L’interleukine 26 (IL-26) a initialement été décrite comme une molécule surexprimée par des lymphocytes humains transformés par un virus. L’IL-26 a ensuite été classée comme cytokine pro-inflammatoire en raison de sa capacité à induire la production de cytokines par les cellules myéloïdes et épithéliales. Toutefois, ses fonctions biologiques restent méconnues, notamment en raison de l’absence d’orthologue chez le rat et la souris. Le laboratoire avait précédemment rapporté que l’IL-26 est surexprimée chez les patients présentant une inflammation hépatique associée à une infection chronique par le virus de l’hépatite C (HCV) et qu’elle participe à l’immunité antivirale, notamment en conférant aux cellules NK la capacité de tuer des hépatocytes infectés par le virus. Les résultats montraient également que l’IL-26 s’accumule dans les hépatocytes infectés en l’absence d’expression du transcrit. Cette observation, suggérant un rôle non conventionnel pour une cytokine associé à une localisation inhabituelle, a constitué la base de ce projet de recherche. Utilisant un modèle original de réplication du virus HCV in vitro, nos travaux montrent que l’IL-26 protège les hépatocytes de l’infection par HCV en inhibant la réplication de l’ARN viral. Cette propriété repose notamment sur la capacité de l’IL-26 à se lier à l’ARN viral. Ces données identifient l’IL-26 comme un nouvel acteur de l’arsenal antiviral, agissant notamment en inhibant la réplication du virus HCV, et suggèrent que cette molécule peut être classée dans la famille des kinocidines. / Initially identified as a molecule over expressed in virus-transformed human T cells, IL-26 has been classified as a pro inflammatory cytokine due to its capacity to induce inflammatory cytokines production by myeloid and epithelial cells. Nevertheless, its biological functions remain largely unknown, in part because of its absence of orthologs in rat and mouse.Our team has previously reported that IL-26, overexpressed in HCV-infected patients, activates NK cells, rendering them able to kill HCV-infected hepatocytes. Results also showed that IL-26 accumulates in hepatocytes of HCV-infected patients, a localization that is not usual for a cytokine. This observation led us to evaluate whether IL-26 may exhibit non-conventional properties. In this study, we demonstrate that IL-26 protects in vitro hepatocytes from HCV infection. Confocal microscopy revealed that IL-26 colocalizes with viral dsRNA. This direct antiviral activity appears to rely on the capacity of IL-26 to bind to viral RNA and to inhibit its replication. Moreover, we investigated the capacity that IL-26 complexed to viral RNA could trigger an antiviral response by immune cells. Collectively, results show that IL-26 may have a manifold protective role during HCV infection: (i) an indirect role via its capacity to confer NK cell the capacity to kill HCV-infected cells, (ii) a direct role through its ability to inhibit viral replication, and (iii) a stimulatory role by rendering viral DNA inflammatory.

H λοίμωξη που προκαλείται απο τον ιό της ηπατίτιδας C HCV είναι μείζον πρόβλημα για την δημόσια υγεία όπως επίσης και η κύρια αιτία της χρόνιας ηπατικής νόσου και ηπατοκυτταρικού καρκινώματος, με περίπου 180 εκατομμύρια μολυσμένα άτομα σε όλο τον κόσμο. Ο HCV είναι ένας RNA ιός θετικής πολικότητας που ανήκει στο γένος Hepacivirus της οικογένειας των Flaviviridae. Έξι γονότυποι ( 1-6 ) είναι γνωστοί, καθένας από τους οποίους μπορεί να υποδιαιρεθεί περαιτέρω σε αρκετές υποτύπους. Το γονιδίωμα του HCV αποτελείται από ένα μεγάλο ανοικτό πλαίσιο ανάγνωσης (ORF), πλαισιωμένο από ιδιαίτερα δομημένες 5 'και 3' αμετάφραστες περιοχές (UTRs). Και οι δύο περιοχές UTRs είναι συντηρημένες και έχουν τον έλεγχο της ιογενούς μετάφρασης και αναπαραγωγής. Το 5' UTR του HCV περιέχει μια περιοχή IRES απο την οποία γινεται η έναρξη της cap ανεξάρτητης μετάφρασης του ιικού RNA. Η HCV IRES εξαρτώμενη μετάφραση του ORF παράγει μια ενιαία πρόδρομη πολυπρωτεΐνη που μεσω μετα-μεταφραστικής τροποποίησης από κυτταρικές και ιικές πρωτεάσες, οδηγεί στην ωρίμανση δομικών και μη δομικών πρωτεϊνών. Αρκετές μελέτες έχουν δείξει ότι διαφορετικές κυτταρικές ή ιικές πρωτεΐνες μπορούν να ρυθμίσουν την ενεργότητα του HCV IRES. Στόχος της παρούσας μελέτης ήταν η κατανόηση της ρύθμισης της HCV IRES εξαρτώμενης μετάφρασης. Πρώτον, προσπαθήσαμε να ορίσουμε το ρόλο των ιικών πρωτεϊνών στην HCV IRES εξαρτώμενη μετάφραση που παραμένει αμφιλεγόμενος. Σαφώς, οι μελέτες μας έδειξαν ότι η HCV NS5A ρυθμίζει αρνητικά την ενεργότητα του IRES με κύτταρο-εξαρτώμενο τρόπο. Επιπρόσθετα, υπάρχουν ισχυρές ενδείξεις ότι η ενεργοποιημένη PKR ρυθμίζει θετικά την ενεργότητα του IRES ενώ η καταστολή της έκφρασης της ενδογενούς PKR έχει το αντίθετο αποτέλεσμα. Επιπλέον, καταλήξαμε στο συμπέρασμα ότι η NS5A μεσολαβεί ανασταλτικά στην IRES-εξαρτώμενη μετάφραση και συνδέεται με την αδρανοποίηση της PKR. Τέλος στην παρούσα εργασία ερευνήσαμε τη ρύθμιση της ενεργότητας του HCV IRES σε συνθήκες χαμηλού οξυγόνου, δεδομένου ότι υποξικές περιοχές εντοπίζονται στον ηπατοκυτταρικό καρκίνο (HCC) και συνδέονται με την ύπαρξη ενός εναλλακτικού προφίλ κυτταρικής μετάφρασης Τα αποτελέσματά μας δείχνουν ότι η HCV IRES-εξαρτώμενη μετάφραση ρυθμίζεται αρνητικά σε υποξικές συνθήκες και μάλιστα με κύτταρο-εξαρτώμενο τρόπο. / HCV infection is a major public health problem and a leading cause of chronic liver disease and hepatocellular carcinoma, with approximately 180 million infected individuals worldwide. HCV is a positive sense RNA virus that belongs to the genus hepacivirus of the Flaviviridae family. Six major HCV genotypes (1–6) are known, each of which can be further subdivided into several subtypes (1a, 1b, 2a, etc). The HCV genome consists of a large open reading frame (ORF), flanked by highly structured 5’ and 3’ untranslated regions (UTRs). Both UTRs are conserved and control viral translation and replication. The HCV 5'-UTR contains an IRES that initiates cap-independent translation of the viral RNA. IRES-mediated translation of the HCV ORF yields a single polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases, giving rise to mature structural and non structural proteins. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. The aim of this study was to understand the regulation of the HCV IRES dependent translation. Firstly, we tried to delineate the role of the viral proteins on HCV IRES dependent translation that still remains controversial. Clearly our studies demonstrated that HCV NS5A down-regulates IRES activity in a cell-type dependent manner. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. In addition, we concluded that the NS5A-mediated inhibitory effect on IRES-dependent translation was linked with the PKR inactivation. Moreover, as it is already reported that localised hypoxic areas are continuously present in HCC due to its high proliferation rate leading to an altered translation pattern, we investigated modulation of HCV IRES activity under low oxygen settings. Our results provided preliminary evidence that HCV-IRES-dependent translation is negatively regulated by low oxygen levels or under hypoxia-mimicking conditions in cell-specific manner.

