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Brandão, Ruben Alexandre Ribeiro. "Characterization of NS5A and NS5B resistance-associated substitutions from genotype 1 HCV infected patients in a Portuguese cohort." Master's thesis, Universidade Nova de Lisboa. Instituto Tecnologia Química e Biológica António Xavier, 2018. http://hdl.handle.net/10362/37051.
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Hepatitis C virus (HCV) is considered to be the leading cause of hepatocellular carcinoma (HCC). During the last years, several highly efficacy regimens of direct-acting antivirals (DAAs) with excellent rates of success became available. However, therapeutic failure may occur in up to 10% of treated individuals.
Our aim was to study the profile of NS5 coding region RASs in DAA-naive genotype 1 HCV infected patients, as well as to ascertain an association between treatment failure and the presence of baseline NS5 RASs. A comparison between LiPA and Sanger sequencing genotyping methods was also assessed.
Plasma RNA from 81 DAA-naïve GT1 HCV infected patients was extracted, followed by an in-house nested RT-PCR of the NS5 coding region. PCR products were purified, leading to Sanger population sequencing on the 3130xl ABI Genetic Analyzer. Sequences were aligned using ChromasPro v1.7.6, and analyzed online in hcv.geno2pheno.org.
NS5A RASs were present in 28,4% (23/81) of all GT1 infected patients. The most commonly detected NS5A RAS was Y93C/H with a prevalence of 9,9% (8/81) in all GT1 infected patients. NS5B RASs showed a prevalence of 14,8% (12/81) in all GT1 infected patients, and were only detected in GT1b, being mainly represented by C316N accounting for 40% (10/25). The combined Q30H+Y93H NS5A RASs, were detected at baseline in one HIV/HCV GT1a co-infected patient who later failed a treatment with sofosbuvir/ledipasvir (SOF/LDV) for 12 weeks. An isolated Y93H mutation was also detected at baseline in a relapsing GT1b mono-infected patient. Overall 38,3% (31/81) of all GT1 HCV infected patients presented NS5 RASs at baseline, in which 58% (18/31) were co-infected with HIV/HCV.
The obtained data supports the usefulness of resistance testing prior to treatment initiation, as a statistical significant association was found between treatment failure and the baseline presence of specific NS5 RASs, namely Y93C/H (p = 0.04).
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Fourar, Monia. "Dynamique structurale de l'ARN polymérase ARN dépendante NS5B : une nouvelle cible pour l'inhibition de la réplication du virus de l'hépatite C." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20137.
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L'une des principales cibles pour la thérapie visant le virus de l'hépatite C (VHC) est l'ARN polymérase dépendante de l'ARN NS5B indispensable à la réplication du génome virale. NS5B est l'une des enzymes clefs du cycle virale de VHC et son activation met en jeu aussi bien des interactions intramoléculaires que des interactions avec des cofacteurs viraux et cellulaires au sein du complexe de réplication. Nous avons développé une nouvelle stratégie d'inhibition de NS5B basée sur l'élaboration de peptides courts dérivés de motifs exposés à la surface de l'enzyme dans le but de cibler les nombreuses interactions impliquées dans l'activation de cette protéine. En associant une analyse fine de la structure cristallographique de NS5B avec de la modélisation moléculaire, nous avons élaboré des peptides courts mimant les motifs « hotspot » de la protéine. Ces peptides ont été évalués sur système réplicon de génotype 1b et nous avons ainsi identifié un peptide leader Moon1 de 15 résidus correspondant à un motif hautement conservé du domaine "thumb". Dans ce travail, nous avons étudié en détail la structure et le mécanisme moléculaire de ce nouvel inhibiteur de NS5B. Moon1 inhibe l'activité polymérase de la forme sauvage de NS5B ainsi que celle de mutants résistants au inhibiteurs nucléosidiques et non nucléosidiques. Nous avons démontré que la fixation de Moon1 entraine un changement de conformation de NS5B et se fait préférentiellement avec NS5B dans une conformation fermée. Ce peptide inhibe spécifiquement l'interaction entre NS5B et l'ARN double brin, indépendamment de la présence d'ions métalliques et de manière dose-dépendante. Moon1 bloque la transition entre l'étape d'initiation de novo de la synthèse d'ARN et l'extension du primer. Nous avons démontré que les résidus essentiels à l'activité de Moon1 sont hautement conservés à travers les différents génotypes et sous-types de VHC. De plus, nous avons établi une séquence minimale pour l'activité de Moon1. Nos travaux permettent de valider l'intérêt d'une stratégie interfaciale ciblant une enzyme clef du cycle du VHC et les interactions intra et intermoléculaires nécessaires à son activationThe non-structural protein RNA-dependent RNA polymerase (RdRp) NS5B plays a key role in hepatitis C virus (HCV) replication and is currently considered as one of the most relevant target to develop safe anti-HCV agents. Although many small molecules have been identified as inhibitors of NS5B, very few are active in clinic. The structure and function of NS5B have been well characterized and as other polymerases, NS5B adopts a typical “right-hand” conformation containing the characteristic fingers, palm and thumb subdomains. The activation of NS5B requires conformational changes involving intramolecular contacts as well interactions with viral proteins and host factors in the replication complex. We developed a new strategy for NS5B inhibition based on short interfacial peptides derived from NS5B surface accessible motifs that target protein-protein interfaces or essential motifs involved in NS5B-activation. Combining the NS5B crystallogaphic structure and molecular modelling, we have designed short peptides derived from NS5B surface “hotspots” that were screened using HCV genotype 1b replicon cell system. We have identified Moon1, a short 15-residu peptide, derived from a well-conserved motif located in the NS5B thumb domain that inhibits HCV replication in the low nanomolar range. Moon1 tightly binds NS5B in a conformational-dependent manner and induces NS5B conformational changes. This peptide specifically inhibits double-stranded RNA/NS5B interactions in a dose-dependent and metal ions-independent manner. Moon1 blocks the transition between RNA de novo initiation and primer-extension. We showed that residues required for Moon-1 anti-polymerase activity are well-conserved among HCV genotypes and subtypes and a minimal Moon1 active motif was established. Taken together, these results demonstrate that NS5B structural dynamics constitute an attractive target for HCV chemotherapeutics and for the design of more specific new antiviral drugs
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Aissa, Larousse Jameleddine. "Etude de la variabilité génétique des régions NS3, NS5A et NS5B du virus de l'hépatite C chez des patients Tunisiens non traités." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0434/document.
