domain of gp41 contains many motifs important for assembly and infectivity. To examine which motifs are responsible for the weak infectivity of LN40, I made chimeras of gp41, as well as point mutations in gp41. The LN40 chimera containing the entire gp41 of B33 restored the most infectivity. Point mutations in the palmitoylation site, Pr55gagbinding region, and dileucine motif at the C-terminus also restored infectivity when combined. Determinants in the gp41 cytoplasmic domain are responsible for the weak infectivity of LN40; however, it is possible that there are contributing determinants in gp120, such as the ability to use low levels of CD4. Here, I examined how changes in the vpu and env genes of HIV-1 can impact infectivity, especially infectivity of macrophages. Changes that adversely impact the virus’ ability to infect macrophages may also impact the overall course of disease. However, the data here show that retaining the ability to infect, and replicate in, macrophages give HIV an advantage. I speculate that retaining the ability to infect macrophages gives the virus a reservoir for later in disease, when CD4+ T cells have been depleted, as well as way of avoiding neutralizing antibodies. This work further defines the importance of macrophages in HIV-1 infectivity and disease.

Around thirty years ago HIV-1 was identified, and from that point the known epidemic has grown to over 30 million infected individuals. Early on in the course of HIV-1 research, viruses were classified as either syncytia inducing, CXCR4-using, T-cell tropic or non-syncytia inducing, CCR5-using, macrophage tropic. Since that time, several groups have shown that this is an oversimplification. There is a great deal of diversity amongst CCR5-using HIV-1 variants. There remains a great deal to be discovered regarding HIV-1 CCR5-tropism and how this affects other aspects of HIV-1 infection. The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. In this thesis, a single amino acid determinant is described in the V1 loop that also modulates macrophage tropism. I identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection. In addition to determinants, this thesis seeks to evaluate the roles of macrophage tropic and non-macrophage tropic envelopes during the course of infection. Non-macrophage tropic virus predominates in immune tissue throughout infection, even in individuals suffering from HIV-associated dementia (HAD) who are known to carry many macrophage tropic viruses. There must be some advantage for these non-macrophage tropic viruses allowing them to persist in immune tissue throughout the disease. This thesis demonstrates that there is no advantage for these viruses to directly infect CD4+ T-cells, nor is there an advantage for them to be preferentially transmitted by dendritic cells to CD4+ T-cells. Given that transmitted/founder (T/F) viruses may preferentially interact with α4β7, and T/F viruses are non-macrophage tropic, I tested whether non-mac viruses could utilize α4β7 to their advantage. These experiments show that macrophage tropism does not play a role in gp120 interactions with α4β7. I evaluated whether there was a distinct disadvantage to macrophage tropic Envs, given their ability to infect dendritic cells and possibly stimulate the innate immune response. Using infected monocyte-derived dendritic cells (MDDCs), it was shown that mac-tropic Envs do not generate a significant immune response. These experiments demonstrate that there does not appear to be any advantage to non-macrophage tropic Envs, and that macrophage tropic Envs are able to infect CD4+ T-cells more efficiently, as well as DCs.

No description available.

Le virus de l’hépatite C (HCV) est un problème majeur de santé publique. Le développement de nouveaux traitements pour lutter contre le HCV a été ralenti par l’absence de modèles d’études in vitro et in vivo convenables. Le but de mon travail de thèse a été, dans un premier temps, de caractériser les facteurs déterminant le tropisme hépatique du HCV. En exprimant des facteurs clés dans une lignée cellulaire humaine non-hépatocytaire, nous avons reconstitué in fine l’ensemble du cycle viral dans ces cellules. L’entrée du virus dans la cellule hôte fait intervenir différents récepteurs d’entrée dont CD81, occludin (OCLN), claudin-1 (CLDN1) et scavenger receptor class B type I (SR-BI). L’expression de ces quatre récepteurs sur cette lignée la rend hautement permissive à l’entrée du virus, mais ne permet pas de rétablir la réplication du virus. L’expression du micro-ARN 122, un micro-RNA important pour l’infection du HCV, dans les cellules exprimant les quatre récepteurs, restaure une forte réplication de l’ARN viral mais ne permet pas de détecter une production de particules infectieuses. L’expression de l’apolipoprotein E (apoE), jouant un rôle primordial dans l’assemblage et la sécrétion, rétablis cette dernière étape du cycle viral du HCV dans la lignée cellulaire humaine non-hépatocytaire. Dans un second temps, j’ai utilisé la stratégie, précédemment établie, pour étudier la spécificité d’espèce de l’infection du HCV dans plusieurs lignées hépatocytaires murines. Nous avons pu rendre ces cellules permissives à l’entrée du HCV et pu détecter une très faible réplication. L’ensemble de mes travaux apportent de nouvelles informations sur la compréhension des facteurs clés nécessaire au cycle viral du HCV dans des cellules murines et humaines. / Hepatitis C virus (HCV) is a global health burden. The development of new therapeutics to treat HCV infection has been hampered by the lack of convenient in vitro and in vivo model systems. The goal of my PhD work was, in a first time, to characterize the factors determining the hepatotropism of HCV. By expressing key factors within a non-hepatic cell line, we reconstituted in fine the full HCV life cycle in those cells. Virus entry into the host cell requires different entry factors which are CD81, occludin (OCLN), claudin-1 (CLDN1) and the scavenger receptor class B type I (SR-BI). The expression of these four factors in this cell line renders it highly permissive to viral entry, but does not allow restoring replication of the virus. The expression of miR-122, a micro-RNA important for HCV infection, into the cell lines expressing the four HCV entry factors restore a strong replication of the HCV RNA but does not allow detecting infectious viral particle production. Further expression of the apolipoprotein E (apoE), which plays a critical role in the assembly and release process, restore the last step of the HCV life cycle in a non-hepatic cell line. In a second part of my PhD, I have used the previously developed strategy to study the species specificity of HCV infection using different mouse hepatoma cell lines. We have been able to render these cell lines permissive to HCV entry and have been able to detect a slight replication. Altogether, my results bring new information on the understanding of key factors important for HCV life cycle in mouse and human cells.