Les étapes précoces de l’infection virale sont des évènements critiques dans le déroulement du cycle viral. Notamment, l’entrée virale est la première étape d’interaction entre un virus et une cellule permettant d’initier, de maintenir et de propager l’infection. De ce fait, cette étape constitue une cible majeure de la réponse immunitaire adaptative de l’hôte. C’est ainsi que cette étude bipartite s’est intéressée aux étapes précoces de l’infection du virus de l’hépatite C (HCV). Dans un premier temps, l’obtention de clones de cellules Huh-7.5 n’exprimant plus le récepteur néonatal des immunoglobulines, le FcRn, a permis d’analyser l’implication de ce récepteur dans la neutralisation du HCV par des anticorps neutralisants. Les résultats obtenus nous informent que le récepteur FcRn n’intervient vraisemblablement pas dans la modulation de la neutralisation du HCV. Dans un second temps, nous avons réalisé une étude préliminaire afin d’approfondir les mécanismes régissant l’endocytose du HCV : à savoir, quelles sont les protéines adaptatrices responsables du déclenchement de l’endocytose dépendante de la clathrine et s’il existe une potentielle voie d’entrée alternative pour ce virus. A ces fins, nous avons opté pour une stratégie basée sur la transfection de siRNA, couplée à l’utilisation des pseudoparticules HCVpp qui constituent une approche encore pertinente appliquée à l’étude de l’entrée du HCV. / The early steps during a viral infection are critical events in the course of the viral cycle. Particularly, the viral entry is the first step allowing the interaction between a virus and a cell to initiate, maintain and propagate an infection. Therefore, this very step is a major target for the host adaptive immunity. This bipartite study is focused on the early steps of the infection by the hepatitis C virus (HCV). First of all, generation of Huh-7.5 clones whose expression of the neonatal Fc receptor, FcRn, has been deleted gave us the opportunity to analyze the involvement of this receptor during the neutralization of HCV by neutralizing antibodies. Regarding the results, it appears unlikely that the FcRn modulates the neutralization of HCV. Then we conducted a preliminary study to further explore the mechanisms underlying the endocytosis of HCV: that is, which are the adaptor proteins that trigger the clathrine-mediated endocytosis and if there is another putative entry pathway for the virus. To these ends we opted for a RNAi based strategy coupled to the use of HCV-derived pseudo-particles (HCVpp) that are still useful and relevant tools dedicated to HCV-entry studies.

A hepatite C, causada pelo vírus da hepatite C (HCV), afeta milhões de pessoas no mundo. A transmissão do HCV é semelhante ao HIV, justificando a alta taxa de prevalência da coinfecção. Pacientes coinfectados HCV/HIV apresentam maior taxa de progressão da fibrose hepática e da mortalidade, em comparação aos pacientes monoinfectados com HCV. Assim, o estudo de genes e/ou moléculas que controlam a resposta imune é pertinente. No presente estudo, avaliamos o papel do antígeno leucocitário humano G (HLA-G), molécula com reconhecida atividade imunomoduladora, capaz de inibir a ativação das células T e a atividade citotóxica das células Natural Killers (NK) e linfócitos T CD8+, além de induzir a formação células T reguladoras. Nós investigamos 216 pacientes monoinfectados pelo HCV, 135 pacientes coinfectados HCV-HIV e 152 indivíduos não infectados. A variabilidade do gene HLA-G foi avaliada por sequenciamento de Sanger e a expressão hepática da molécula por imunoistoquímica. A expressão de HLA-G foi observada somente no tecido hepático dos pacientes, principalmente nos hepatócitos. O aumento de expressão de HLA-G foi associado com avanço da fibrose e da atividade necroinflamatória no fígado de ambos os grupos de pacientes. Idade igual ou superior a 40 anos e a cor de pele não-branca também foram associados com aumento da expressão hepática da molécula nos pacientes HCV. Outros fatores do hospedeiro analisados como gênero e genótipo do HCV não foram associados com o nível de expressão de HLA-G no fígado. A frequência do alelo HLA-G*01:01:01:01 estava aumentada nos pacientes HCV e do alelo G*01:05N diminuída nos pacientes coinfectados HCV-HIV, porém, não houveram associações significantes entre a variabilidade genética de HLA-G e a expressão hepática de HLA-G. O presente estudo contribui para a