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Introduction : Le virus de l’hépatite C (VHC), est l’une des premières causes de pathologie hépatique dans le monde. Ce virus à ARN est responsable de l’hépatite C qui aboutit au développement de la cirrhose et du cancer du foie. Selon l’Organisation Mondiale de la Santé, le VHC infecte actuellement plus de 170 millions de personnes dans le monde, soit 3% de la population. L’hépatite C chronique connait toujours en Tunisie un taux de guérison faible pour le génotype 1 car le traitement standard actuellement disponible est la bithérapie interféron pégylé associé à la ribavirine. A l’heure actuelle, le développement de différentes molécules ciblant spécifiquement le VHC, appelées les antiviraux à action directe (AAD), apparait comme une potentielle révolution dans le traitement de l’infection par le VHC.Ces AAD comprennent les inhibiteurs de protéase (IP), les inhibiteurs nucléos(t)idiques (IN) et les inhibiteurs non-nucléosidiques (INN) de la polymérase NS5B ainsi que les inhibiteurs de la protéine NS5A. La quasi-espèce virale est formée d’un mélange complexe de variants viraux parmi lesquels se trouvent des variants associés à des degrés variables à la résistance aux AAD. Ces variants peuvent donc exister naturellement en absence de toute pression médicamenteuse et sont susceptibles d’avoir un impact sur la réponse aux différents traitements par AAD. Notre objectif était de déterminer la prévalence des variants associés à la résistance dans les souches tunisiennes circulantes en préambule à l’introduction deces molécules en Tunisie. Méthodes : L’amplification et le séquençage direct de la protéase NS3, de la polymérase NS5B ainsi que la région NS5A ont été effectuées chez 149 patients tunisiens naïfs de traitement et infectés par le VHC de génotype 1 (génotype 1b = 142 ; génotype 1a = 7). Résultats : Douze séquences NS3 (12/131 ; 9,2%) ont montré des mutations connes pour conférer une résistance aux IP. Une seule séquence (1/95 ; 1,1%) a montré la mutation V321I connue pour conférer une résistance aux IN-NS5B. Trente quatre séquences (34/95 ; 35,8%) ont montré des mutations connues pour diminuer la sensibilité des INN-NS5B. Une seule séquence de génotype 1a (1/7 ; 14,3%) et 17 séquences de génotype 1b (17/112 ; 16,2%) ont montré des mutations connues pour conférer une résistance au inhibiteurs de la protéine NS5A. Conclusions : Notre étude a permis de mettre en évidence la présence de substitutions conférant une diminution de la sensibilité aux AAD chez des patients tunisiens naïfs de tout traitement anti-VHC. Des études in situ seront nécessaires pour évaluer l’impact de ces mutations sur la réponse au traitementIntroduction: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. This RNA virus is responsible for hepatitis C, which leads to the development of cirrhosis and liver cancer. According to the World Health Organization, HCV infects more than 170 million people worldwide, about 3% of the population. Chronic hepatitis C still know in Tunisia low cure rates for genotype 1, because the currently standard treatment available is combination therapy of pegylated interferon plus ribavirin. At present, the development of different molecules that specifically target HCV, called direct-acting antivirals (DAA) appears as a potential revolution in the treatment of HCV infection. These DAA include protease inhibitors (PI), nucleos(t)ide (NI) and non-nucleoside inhibitors (NNI) for NS5B polymerase and NS5A inhibitors. The viral quasispecies is formed by a complex mixture of viral variants including variants associated with variable degrees of resistance to DAA. These variants may therefore exist naturally in absence of drug pressure and may affect response to different treatments by DAA. Our objective was to determine the prevalence of variants associated with resistance in circulating Tunisian strains preamble to the introduction of these molecules in Tunisia. Methods: Amplification and direct sequencing of NS3 protease, NS5B polymerase and NS5A region were performed in 149 Tunisian naïve patients infected with HCV genotype 1 (genotype 1b = 142; genotype 1a = 7) . Results: Twelve sequences NS3 (12/131; 9.2%) showed mutations known to confer resistance to PI. One sequence (1/95; 1.1%) showed the V321I mutation known to confer resistance to NS5B-IN. Thirty four sequences (34/95; 35.8%) showed mutations known to reduce the sensitivity of NS5B-INN. One genotype 1a sequence (1/7; 14.3%) and 17 genotype 1b sequences (17/112; 16.2%) showed mutations known to confer resistance to NS5A inhibitors.Conclusions: Our study highlighted the presence of substitutions conferring decreased susceptibility to DAA in naïve patients infected with HCV genotype 1. Field studies will be needed to evaluate the impact of these mutations on the treatment response
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Meguellati, Amel. "Synthèse de biomolécules agissant comme inhibiteurs de l'ARN polymérase ARN dépendante du virus de l'hépatite C et développement de nouveaux surfactants comme stabilisants des protéines membranaires par réseaux de ponts salins." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GRENV001.
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Le projet de thèse se focalise sur la synthèse de biomolécules et se subdivise en deux parties. La première partie concerne la conception et la synthèse de dérivés de produits naturels d'intérêt thérapeutique nommés aurones en vue de mettre au point de nouvelles molécules à activité antivirale. Récemment, les aurones ont été identifiées comme étant des inhibiteurs de l'ARN-polymérase ARN-dépendante (NS5B) du virus de l'hépatite C (VHC). Cette enzyme, présente chez le virus mais absente chez l'homme, joue un rôle central dans la réplication virale. Suite à ces résultats antérieurs, les efforts ont été poursuivis et, dans le cadre de cette thèse, nous avons entrepris,d'une part, la synthèse d'analogues originaux dont le cycle B des aurones a été remplacé par des hétérocycles et, d'autre part, la synthèse depseudodimères d'aurones dans le but d'affiner les exigences structurales pour améliorer l'effet inhibiteur.L'activité a été évaluée selon des tests enzymatiques et cellulaires et a permis d'identifier quelques candidats doués d'une bonne activité inhibitrice et d'une faible toxicité. La deuxième partie du projet de thèse, sans lien avec la première partie,concerne des aspects plus fondamentaux et porte sur la synthèse de nouveaux surfactants agissant comme agents stabilisants lors des procédures d'extraction et de cristallisation des protéines membranaires. Les surfactants sont des composants clés dans le domaine de la biologie structurale et de la biochimie des protéines membranaire. Ils sont nécessaires pour maintenir les protéines membranaires dans leur état fonctionnel après extraction. La grande majorité des protéines membranaires est riche en résidus basiques à l'interface. Sur la base de cette caractéristique, une nouvelle famille de surfactants est développée et testée sur des protéines membranaires appartenant aux pompes d'efflux ABC multi-résistantesThe PhD project focuses on biomolecules and is divided into two parts. The first part concerns the design and synthesis of natural product derivatives with therapeutic interest in order to develop new molecules with antiviral activity. Recently, aurones were identified as new inhibitors of hepatitis C virus (HCV) NS5B polymerase. Following these results, efforts were continuedand we undertook, on the one hand,the synthesis of original analogues in which the aurone B-ring was replaced by a heterocyclic rings and, on the other hand, the synthesis of aurone pseudodimers in order to refine the structural requirements to improve the inhibitory effect. The potent NS5B inhibitory activity combined with their low toxicity make aurones attractive drug candidates against HCV infection. The second part of the PhD thesis is unrelated to the first part and concerns more fundamental aspects. It focused on the synthesis of new surfactants acting as stabilizing agents during extraction of membrane proteins (PM). Surfactants are required for maintaining PM in their functional state after extraction from membrane lipid matrix. The vast majority of PM shares a net enrichment in basic residues at the interface between membrane and cytoplasm, a property known as the positive inside rule. Based on this feature, a new family of surfactants is developed and tested on membrane proteins belonging to the multidrug ABC efflux pumps family
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Valdau, Olga Verfasser], Dieter [Akademischer Betreuer] [Willbold, and J. [Akademischer Betreuer] Bode. "Untersuchungen zur Rekonstruktion von c-Src-NS5A-NS5B sowie EDD E3-β-Catenin – zweier krankheitsrelevanter Proteinkomplexe / Olga Valdau. Gutachter: Dieter Willbold ; J. Bode." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/1077866798/34.
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Valdau, Olga [Verfasser], Dieter [Akademischer Betreuer] Willbold, and J. [Akademischer Betreuer] Bode. "Untersuchungen zur Rekonstruktion von c-Src-NS5A-NS5B sowie EDD E3-β-Catenin – zweier krankheitsrelevanter Proteinkomplexe / Olga Valdau. Gutachter: Dieter Willbold ; J. Bode." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/1077866798/34.
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Mamigonian, Bessa Luíza. "Investigation of the hepatitis C virus RNA polymerase NS5B in solution by nuclear magnetic resonance and its interaction with intrinsically disordered domain 2 of the NS5A protein." Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10117/document.
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NS5B est l’ARN polymérase du virus de l’hépatite C (VHC). Sa structure a beaucoup été étudiée par radiocristallographie, elle contient trois sous-domaines appelés doigts, paume et pouce. Cependant, les études structurales de cette protéine en solution sont très limitées. La résonance magnétique nucléaire (RMN) a été utilisée pour étudier NS5B en solution ainsi que son interaction avec différents partenaires. L’emploi d’un échantillon de NS5B (65kDa) perdeuterée et sélectivement enrichie au niveau des méthyles 1 des résidus d’isoleucines a permis d’obtenir un spectre simplifié et de bonne qualité. Cette étude a confirmé la présence d’une dynamique particulière dans le pouce et a permis de mettre en évidence des effets à longues distances que se transmettent aux autres sous-domaines. Cette approche a alors été utilisée pour étudier l’interaction entre NS5B et le domaine 2 de la protéine NS5A (NS5A-D2) du VHC. Celui-ci est un domaine intrinsèquement désordonné qui interagit directement avec NS5B in vitro. Nous avons identifié que NS5A-D2 se lie via deux sites d’interaction sur le sous-domaine du pouce. Puisqu’un de ces sites est le site de liaison de l’inhibiteur allostérique filibuvir, nous avons étudié la liaison de cette molécule à la polymérase. Sa liaison cause des effets à longues distances tout au long de NS5B. Enfin, nous avons caractérisé la liaison d’un ARN simple brin à NS5B et nous avons identifié que NS5A-D2 et filibuvir réduisent mais ne suppriment pas l’interaction de NS5B avec l’ARN. L’analyse de NS5B par RMN en solution a permis d’étudier des interactions et d’accéder à des paramètres dynamiques très complémentaires des études cristallographiquesNS5B is the hepatitis C virus (HCV) RNA-dependent RNA polymerase. This protein has been extensively studied by X-ray crystallography and shows an organization in three subdomains called fingers, palm and thumb. Whereas static crystallographic data are abundant, structural studies of this protein in solution are limited. Nuclear magnetic resonance (NMR) spectroscopy was used to study the 65 kDa NS5B in solution as well as its interaction with binding partners. It was characterized using selective isotopic labeling of isoleucine side-chain methyl groups, which gives rise to a simplified NMR spectrum with an improved signal-to-noise ratio. This characterization confirmed the presence of particular dynamics in the subdomains, especially in the thumb, as well as long-range effects that are transmitted through to other subdomains. Furthermore, this system was used to investigate the binding of the domain 2 of NS5A (NS5A-D2), a disordered domain of another HCV protein that has been shown to directly interact with NS5B in vitro. With paramagnetic relaxation enhancement experiments we showed that NS5A-D2 binds to NS5B via, at least, two binding sites on the thumb subdomain. As one of these sites was the binding site of allosteric inhibitor filibuvir, we characterized the binding of this small molecule to NS5B by NMR and found long-range effects of its binding throughout the polymerase. Finally, we studied the binding of a small RNA template strand to NS5B and found that both NS5A-D2 and filibuvir reduce but do not abolish the interaction between the polymerase and RNA. In sum, NMR spectroscopy was used to study dynamic properties of NS5B and its interactions with binding partners
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Powdrill, Megan. "Characterization of the hepatitis C virus NS5b RNA-dependent RNA polymerase: novel inhibitors and antiviral resistance." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107791.
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The hepatitis C virus polymerase NS5b is required for replication of the viral genome, making it an attractive target for antiviral development. The polymerase contains no proof-reading activity and generates viral variants during replication with a high degree of genetic heterogeneity, complicating the development of effective antiviral therapies since resistance mutations are readily selected under drug pressure. A successful treatment regimen will likely require a combination therapy that can suppress the emergence of resistance. Here, we have described the mechanism of action of a novel class of polymerase active site inhibitors, pyrophosphate analogues. We studied interactions between these compounds and NS5b in the presence of the resistance mutations G152E and P156L and have identified interactions leading to resistance. Additionally, we have combined the pyrophosphate analogues with a second class of polymerase active site inhibitors, nucleoside analogue inhibitors (NIs). We found that the combination can interfere with excision, a potential mechanism of resistance to NIs. We have also examined fidelity of the polymerase to better understand its contribution to variability in the viral genome. Our biochemical findings suggest that the efficiency of nucleotide mismatch formation during replication influences the prevalence of resistance mutations within the viral quasispecies population. This is supported by deep-sequencing data from an HCV-infected patient cohort. Based on these findings, we have developed a mathematical model showing that combining inhibitors selecting for resistance mutations generated through difficult-to-form nucleotide mismatches could delay the onset of resistance.We extended this study by performing transient kinetic assays to characterize incorporation of NIs by NS5b and compared this to the efficiency of mismatched nucleotide incorporation. These studies demonstrate that current NIs incorporate more efficiently than mismatched nucleotides. The incorporation efficiency of the guanosine analogue ribavirin was low as compared to other NIs tested and also as compared to G:U and U:G mismatches examined in our fidelity study, suggesting its incorporation during RNA synthesis does not cause error catastrophe. Overall, these studies provide a greater understanding of the mechanism of action of polymerase inhibitors, and of the role of the polymerase in the development of antiviral resistance.La polymérase NS5b du virus de l'hépatite C est nécessaire pour la réplication du génome viral et représente donc une cible importante pour la découverte et le développement de nouveaux médicaments. La polymérase contient aucune activité de relecture et génère des variantes du virus avec un haut degré d'hétérogénéité génétique lors de sa réplication. Ceci nuit au développement de traitements antiviraux efficaces puisque les mutations de résistance sont facilement sélectionnées sous pression de médicaments. Un traitement efficace exigera probablement une combinaison thérapeutique qui pourrait empêcher la résistance. Ici, nous avons décrit le mécanisme d'action d'une nouvelle classe d'inhibiteurs du site actif de la polymérase, les analogues du pyrophosphate. Nous avons étudié les interactions entre ces inhibiteurs et NS5b, en présence des mutations de résistance G152E et P156L en plus d'identifier des interactions conduisant à la résistance. De plus, nous avons combiné les analogues du pyrophosphate avec une deuxième classe d'inhibiteurs du site actif de la polymérase, les inhibiteurs nucléotidiques (INs). Nous avons constaté que la combinaison peut interférer avec l'excision, un mécanisme potentiel de résistance aux INs. Nous avons également examiné la fidélité de la polymérase pour mieux comprendre sa contribution à la variabilité du génome viral. Nos résultats biochimiques suggèrent que l'efficacité de la formation de décalage lors de la réplication influence la prévalence des mutations de résistance au sein de la population virale quasi-espèces. Ceci est soutenu par les données obtenues suite au séquençage à très haut débit d'une cohorte de patients infectés par le VHC. Basé sur ces résultats, nous avons développé un modèle mathématique démontrant que la combinaison d'inhibiteurs qui sélectionnent des mutations de résistance générées par des mésappariements nucléotidiques difficiles à former pourrait retarder l'apparition de la résistance. Nous avons poursuivi cette étude en caractérisant l'incorporation des INs par NS5b et en comparant cela à l'efficacité de l'incorporation de nucléotides dépareillés. Ces études démontrent que les INs actuelles sont incorporées avec plus d'efficacité que les nucléotides dépareillés. L'efficacité d'incorporation de l'analogue ribavirine était faible par rapport aux autres INs testés et aussi par rapport aux mésappariements G: U et U: G examinés dans notre étude de fidélité. Ceci suggère que l'incorporation de la ribavirine lors de la synthèse d'ARN ne provoque pas d'erreur catastrophique. Globalement, ces études nous mènent à une meilleure compréhension du mécanisme d'action des inhibiteurs de la polymérase NS5b, et du rôle de la polymérase dans le développement de la résistance aux antiviraux.
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Dahl, Göran. "Kinetic studies of NS3 and NS5B from Hepatitis C virus : Implications and applications for drug discovery." Doctoral thesis, Uppsala universitet, Institutionen för biokemi och organisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-98868.
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The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design. Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.
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Choi, Yook-Wah. "Structural and functional characterization of human DDX5 and its interaction with NS5B of hepatitis C virus." University of the Western Cape, 2011. http://hdl.handle.net/11394/5299.
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Philosophiae Doctor - PhDHepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.
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Zlatev, Ivan. "Synthèse et étude d'analogues de dinucléosides phosphoramidates - inhibiteurs de la polymérase NS5B du Virus de l’Hépatite C." Montpellier 2, 2008. http://www.theses.fr/2008MON20129.