ampliar os conhecimentos acerca da participação da molécula HLA-G na hepatite C crônica, associado ou não com infecção pelo HIV. / Hepatitis C, caused by the hepatitis C virus (HCV), affects millions of people worldwide. The transmission of HCV is similar to HIV, which explains the high prevalence of coinfection. HCV-HIV coinfected patients have higher rate of liver fibrosis progression and mortality when compared to HCV monoinfected patients. Thus, the study of genes and/or molecules that control the immune response is relevant. In the present study, we evaluated the role of human leukocyte antigen G (HLA-G), a molecule known by its immunomodulatory activity, which is capable to inhibit T cell activation and cytotoxic activity of natural killer (NK) cells and CD8+ T cells, in addition to inducing the formation of regulatory T cells. We studied 216 HCV patients, 135 HIV-HCV coinfected patients and 152 uninfected individual. The variability of the HLA-G gene was evaluated by Sanger sequencing and the hepatic expression of the molecule by immunohistochemistry. The HLA-G expression was observed only in liver tissue of patients, mainly in hepatocytes. The increased HLA-G expression was associated with increased liver fibrosis and necroinflammatory activity in both groups of patients. The age greater than or equal to 40 years and the non-white skin color were also associated with increased hepatic expression of the molecule in the HCV patients. Other host factors analyzed as gender and HCV genotype were not associated with the level of HLA-G expression in the liver. The frequency of HLA-G*01:01:01:01 allele was increased in HCV patients and G*01:05N decreased in HCV-HIV coinfected patients, however, there was no significant association between the genetic variability of HLA-G and HLA-G liver expression. The present study contributes to expand the knowledge regarding the participation of HLA-G in chronic C hepatitis, associated or not with the HIV infection.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-04-27T18:05:38Z No. of bitstreams: 2 Dissertação - Nativa Helena Alves Del-Rios - 2011.pdf: 2685862 bytes, checksum: a88d60dfeef4b6b920f7a23cc00881a3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Erika Demachki (erikademachki@gmail.com), reason: on 2017-04-27T18:05:52Z (GMT) / Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-04-27T18:06:12Z No. of bitstreams: 2 Dissertação - Nativa Helena Alves Del-Rios - 2011.pdf: 2685862 bytes, checksum: a88d60dfeef4b6b920f7a23cc00881a3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-03T11:47:33Z (GMT) No. of bitstreams: 2 Dissertação - Nativa Helena Alves Del-Rios - 2011.pdf: 2685862 bytes, checksum: a88d60dfeef4b6b920f7a23cc00881a3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-03T11:47:33Z (GMT). No. of bitstreams: 2 Dissertação - Nativa Helena Alves Del-Rios - 2011.pdf: 2685862 bytes, checksum: a88d60dfeef4b6b920f7a23cc00881a3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2011-03-30 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are characterized by causing chronic infections in the host. The advent of potent antiretroviral therapy has resulted in a significant reduction in the incidence of opportunist infections, and thus greater life expectancy for HIV positive patients. However, the liver disease appears as a major cause of morbidity and mortality among these patients, especially those related to hepatitis C virus. Co-infection with HCV/HIV induces a worse prognosis for both infections, which may lead to the development of AIDS, a faster rapid evolution to chronic active hepatitis and / or liver cirrhosis and death. This study aimed to investigate the epidemiology and molecular profile HCV infection in HIV-infected individuals with no prior antiretroviral therapy, seen in the referral hospital for the treatment of infectious diseases (Hospital for Tropical Diseases - Anuar Auad / HDT) in Goiania, Goiás. A total of 505 treatment naïve individuals and were referred to the HDT, from April 2009 to April 2010 were interviewed and underwent blood collection. All sera were tested for antibodies to HCV (anti-HCV) and for HCV RNA by polymerase chain reaction (PCR). Genotyping was performed by reverse hybridization by the Line Probe Assay (LiPA) method. The prevalence of anti-HCV was 4.6% (95% CI: 3.0 to 6.8). The viral RNA was detected in 65.2% (15/23) of anti-HCV positive samples. The genotypes