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Avec plus de 3 % de la population mondiale actuellement infectée, l'hépatite C est une des plus graves maladies infectieuses. La recherche et la mise au point de nouvelles molécules, capables de traiter ou de conduire à l'éradication de cette maladie, sont donc d'une grande importance. Nous présentons dans ce manuscrit le développement et la synthèse de deux séries de dinucléosides phosphoramidates de type 2'-O-méthylguanosin-3'-yl-cytidin-5'-yle et de type 2'-O-méthylguanosin-3'-yl-3'-désoxycytidin-5'-yle, utilisés comme inhibiteurs de la polymérase NS5B du VHC. Ces composés ont été évalués in vitro sur une polymérase purifiée et en culture cellulaire contenant un réplicon sub-génomique du virus. Ils ont montré un modeste caractère inhibiteur de la réplication du VHCWith more than 3% of the world's population chronically infected, hepatitis C is nowadays one of the leading infectious diseases. The research and development of novel antiviral molecules is hence of great importance. We describe in this manuscript the development and the synthesis of two major series of phosphoramidate dinucleosides 2'-O-methylguanosin-3'-yl-cytidin-5'-yle and 2'-O-methylguanosin-3'-yl-3'-désoxycytidin-5'-yle, used as HCV polymerase inhibitors. The target compounds were evaluated in vitro on a purified recombinant NS5B polymerase and in cells containing a HCV sub-genomic replica. Tested compounds exhibited modest inhibitory activity towards HCV replication
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Nakatani, Sueli Massumi. "Genotipagem do vírus da hepatite C por PCR em tempo real com base na análise da região NS5B." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5147/tde-30012009-162159/.
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A genotipagem do vírus da hepatite C (VHC) é a principal ferramenta para prognóstico e tempo de tratamento. Dependendo do genótipo infectado existem diferentes esquemas e tempo de tratamento. O objetivo deste trabalho foi desenvolver padronizar e validar um método de genotipagem por PCR em tempo real com base na análise da região NS5B. Esta região apresenta um grau de polimorfismo que permite identificar de modo mais acurado tanto os tipos como os subtipos do VHC. Para isto foram desenhados dois conjuntos de primers e sondas. Neste trabalho desenvolvemos um método one-step modificado em uma reação triplex em que ocorre a identificação dos genótipos (1a, 1b, 3a) e em outro set a identificação dos genótipos (2a, 2b, 2c). Os resultados obtidos pelo método de genotipagem em tempo real concordaram em 100% com os resultados de seqüênciamento da região NS5B quando excluímos amostras que foram identificados como mistura de genótipos no método desenvolvido e classificados somente como um único genótipo no seqüênciamento. Houve uma boa concordância entre o método desenvolvido e o seqüênciamento da região NS5B pelo coeficiente de Kappa (k= 0,6222; p=0,0020). O método de desenvolvido conseguiu detectar 97,93% (190/194) do genótipo 1, 86,11% (31/36) do genótipo 2 e 100% (80/80) do genótipo 3. A média da sensibilidade foi de 97%. Quando comparamos a genotipagem por PCR em tempo real e LiPA nas 310 amostras analisadas não houve resultados discordantes em relação ao genótipo. Entretanto, 26,24% (79/301) das amostras analisadas apresentaram resultados discordantes em relação ao subtipo quando comparados os dois métodos. Foi determinada a sensibilidade analítica do método através de um ponto do painel da OptiQuant que foi diluído de modo seriado e a sensibilidade relativa foi realizada através de amostras de plasma de pacientes com carga viral determinada pelo Cobas Amplicor. O limite mínimo de detecção determinado foi de 125UI/ml para o genótipo 3a, 250 UI/ml para o genótipo 1b e 2b e 500 UI/ml para o genótipo 1a. Somados a isso, o método desenvolvido tem um custo de R$ 58,00, um valor nove vezes menor que o método comercial utilizado em nosso laboratório. Além disso, no método desenvolvido, o tempo trabalhado cai para menos de 2 horas sem a necessidade de manipulação constante em comparação com LiPA que necessita em torno de 16 horas, devido ao vários passos de hibridizações e lavagens. No presente trabalho o método de genotipagem por PCR em tempo real para o VHC mostrou-se eficiente e capaz de identificar de um modo acurado os diferentes genótipos e seus subtipos e contribuir para um entendimento melhor do verdadeiro papel dos genótipos e seus subtipos e da variabilidade genética na história natural da infecção do VHC.Hepatitis C virus (HCV) genotyping is the most significant predictor of response to antiviral therapy. Depending on the infecting HCV genotyping different antiviral regimens have been proposed as well as the length of different treatment. The aim of this study was to develop and evaluate a new real time PCR of HCV genotyping based in NS5B region. This region has sequencing heterogeneity and can accurately identify both type and subtype of HCV. Furthermore, we compared the real time PCR with LiPA and sequencing of NS5B region. We developed a new one-step modified method in triplex reaction where we identified in two sets genotypes (1a, 1b, 3a) and (2a, 2b, 2c). Results obtained by real time PCR agreed 100% with those obtained by NS5B sequencing when excluded samples with mixed of HCV genotypes identified by real time PCR genotyping and in NS5B sequencing all samples were classified only as only one genotype. We found a good concordance for the analysis of genotype concordance between genotyping by real time and sequencing of NS5B region through the coefficient kappa (k= 0,6222; p=0,0020). The method developed detected 97,93% (190/194) of genotype 1, 86,11% (31/36) of genotype 2 and 100% (80/80) of genotype 3, with the overall sensitivity of this new method being 97%. Among 310 samples only two samples had discordant results at type level when comparing real time PCR and LiPA. However, 26,24% (79/301) had discordant results at subtype level when comparing LiPA and real time PCR genotyping of HCV. In order to measure the analytical sensitivity of the real time assay, one member of the panel OptiQuant HCV RNA was diluted. The relative sensitivity was determined by analysis the clinical specimens based upon the initial HCV RNA concentration determined by Cobas Amplicor. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotype 1b and 2b, and 500 IU/ml for genotype 1a. Finally, the cost of each reaction are about R$ 58,00 nine fold lower than the commercial method available in Brazil. Manipulation time of real time PCR genotyping is about 2 hours, in comparison to LiPA that requires about 16 hours due to various hybridization steps and washing. This study was demonstrated an efficient method of identification in a accurate way. HCV genotyping which is important to understand the role of genotypes and subtypes, as well as of genomic variability in the natural history of HCV infection.
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Castilho, Magda Cristina Bernardino. "Avaliação da presença de mutações de resistência no gene da NS5B e do prognóstico da infecção pelo HCV através da IL-28B em pacientes monoinfectados com HCV no RJ." Universidade do Estado do Rio de Janeiro, 2013. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=6047.
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Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão.It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.
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Khalil, Yasmin. "Study on Hepatitis C virus (HCV) subtypes in Sweden before and after the universal screening of blood donors." Thesis, Södertörn University College, School of Life Sciences, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-3354.
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Since the discovery in 1989 of hepatitis C virus (HCV) as the infectious agent responsible for the vast majority of post-transfusion non-A non-B hepatitis, blood transfusions are no longer a source for HCV transmission in Sweden. Anti-HCV testing was implemented for all blood donations in 1992. Since then intravenous drug use (IDU) has become the major route of transmission in the western world. Six genotypes and more than 80 subtypes of HCV have now been identified world-wide. These genotypes and subtypes are determined by genetic divergences between the HCV strains. Subtypes 1a, 1b, 2b, 2c, and 3a have global spread, while the other subtypes have a more limited geographical distribution. Little was known on the prevalence of HCV among blood donors and on which genotypes and subtypes of HCV were circulating in Sweden before the testing of all blood donations was implemented. The prevalence of anti-HCV was therefore investigated in sera sent to the Swedish Institute for Infectious Disease Control (SMI) from 412 patients; 241 were sampled between 1970 and 1991 before the universal screening in 1992, while 171 were sampled between 1992 and 2002. The samples derived from 193 (47%) blood donors, (104 sampled before, and 89 after 1992), and from seven other groups of patients. Two groups had suspected known routes of infection, intravenous drug use (IDU) 33 patients and hemodialysis, 16 patients, while it was unknown for the other patients. Anti-HCV was detected in 120 (29%) samples. The highest frequency was found among IDUs, (91%). Before general screening was implemented, 2.8% of the blood donors were positive for hepatitis C, whereas 28% of those sampled after 1992 were anti-HCV positive. Those latter samples were sent to SMI due to anti-HCV reactivity in a primary test at the blood centre. HCV RNA could be detected by PCR in 56 (47%) of the anti-HCV positive samples, the subtype could be determined by sequencing in 45 (80%) of those. The subtypes found were 1a in 31 %, 1b in 18%, 2b in 22%, and 3a in 27%. One sample was of subtype 2c. There was a tendency of increase of genotype 2 and a decrease in subtype 1a with time. 1a was found in 38% of the samples collected before 1992, while it was only found in 19% of the samples from 1992 or later. On the other hand genotype 2 was found in 17% sera sampled before 1992 and in 37% of the samples collected 1992 or later. It is not known if this genotype has recently been introduced into Sweden. Further analysis on larger series of samples is needed to confirm these preliminary results.
AcknowledgmentsI would like to express my gratitude to several people who have been supportive in different ways throughout this project.First of all, I want to thank my supervisor Helene Norder, for giving me the possibility to do my diploma thesis at the Department of Virology, Swedish Institute for Infectious Disease control (SMI) and for helping me during this study and for the many insightful conversations during the design and development stages of the application, and also for the many helpful comments and suggestions on the text of the thesis.I want to express my appreciation to my laboratory supervisor Regina Wallin, Camilla Jern and Josefine Ederth for helping me during the procedure for this study. Then, I want to thank my examiner Magnus Johansson from the Södertörns university collegefor his advice on writing this paper. Finally, I would like to thank my family and specially my mother Bahar Hamid for always supporting me during my whole life.Last, but not least, I would like to thank my friends Annika Andersson and Yourdons Yemane for being encouraging, understanding and always supportive.
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Uengwetwanit, Tanaporn [Verfasser], Wolfgang [Akademischer Betreuer] Sippl, Gabriele [Akademischer Betreuer] Costantino, and Gerhard [Akademischer Betreuer] Wolber. "In silico screening of inhibitors and conformational analysis of HCV NS5B polymerase / Tanaporn Uengwetwanit. Betreuer: Wolfgang Sippl ; Gabriele Costantino ; Gerhard Wolber." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2014. http://d-nb.info/1054636761/34.
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Harrus, Déborah. "Compréhension des déterminants moléculaires de l'activation de la réplication du virus de l'hépatite C par corrélation d'informations biochimiques et structurales sur sa polymérase NS5B." Paris 11, 2010. http://www.theses.fr/2010PA114861.
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Le virus de l'hépatite C (VHC) est un virus variable classifié en génotypes, qui diffèrent par leur distribution géographique, la sévérité des dommages causés au foie, et leur réponse aux traitements. L'ARN polymérase dépendante de l'ARN NS5B est une cible de choix pour les inhibiteurs du VHC. Les structures cristallographiques décrivent son organisation en 3 domaines appelés "doigts", "paume" et "pouce", le site catalytique se situant à la jonction entre ces trois domaines. Un segment C-Terminal de 40 acides aminés appelé "connecteur" part du pouce et relie les 530 résidus N-terminaux à une ancre transmembranaire de 21 résidus. La position du connecteur, replié au sein de la crevasse catalytique, est critique pour l'initiation de la synthèse d'ARN mais inhibitrice pour le processus d'élongation puisqu'il bloque l'enzyme en forme "pouce fermé". L'objectif de ce travail a été de réunir des informations biochimiques et structurales sur NS5B afin de mieux comprendre les mécanismes de la synthèse d'ARN de novo par le VHC, et plus spécifiquement les changements de conformation qui ont lieu lors de la transition entre les étapes d'initiation de d'élongation. Nous proposons un schéma explicatif de la séquence d'événements qui permettent la synthèse d'ARN. Celle-ci débute par la reconnaissance et la fixation de NS5B sur le génome viral, suivi par la fixation des deux premiers nucléotides incorporés, puis un repositionnement du dinucléotide néo-synthétisé grâce à des changements de conformation de NS5B pour permettre la poursuite de la synthèse d'ARN. La description de ce mécanisme constitue une avancée importante dans la compréhension du fonctionnement de VHC-NS5BHepatitis C virus (HCV) is a highly varaible virus, classified in genotypes that differ in their geographical distribution, the seriousness of the liver disease they cause, and response et the available treatment. RNA-dependant RNA polymerase NS5B is a choice target for specific inhibitors of HCV. Its organization can be described as a catalytic domain comprising the 530 N-terminal residues connected by a 40-residue linker to a C-terminal 21-residue transmembrane anchor. The linker occludes the catalytic cleft in the crystal structures of NS5B, a conformation likely conducive to initiation of RNA synthesis but clearly inhibitory to elongation, both because of direct steric hindrance and because it locks NS5B in a closed conformation. The main objective of our research was the understanding of the molecular mechanisms of de novo RNA synthesis by HCV, and more specially the conformation changes that occurs during the transition between the initiation and the elongation steps. We proposed a diagram explaining the sequence of events alllowing RNA synthesis. It begins with NS5B's recognition of the viral genome, followed by the fixation of the first two incorporated nucleotides, and so this neo-synthesized dinucleotide repositioning thanks to NS5B's conformational changes to allow the further RNA elongation. This mechanism description highly improves our understanding of HCV-NS5B
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Cavalheiro, Norma de Paula. ""Hepatite C: transmissão entre casais"." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/5/5134/tde-14062004-144045/.
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RESUMO Cavalheiro, NP. Hepatite C: transmissão entre casais. São Paulo, 2004. 111p. Tese (Doutorado) - Faculdade de Medicina, Universidade de São Paulo. Introdução: A ocorrência e eficiência da transmissão sexual do VHC na ausência de outros fatores de risco ainda é muito controversa. Foram investigados e analisados 24 casais, ambos cônjuges infectados com o VHC. Destes, 22 apresentaram o mesmo subtipo viral e a análise filogenética de parte da região NS5b mostrou altos índices de homologia nas seqüências entre os vírus dos casais infectados. Objetivo: Análise da transmissão da Hepatite C entre casais heterossexuais. Método: O estudo recrutou 45 casais. Destes, 24 foram selecionados e incluídos na pesquisa, com diagnóstico clínico e laboratorial para a Hepatite C crônica. A infecção foi diagnosticada por testes imunoenzimáticos de terceira geração e pela presença da partícula viral circulante detectada pela PCR. As amostras de sangue foram coletadas entre os anos de 1999 e 2002. Foram seqüenciadas partes das regiões 5NC e NS5b do VHC, para determinação dos subtipos virais. Os testes utilizados para as PCRs estão disponíveis para pesquisa e foram respectivamente TRUGENE 5NC Test (Bayer Health Care Diagnostics, Tarrytown, NY, USA) e Titan One Tube RT-PCR Kits (Roche Molecular, Mannheim, Germany). Para as PCRs dos seqüenciamentos foi utilizado o kit CLIP sequencing test (Bayer Health Care Diagnostics, Tarrytown, NY, USA). As seqüências foram analisadas com o sistema de seqüenciamento Open Gene DNA, software na Versão 3.1, biblioteca específica (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Para o alinhamento das seqüências, referentes a região NS5b, foi utilizado o programa Clustal W (Clustal W Multiple Sequence Alignment Program, v1.7, June 1997) e a árvore filogenética foi gerada pelo método Neighbor Joining. Um questionário padrão e entrevistas foram usados para coleta de dados sobre fatores de risco para aquisição da doença e comportamento sexual. Os pacientes desta pesquisa foram recrutados no Ambulatório de Hepatite do Hospital das Clínicas da Universidade de São Paulo e Ambulatório de Hepatite do Hospital Guilherme Álvaro, da cidade de Santos. Resultados: Entre os 24 casais selecionados, 22 apresentaram o mesmo subtipo viral e altas porcentagens de homologia (região NS5b) entre 93,0% e 99,4%. Os subtipos HCV apresentados foram dois (9,1%) casais infectados por 1a, nove (40,9%) com subtipo 1b, um (4,6%) com subtipo 2b e dez (45,5%) dos casais pelo subtipo HCV 3a. Os dois casais discordantes apresentaram índices de 70,1% e 82,2% e foram infectados pelos subtipos 2b e 1b, e 1b e 1a respectivamente. A média de tempo de convivência foi de 22,4 anos, variando de 2 a 45 anos e a renda per capta anual foi em média US$2,270/ano. Com base nos questionários e entrevistas os fatores de risco apresentados pelos casais foram: 9 (37,5%) transfusão de sangue, 17 (70,8%) U.D. endovenosa e 15 (62,5%) U.D. inalatória, 4 (16,7%) acupuntura e 5 (20,8%) tatuagem. O compartilhar de utensílios de higiene pessoal relatado pelos casais apresentou altos índices e 6 (25,0%) dos casais assumiram o uso comum de escova de dente, 16 (66,7%) lâmina de barbear, 21 (87,5%) cortador de unhas e 14 (58,3%) alicate de manicure. Os dois casais discordantes relataram fatores de risco como transfusão de sangue e U.D. Conclusão: A alta similaridade encontrada entre as cadeias genômicas do VHC pode dar suporte a hipótese de transmissão do VHC entre esses casais. O uso compartilhado de utensílios de higiene pessoal e o tempo de convivência tornam difícil a interpretação dos dados. O uso compartilhado de utensílios de higiene pessoal pode dificultar a interpretação dos dados em relação à transmissão sexual do VHC. A hipótese do sentido mais provável de transmissão do VHC, de homem para mulher, foi reforçada neste trabalho.ABSTRACT Cavalheiro, NP. Hepatitis C: transmission between couples. São Paulo, 2004. 