identified were 1 (subtypes 1a and 1b) and 3 (subtype 3a). The age > 40 years, living in other states or Goiania city, surgery, injecting and non-injecting drug and anti-HBc positive (antibody to core antigen of hepatitis B virus) were associated with HCV infection after logistic regression. The data presented shows the vulnerability of the HIV sropositive population to acquisition of infectious diseases such as HCV infection. Thus, the information obtained will be essential for planning public health interventions, preventing and control of hepatitis C in this population. / Os vírus da hepatite C (HCV) e da imunodeficiência humana (HIV) causam infecções crônicas no hospedeiro. O advento da terapia antiretroviral potente trouxe uma redução da incidência de infecções oportunistas, e consequentemente, uma maior expectativa de vida aos pacientes HIV soropositivos. No entanto, as hepatopatias surgem como uma das principais causas de morbimortalidade entre esses pacientes, principalmente aquelas relacionadas ao vírus C. A coinfecção HCV/HIV induz a um pior prognóstico de ambas as infecções, podendo levar ao desenvolvimento da Aids, evolução mais rápida para hepatite crônica ativa e/ou cirrose hepática e morte. Este estudo teve como objetivo investigar o perfil epidemiológico e molecular da infecção pelo HCV em indivíduos infectados pelo HIV, sem tratamento antiretroviral prévio, atendidos no Hospital de referência para o tratamento de doenças infecciosas (Hospital de Doenças Tropicais - Anuar Auad / HDT) em Goiânia, Goiás. Um total de 505 indivíduos, virgens de tratamento, encaminhados ao HDT, no período de abril/2009 a abril/2010, foram entrevistados e submetidos à coleta de sangue. As amostras (soros) foram testadas para a detecção de anticorpos para o HCV (ELISA/LIA) e submetidas à identificação do RNA-HCV pela reação em cadeia da polimerase (PCR). A genotipagem foi realizada por hibridização reversa, pelo método Line Probe Assay (LiPA). A prevalência para anti-HCV foi de 4,6% (IC 95%: 3,0-6,8). O RNA viral foi detectado em 15 amostras, sendo todas elas anti-HCV positivas. Foram identificados os genótipos 1 e 3, com predomínio do subtipo 1a, seguido dos subtipos 1b e 3a. As variáveis idade superior a 40 anos, ser procedente de Goiânia ou outros estados, cirurgia, uso de drogas injetáveis e não-injetáveis, história de prisão e positividade ao anti-HBc foram associados à infecção pelo vírus da hepatite C, após regressão logística. Os dados apresentados revelam a vulnerabilidade da população HIV soropositiva à aquisição de doenças infecciosas como a infecção pelo HCV. Assim, as informações obtidas serão essenciais para o planejamento de ações de saúde pública para a prevenção e controle da hepatite C nessa população.

Made available in DSpace on 2014-06-12T18:32:19Z (GMT). No. of bitstreams: 2 arquivo8105_1.pdf: 1882884 bytes, checksum: e39c1a843287ccbea7f187b84dbe1593 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / A ocorrência de co-infecção pelo HIV e hepatites B e C tem sido relatada desde a era- HAART (do inglês Highly Active Antinetrovial Therapy), quando a mortalidade nas pessoas infectadas pelo HIV começou diminuir. Como conseqüência do fato de terem as mesmas rotas de transmissão, a co-infecção do HBV ou HCV em pessoas infectadas pelo HIV tem aumentado e tornou-se um problema de saúde pública. No Brasil, a prevalência média da coinfecção HIV e hepatites, encontrada pelo Ministério da Saúde é em torno de 40%, com a maioria em grupos de usuários de drogas. Freqüências variáveis de co-infecção têm sido relatadas, dependendo da população e da região estudada. O objetivo principal deste estudo foi identificar a freqüência de marcadores sorológicos para hepatite B e C em pacientes infectados pelo HIV, acompanhados em um hospital escola e os possíveis fatores associados à presença de tais marcadores. Quatrocentos e vinte e nove pacientes foram estudados, de ambos os sexos e com idade variando entre 18 a 77 anos. Os participantes respondiam um questionário específico, com características sócio-demográficas e tinham uma amostra de sangue testada para os marcadores HBsAg, Anti-HBc total e Anti-HCV, utilizando a técnica MEIA-Axym-Abbott. A freqüência encontrada de marcadores foi 10,3% para o HBsAg, 38,7% para o Anti-HBc total e 10,7% para o Anti-HCV. Dentre os pacientes, 1,4% possuíam tanto HBsAg quanto Anti-HCV positivos. Não houve associação significante