111p. Thesis (Doctoral) - Faculdade de Medicina, Universidade de São Paulo. Introduction: The occurrence and the efficiency of HCV sexual transmission in the absence of other risk factors are still very controversial. I investigated and analyzed 24 couples, both infected with HCV, of whom 22 shared the same viral subtype. A phylogenetic analysis of NS5b region showed high sequence homology among the infected couples. Objective: Analysis of the Hepatitis C transmission between heterosexuals couples. Methods: The study recruited 45 couples, 24 were included, with anti-HCV positive and clinical diagnosis of active chronic hepatitis. HCV infection was diagnosed by positivity of serum samples for anti HCV (third-version enzyme immunoassay) and by circulating HCV-RNA detected by Polymerase Chain Reaction (PCR). All blood samples were collected between 1999 and 2002. Sequencing of the 5NC region was performed utilizing the research available TRUGENE HCV 5NC Test (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Sequencing of the NS5B region was performed by RT-PCR amplification with Titan One Tube RT-PCR Kits (Roche Molecular, Mannheim, Germany) and CLIP sequencing using a prototype NS5B genotyping assay (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Sequence analysis was completed using the Open Gene DNA Sequencing System, Gene Objects software package (Version 3.1), and Gene Librarian module (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Multiple sequence alignments of the NS5B region were performed with Clustal W (Clustal W Multiple Sequence Alignment Program, v1.7, June 1997), and phylogenetic trees were generated using the Neighbor Joining Method. A standardized questionnaire and interview was used to collect data concerning risk factors and sexual behaviors. Follow up of all subjects was conducted at the hepatitis clinic of the Clinical Hospital of the University of Sao Paulo and at the Hospital Guilherme Alvaro in the city of Santos, in the state of Sao Paulo, Brazil. Results: Among the 24 couples, 22 had matching viral subtypes with homology scores (NS5b) ranging from 93.0% to 99.4%. Of the 22 couples with matching subtype, two (9.1%) where infected with subtype 1a, nine (40.9%) with subtype 1b, one (4.6%) with subtype 2b and ten (45.5%) with subtype 3a. The two couples that did not show matching viral subtypes had scores of 70.1% and 82.2%, and were infected with subtypes 2b and 1b, and 1b and 1a, respectively. The average of duration of marriage was 22.4 years (range 2-45 years) and the per capita income was an average of US$2,270/year. Based on the questionnaire and interviews, cause of infection of the 24 couples could be attributed to: blood transfusions 9 (37.5%), drug use, I.V. 17(70.8%) and inhalation 15 (62.5%), acupuncture 4 (16.7%) and tattooing 5 (20.8%). Shared hygienic utensils showed a much higher correlation of possible route of transmission, and are better explained by the sequence homology data than by the other associated risk factors. A total of 6 (25.0%) couples shared tooth brushes, 16 (66.7%) shared shaving blades, 21 (87.5%) shared nail clippers and 14 (58.3%) shared manicure cutters. The two couples that had different subtypes, both of them related transfusion blood and I.V. drug use. Conclusions: The high similarity found among the genome chains of HCV supports the hypothesis of transmission between these couples. The shared use of personal hygiene utensils and the amount of time spent living together made it difficult to interpret the data. Also, the shared use of personal hygiene utensils can make it difficult to interpret the data in relation to the sexual transmission of HCV. The hypothesis in relation to the direction of the HCV transmission, from man to woman, was reinforced in this work.
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Dünnes, Nadia [Verfasser]. "Analyse der Interaktion von microRNA-122-Protein-Komplexen mit der NS5B-kodierenden Region und der 3´-untranslatierten Region der Hepatitis C Virus-RNA / Nadia Dünnes." Gießen : Universitätsbibliothek, 2016. http://d-nb.info/1118289773/34.
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Taylor, Annette Irene. "The intracellular localisation and membrane-altering properties of hepititis C virus proteins NS4B and NS5A." Thesis, University of Glasgow, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274768.
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Abdurakhmanov, Eldar. "Discovery and evaluation of direct acting antivirals against hepatitis C virus." Doctoral thesis, Uppsala universitet, Biokemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-265299.
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Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued. In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants. By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3. In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV. An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate. Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.
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Lee, M. S. "Classical trajectory studies of transport properties for Ar-Nsub(2) and He-Nsub(2) mixtures." Thesis, University of Newcastle Upon Tyne, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370624.
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Meyer, Aline Katharina [Verfasser], and Christoph [Akademischer Betreuer] Sarrazin. "Bedeutung eines prädizierten Leuzinzippermotivs im NS4B-Protein des Hepatitis-C-Virus für NS4B-Proteininteraktionen / Aline Katharina Meyer. Betreuer: Christoph Sarrazin." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2013. http://d-nb.info/105290498X/34.
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Grimm, Christian [Verfasser], Robert [Gutachter] Tampé, and Christoph [Gutachter] Welsch. "Charakterisierung des Lipidbindungsverhaltens und der Proteinfaltung von HCV NS5A unter Einfluss des NS5A-Inhibitors Daclatasvir / Christian Grimm ; Gutachter: Robert Tampé, Christoph Welsch." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2021. http://d-nb.info/1239730276/34.
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Huang, Chao-Kun. "Turbulence and cavitation : applications in the NSMB and OpenFOAM solvers." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAD035/document.
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L'objectif de ce travail de thèse concerne l'étude et la mise en œuvre de deux modèles de cavitation dans le solveur NSMB (Navier-Stokes-Multi-Blocks): les modèles HEM (Homogeneous Equilibrium Model) et une équation pour le taux de vide: le modèle à transport de taux de vide (TTV). Le phénomène de cavitation est modélisé par différentes équations d'état de mélange liquide-vapeur (EOS). Des simulations numériques sont réalisées sur des écoulements diphasiques compressibles unidimensionnels et bidimensionnels avec des conditions d'interface et comparées à des solutions de référence. De plus, la méthode TTV basée sur le taux de vide incluant les termes source pour la vaporisation et la condensation dans le logiciel libre open source OpenFOAM est également présentée sur la géométrie Venturi pour capturer le phénomène du jet réentrant. La modélisation de la turbulence joue un rôle majeur dans la capture des comportements instationnaires et un limiteur est introduit pour réduire la viscosité turbulente afin de mieux prédire la structure à deux phases. Une comparaison de divers modèles de cavitation couplés avec des modèles de turbulence est étudiée. Les résultats computationnels sont comparés aux données expérimentales existantesThe objective of this thesis work concerns the study and implement of two cavitation models in the NSMB (Navier-Stokes-Multi-Blocks) flow solver: the Homogeneous Equilibrium Models (HEM) and a void ratio Transport-based Equation Model (TEM). The cavitation phenomenon is modeled by different liquid-vapor mixture equation of state (EOS). Numerical simulation are performed on some one- and two-dimensional compressible two-phase flows with interface conditions and compared with reference solutions. Moreover, The TEM based method for the void ratio including the source terms for vaporization and condensation in the free, open source software OpenFOAM is also presented on the Venturi geometry to capture the re-entrant jet phenomenon. The turbulence modeling plays a major role in the capture of unsteady behaviors and a limiter is introduced to reduce the eddy-viscosity to better predict the two-phase structure. A comparison of various cavitation models coupled with turbulence models are investigated. Computational results are compared with existing experimental data
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Lundin, Marika. "Topology and membrane rearrangements of the hepatitis C virus protein NS4B /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-927-0/.