estatisticamente entre as variáveis parceiro homossexual, uso de drogas endovenosas, ingesta de álcool, tatuagem ou piercing, cirurgia, procedimentos invasivos e hemotransfusão e a infecção pelo HBV, expressa pela positividade do HBsAg. A única variável que mostrou associação com infecção pelo HBV foi uso de drogas inalatórias. Nenhuma destas variáveis, incluindo, parceiro homossexual, uso de drogas endovenosas, uso de drogas inalatórias, ingesta de álcool, tatuagem ou piercing, cirurgia, procedimentos invasivos e hemotransfusão tiveram associação significativa estatisticamente com a presença do Anti-HCV. Este estudo encontrou freqüências comparáveis com outros relatados no Brasil, mas com freqüências de coinfeccção menores que aqueles das regiões Sul e Sudeste. Entretanto, nenhuma associação específica com comportamentos de risco foi encontrada neste estudo, mostrando importante diferença quando comparado com estudos realizados em outras regiões do Brasil

Le génome du virus de l’Hépatite C est constitué d’un ARN monocaténaire linéaire de polarité positive (+). Les interactions ARN-ARN prennent une place essentielle dans la régulation du cycle viral.La synthèse de l’ARN est réalisée par l’ARN-polymérase ARN-dépendante (RdRp) codée par le virus. Elle serait initiée à l’extrémité 3’ des molécules à répliquer. Un ARN génomique complémentaire de polarité négative (-) est d’abord synthétisé. Il sert ensuite de matrice pour la production des brins génomiques. Les mécanismes qui président au recrutement de la polymérase et à l’initiation de la synthèse d’ARN restent, aujourd’hui, mal connus.Les structures ARN présentes aux extrémités 3’ et leurs rôles ont donc étés étudiés au cours des travaux de cette thèse. Au niveau de l’extrémité 3’ de l’ARN (+), la dimérisation a été montrée indispensable à la réplication du virus in cellulo. Ces travaux ont par la suite permis de caractériser par gel retard et cryo-microscopie la dimérisation des ARN génomiques en solution. Au niveau de l’extrémité 3’ de l’ARN (-), la dimérisation de deux molécules a également pu être caractérisée par des approches biochimiques et biophysiques. Par ailleurs la présence d’un G-quadruplex introduit un remaniement conformationnel qui se révèle indispensable à une synthèse performante de l’ARN. De manière similaire au brin génomique, la dynamique structurale résultante de ces interactions semble donc nécessaire à une réplication efficace de l’ARN par la RdRp.Les résultats obtenus soulignent l’importance de la dimérisation et des variations conformationnelles prisent aux extrémités 3’ pour la réplication de l’ARN. Ces données ouvrent alors la voie vers de nouvelles perspectives quant à la compréhension des mécanismes qui président au fonctionnement de la polymérase du VHC. / The hepatitis C virus genome consists of a linear positive sens (+) single-stranded RNA. RNA-RNA interactions play an essential role in the regulation of the viral cycle.RNA synthesis is performed by the RNA-dependent RNA-polymerase (RdRp) encoded by the virus. It would be initiated at the 3 'end of the molecule to be replicated. A complementary genomic RNA of negative polarity (-) is first synthesized. It then serves as a matrix for the production of genomic strands. The mechanisms that govern the recruitment of the polymerase and the initiation of RNA synthesis remain poorly understood today.The RNA structures found at the 3 'ends and their roles have therefore been studied during the work of this thesis. At the 3 'end of the (+) RNA, dimerization was shown to be essential for replication of the virus in cellulo. This work made it possible to characterize by gel shift assay and cryo-microscopy the dimerization of the genomic RNAs in solution. At the 3 'end of (-) RNA, the dimerization of two molecules could also be characterized by biochemical and biophysical approaches. Moreover, the presence of a G-quadruplex introduces a conformational reshuffle which proves to be indispensable for an efficient RNA synthesis. Similarly to the genomic strand, the resulting structural dynamics of these interactions appear to be necessary for efficient RNA replication by the RdRp.The results obtained here underline the importance of dimerization and conformational variations at the 3 'ends for RNA replication. These data then open the way to new perspectives on understanding the mechanisms that govern the functioning of HCV polymerase.