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Millies, Benedikt [Verfasser]. "Entwicklung neuer nicht-kompetitiver Inhibitoren flaviviraler NS2B/NS3-Proteasen / Benedikt Millies." Mainz : Universitätsbibliothek Mainz, 2020. http://d-nb.info/1202452345/34.
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Gretton, Sarah N. "Topology and biophysical characterisation of the hepatitis C virus NS4B protein." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433078.
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CORREIA, CAIO S. de C. "Estudo da emissão/absorção de Nsub(2)O da bacia Amazônica." reponame:Repositório Institucional do IPEN, 2013. http://repositorio.ipen.br:8080/xmlui/handle/123456789/25355.
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Dissertação (Mestrado em Tecnologia Nuclear)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Lorente, Espín Oscar. "Hawking radiation in NS5 and little string theory." Doctoral thesis, Universitat Politècnica de Catalunya, 2012. http://hdl.handle.net/10803/96780.
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In this thesis we have focused on semi-classical methods that enables us to obtain non-thermal spectra for the vast majority of black holes. This fact is due to taking into account the backreaction of the metric, when the black hole emits, imposing energy conservation. Specifically we have studied NS5 and Little String Theory (LST) black holes. We have calculated the Hawking radiation for both models, obtaining a non-thermal spectrum for NS5, whereas a purely thermal spectrum for LST. This last fact is due to the peculiar behavior of LST, whose temperature is independent of its mass. After a brief outline in Chapter 1 about properties of black holes, where we have introduced the information loss paradox, we have reviewed in the Chapter 2 how curved space-times, e.g. black hole backgrounds, create particles. Hawking demonstrated that black holes has temperature thus emit thermal radiation, and calculated its flux without taking into account the back-reaction of the metric. Afterwards we have presented two semi-classical methods, i.e. the tunneling approach and the complex path method, that somehow solve the information loss paradox stated by the work of Hawking. In Chapter 3 we have applied both semi-classical methods plus the covariant anomaly method in NS5 and Little String Theory (LST) black holes. We have calculated some thermodynamical quantities as the temperature and the entropy; furthermore, after reducing the ten-dimensional theory to a two-dimensional effective theory, we have calculated the emission rate and the corresponding fluxes taking into account the back-reaction of the metric. In Chapter 4 we have calculated the emission of fermions by NS5 and LST obtaining identical results as for scalar particles. In Chapter 5 we have presented a novel method in order to introduce quantum perturbations directly in the black hole metric, that accounts for back-reaction effects. This method has been applied in a general stationary spherically symmetric metric, recovering similar results as in the semi-classical methods presented in the previous chapters. Moreover, when we have applied this method in LST black hole we have obtained similar results as in string one-loop theory. Finally, in Chapter 6 we have calculated and compared some thermodynamical quantities as the entropy, using both Einstein frame and conformal frame.En aquesta tesi hem estudiat mètodes semiclàssics que ens permeten obtenir espectres no tèrmics en la majoria de forats negres. Aquest fet és degut a tenir en compte l'autoreacció de la mètrica quan el forat negre emet radiació, tot imposant la conservació de l'energia. Concretament hem estudiat els forats negres NS5 i Little String Theory (LST). Hem calculat la radiació de Hawking en tot dos models, obtenint un espectre no tèrmic per a NS5, mentres que hem obtingut un espectre purament tèrmic en LST. Aquest darrer fet és degut al comportament especial de LST, en què la temperatura no depèn de la seva massa. Després d'una breu introducció de les propietats dels forats negres al capítol 1, on hem introduït la paradoxa de la pèrdua d'informació, hem vist al capítol 2 com els espais-temps corbs, per exemple l'entorn d'un forat negre, creen partícules. Hawking va demostrar que els forats negres tenen temperatura, per tant emeten radiació tèrmica, i va calcular el flux de partícules emès per un forat negre sense tenir en compte l'autoreacció de la mètrica. A continuació hem presentat dos mètodes semiclàssics, a saber, l'aproximació de tunneling i el mètode de camins complexos, que d'alguna manera resolen la paradoxa de la pèrdua d'informació plantejada en el treball de Hawking. En el capítol 3 hem aplicat els dos anteriors mètodes semiclàssics anteriors a més del mètode de l'anomalia covariant, tant en forats negres NS5 com en LST. Hem calculat quantitats termodinàmiques com la temperatura i l'entropia; a més, després de reduir la teoria de dimensió deu a una teoria efectiva en dos dimensions, hem calculat el ritme d'emissió i els fluxes corresponents tenint en compte l'autoreacció de la mètrica. En el capítol 4 hem calculat l'emissió de fermions en NS5 i LST obtenint idèntics resultats que pel cas de partícules escalars. En el capítol 5 hem presentat un nou mètode que introdueix directament un tipus de perturbació quàntica en la mètrica del forat negre i que té en compte els efectes d'autoreacció de la mètrica. Aquest mètode ha estat aplicat a una mètrica estacionària general amb simetria esfèrica, i hem obtingut els mateixos resultats que fent servir els mètodes semiclàssics presentats en els anteriors capítols. A més, quan hem aplicat aquest nou mètode al forat negre LST, hem obtingut resultats similiars que fent servir teoria de cordes one-loop. Finalment, en el capítol 6 hem calculat i comparat algunes quantitats termodinàmiques com l'entropia, utilitzant tant el marc d'Einstein com el marc conforme.
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Duarte, Tharlley Rodrigo Eugenio. "Diagnóstico e filogenia molecular dos vírus da febre amarela a partir de amostras humanas negativas para os vírus dengue." Universidade Federal de Goiás, 2018. http://repositorio.bc.ufg.br/tede/handle/tede/8475.
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Brazil is the largest arbovirus granary in the world and presents the largest endemic area of yellow fever (YF). The Ministry of Health reported in 1170 suspected cases of yellow fever, of which 847 are under investigation, 93 were discarded and 230 were confirmed, being in the states of Minas Gerais (201), Espírito Santo (25) and São Paulo (4). Of the total number of cases reported, 186 died, 104 of which remained in the investigation, 79 deaths were confirmed and 3 were discarded. The case fatality rate among confirmed cases was 34.3%. For YF there is no specific treatment, however, vaccination is effective being the only and best way of prevention. Precisely because of this factor and others involved the diagnosis of YF is not made in the health system except by a relevant suspicion. The problem is that in many cases viruses go unnoticed in cases of Dengue virus infection because of cross reactivity between members of the genus Flavívirus or because of non-specific symptoms. The present study analyzed 118 samples that were screened for suspected Dengue Virus infections (VDEN) for the year 2011 to 2013, but discarded for this virus because they gave negative results to the viral agent through serological and molecular tests in the municipality of Goiânia, Goiás. Samples were sent to the Virology Laboratory of the Federal University of Goiás Regional Jataí and analyzed by molecular methods such as RT-PCR and Nested-PCR followed by verification of the amplicon by agarose gel electrophoresis. Among the 118 negative samples for the DENV virus, three of the samples were positive for the Yellow Fever Virus (YVF) according to the production of amplicons of 253 bp of the NS5 region and confirmation of the identity of the amplicon by nucleotide sequencing. The sequences obtained were submitted to BLAST for identity confirmation. The translated sequence was analyzed by MEGA software version 7.0. For phylogenetic analysis, the best model was previously determined and then the tree was constructed with 53 sequences of all Yellow Fever viruses present in databases for a comparative analysis. The sequences found were compared to sequences from the VFA recorded in the Genbank database and were identified as referring to a portion of the NS5 nonstructural protein, position 216 to 296 of this protein, conferring 81 amino acids. The representative tree demonstrated that the sequences submitted were directly related to Senegal taxa. The robustness of the phylogenetic method was by Bootstrap 2000 replicates using the best JTT + G model, Maximum Likelihood. The Tajima test applied yielded a value of D = 1.159570, thus demonstrating that there was no population expansion of the taxa analyzed, considering that they have a significant degree of conservation during evolution. From the results obtained in the study, it can be affirmed that there was already an YFV circulation in the year 2013, at least of patients seen in the central region of Brazil, even before the last outbreak in 2017.