Background and Aims: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation–induced T cell dysfunctions during HCV infection remain elusive. Approach and Results: Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+. In contrast, the expression of stem cell–like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. Conclusions: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-15Bitstream added on 2014-06-13T21:03:48Z : No. of bitstreams: 1 provazzi_pjs_dr_sjrp.pdf: 1081709 bytes, checksum: 9a35b9ad50fc4eed266a481d830f02de (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A proteína NS3 apresenta dois domínios e é bifuncional. Apresenta três funções enzimáticas que são; 1) atividade de protease; 2) NTPase e 3) helicase. A função protease relaciona-se a tradução da proteína precursora e as funções NTPase e helicase tem grande participação na replicação do material genético viral. Trata-se de uma molécula essencial para o processamento da poliproteína precursora e também para a replicação viral e portanto, um dos principais alvos para o desenvolvimento de drogas antivirais. No domínio Protease foram evidenciadas substituições na tríade catalítica e na região de ligação ao íon zinco nos pacientes avaliados. Estas substituições, quando somadas podem explicar a resposta ao tratamento. Também foram visualizadas alterações na porção Helicase da NS3. As substituições ocorreram nos sítios de ligação ao ATP e ao RNA. Outros resíduos da Helicase relevantes para o desenvolvimento de inibidores, como R2133 e F258 e F264 não apresentaram substituições, evidenciando tratarem-se de aminoácidos conservados nessa região. Os resultados obtidos nesse trabalho fornecem informações sobre o perfil genético do vírus HCV do genótipo 3 especificamente da região codificadora da proteína NS3, permitindo o conhecimento do genoma viral e a identificação de regiões para ligação de possíveis inibidores. Este projeto certifica que a modelagem é uma ferramenta útil para a biologia estrutural e funcional, e que os modelos obtidos aqui contribuem para o desenho de novas drogas anti-virais específicas para o genótipo 3 do vírus HCV / The NS3 protein has two domains and is bifuntional. It presents three functions: 1) protease activity, 2) NTPase and 3) helicase. The protease function is related to the translation of the poliprotein precursor and functions NTPase and helicase has great participation in the replication of the viral genetic material. So. The NS3 is considered the major target for the development of antiviral drugs. In the Protease portion substitutions were evidenced in catalytic triad and the zinc ion binding sites, in the patients evaluated. These substitutions, when added up can explain the response to treatment. Also were observed changes in Helicase portion of NS3. The substitutions took place on ATP and RNA binding sites. Other residues of Helicase relevant to the development of inhibitors, as R2133 and F258 and F264, showed no substitutions, highlighting the great conservation of amino acids in this region. The results obtained in this work provide information on the genetic profile of the HCV virus genotype 3, specifically the region of NS3 protein, allowing the knowledge of the viral genome and the identification of regions for possible connection of inhibitors. This project certifies that the modeling is a useful tool for structural biology and functional, and that the models obtained here contribute to the design of new anti-viral drugs specific to the genotype 3 of HCV virus

Clinical laboratory test results are integral to patient management. Important aspects of laboratory tests’ contributions are the use of the test information and the role they have in facilitating efficient and effective use of healthcare resources. Methods of measuring those contributions were examined using quantitative HCV RNA test results (HCV VL) in therapeutic management decisions as a model. Test precision is important in those decisions; therefore, the clinical use was evaluated by studying the impact that knowledge of inherent assay imprecision had on clinicians’ decisions. A survey describing a simulated patient at a decision point for HCV triple-combination therapy management was sent to 1491 hepatology clinicians. Participants saw HCV RNA results at five different levels and were asked to choose to: continue therapy, discontinue therapy, or repeat the test. Test results were presented both with and without the 95% confidence intervals (CIs). Three of the VLs had CIs that overlapped the therapeutic decision level. Participants saw both sets of results in random order. Demographics and practice preferences were also surveyed. One-hundred-thirty-eight responses were received. Adherence to clinical guidelines was demonstrated in self-reported behaviors and in most decisions. However, participants chose to repeat the test up to 37% of the time. The impact of the knowledge of assay imprecision did not have a statistically significant effect on clinicians’ decisions. To determine economic value, an analytic decision-tree model was developed. Transition probabilities, costs, and Quality of Life values were derived from published literature. Survey respondents’ decisions were used as model inputs. Across all HCV VL levels, the calculated test value was approximately $2600, with up to $17,000 in treatment-related cost savings per patient at higher HCV VLs. The test value prevailed regardless of the presence or absence of CIs, and despite repeat testing. The calculated value in cost savings/patient was up to 100 times the investment for HCV VL testing. Laboratory tests are investments in efficient uses of healthcare resources. Proper interpretation and use of their information is integral to that value. This type of analysis can inform institutional decisions and higher level policy discussions.