O Brasil é o maior celeiro de arbovírus do mundo e apresenta a maior área endêmica de febre amarela (FA). O Ministério da Saúde notificou, em 2017, 1170 casos suspeitos de febre amarela, sendo que desses 847 estão em investigação, 93 foram descartados e 230 foram confirmados, sendo nos estados de Minas Gerais (201), Espírito Santo (25) e São Paulo (4). Do total de casos notificados, 186 evoluíram para óbito, sendo que 104 óbitos permanecem em investigação, 79 óbitos foram confirmados e 3 foram descartados. A taxa de letalidade entre os casos confirmados foi de 34,3%. Para FA não há tratamento específico, no entanto, a vacinação é eficaz sendo a única e melhor maneira de prevenção. Justamente por esse fator e outros envolvidos o diagnóstico da FA não é feito no sistema de saúde a não ser por uma suspeita relevante. O problema é que em muitos casos os vírus passam despercebidos em casos de infecção pelo vírus Dengue por apresentar reatividade cruzada entre membros do gênero Flavívirus ou por apresentar sintomas inespecíficos. O presente estudo analisou 118 amostras que foram triadas para infecções suspeitas de Vírus da Dengue (VDEN) referentes ao ano de 2011 a 2013, porém descartadas para esta virose por terem dado resultados negativos para o agente viral através de testes sorológicos e moleculares no município de Goiânia, Goiás. As amostras foram encaminhadas para o Laboratório de Virologia da Universidade Federal de Goiás Regional Jataí e analisadas por métodos moleculares, como o de RT-PCR e Nested-PCR seguida da verificação do amplicon por eletroforese em gel de Agarose. Dentre as 118 amostras negativas para os vírus DENV, três das amostras foram positivas para o Vírus da Febre Amarela (VFA) conforme produção de amplicons de 253 pb da região NS5 e confirmação da identidade do amplicon por sequenciamento nucleotídico. As sequências obtidas foram submetidas ao BLAST para confirmação da identidade. A sequência traduzida foi analisada pelo software MEGA versão 7.0. Para análise filogenética foi determinado previamente o melhor modelo e em seguida a árvore foi construída com 53 sequências de todos os vírus da Febre Amarela presentes em bancos de dados para uma análise comparativa. As sequências encontradas foram comparadas com sequências do VFA registradas no banco de dados Genbank e identificouse que se refere a uma porção da proteína não estrutural NS5, posição 216 a 296 desta proteína, conferindo 81 aminoácidos. A árvore representativa demonstrou que as sequências submetidas estavam diretamente relacionadas com táxons do Senegal. A robustez do método filogenético foi por Bootstrap 2000 réplicas utilizando o melhor modelo JTT+G, Maximum Likelihood (Máxima probabilidade). O teste D de Tajima aplicado gerou um valor de D= 1.159570, demonstrando assim que não houve expansão populacional dos táxons analisados, considerando que os mesmos possuem significativo grau de conservação durante a evolução. A partir dos resultados obtidos no estudo, pode-se afirmar que já havia circulação do VFA no ano de 2013, pelo menos de pacientes atendidos na região central do Brasil, antes mesmo do último surto em 2017.
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Taveneau, Cyntia. "Modélisation, purification et caractérisation des modules et domaines de la PI4KA humaine." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA114827/document.
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La phosphatidylinositol-4-kinase de type IIIα est une kinase de lipide eukaryote ubiquitaire qui synthétise le phosphatidylinositol-4-phosphate PtdIns(4)P de la membrane plasmique. Ce phosphoinositide est d’autant plus important qu’il tient un rôle majeur dans différentes voies de signalisation cellulaire, le traffic vésiculaire ainsi que dans l’identité des organelles. De plus, la PIK4A humaine est un facteur essentiel pour la réplication du virus de l’hépatite C (VHC). En effet, le recrutement du complexe de réplication du VHC par la protéine virale NS5A à la membrane du reticulum endoplasmique permet la formation d’un réseau membranaire à l’origine de la structuration des complexes de replication viraux.La PI4KA est une kinase imposante (2102 résidus, 240 kDa pour la PI4KA humaine) qui possède un domaine kinase C-terminal d’environ 400 résidus précédé d’un domaine formé de répétitions Armadillo pour lequel aucune fonction n’a été determinée. Le rôle ainsi que le repliement des 1500 résidus N-terminaux de PI4KA ne sont pas connus à ce jour.Afin d’en savoir plus sur la structure tri-dimensionnelle de la PI4KA humaine, nous avons utilisé des outils bio-informatiques afin de délimiter et de modéliser les modules et domaines la composant. Nous avons pu ainsi les exprimer et les produire en bactérie et en cellules d’insecte afin de vérifier nos hypothèses. Nous avons pu conclure que PI4KA est composée de deux modules. Le module N-terminal (1100 résidus), est composé de deux domaines dont un solénoïde α. Les résultats obtenus par diffusion des rayons X aux petits angles (SAXS) nous permettent de définir leur agencement potentiel. Le second module (1000 résidus), le module C-terminal, est l’enzyme-core. Son analyse nous a permis d’identifier une similitude remarquable avec les sérine/thréonine kinases PIKKs, comme mTor, apparentées aux phosphatidylinositol-3-kinases. Nous avons défini au début du module C-terminal de PI4KA trois domaines putatifs que nous avons nommés DI, DII et DIII. Nos collaborateurs ont montré qu'ils sont essentiels à l’activité kinase de la protéine ainsi qu’à la replication du VHC. Le domaine DI a été caractérisé et a permis la validation d’une nouvelle paramétrisation de la molécule de N, N-dimethyl-dodecylamine oxide (LDAO) pour des simulations de dynamique moléculaire. Enfin, la PI4KA humaine dans son entier a été exprimée en cellules d’insecte puis purifiée, et un premier test d’interaction avec les membranes a été initiéThe eukaryotic lipid kinase phosphatidylinositol 4-kinase III alpha is a ubiquitous enzyme that synthesizes the plasma membrane pool of phosphatidylinositol 4-phosphate. This important phosphoinositide has key roles in different signalization pathways, vesicular traffic and cellular compartment identity. Moreover, PI4KA is an essential factor for hepatitis C virus (HCV) replication. Indeed, PI4KA's interaction with the non-structural HCV protein NS5A at the endoplasmic reticulum membrane leads to formation of a “membranous web” giving to the membrane the signature necessary to the formation of viral replication machineryPI4KA is a large protein (2102 residues, 240 kDa for human PI4KA) with the kinase domain making up the ca 400 C-terminal residues preceded by an Armadillo domain for which no function is known. There is essentially no structural information about the 1500 N-terminal residues and no clue as to the function of most of this region of PI4KA.We use computational methods in order to delineate fragments of human PI4KA amenable to soluble production in Escherichia coli and insect cells. We clone and express these fragments and evaluate the soluble fraction of each construction. Our results further suggest that PI4KA can be described as a two-module protein. The N-terminal module (1100 residues), is composed of two domains which one is an alpha solenoid. Their potential arrangement was defined by small angle X-ray scattering (SAXS).The second module (1000 residues), the C-terminal module, is the core enzyme. Its analysis leads us to identify similarities with the serine/threonine kinases PIKKs, as mTor, homologous to phosphatidylinositol-3-kinases. Three putative domains were delineate at the beginning of this C-terminal module. We name the DI, DII and DIII. Our collaborators have shown their necessity to the kinase activity of PI4KA and the HCV replication. DI domain was characterized and allowed the validation of a new parametrization of the N, N-dimethyl-dodecylamine oxide molecule (LDAO) for simulation of molecular dynamics. Finally, the full-length human PI4KA was expressed in insect cells, purified and a first interaction experiment with membranes have been initiated
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Condotta, Stephanie Anne. "Molecular and cellular studies of the West Nile virus NS2B/NS3 protease." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/26994.
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West Nile virus (WNV) is the most widely distributed arthropod-borne virus globally. It can cause a potentially fatal infection and has become a public health concern in North America since its introduction in 1999. Currently, there are no vaccines or treatments available for human WNV infections. As such, it is important to understand the virus life cycle, in order to develop effective therapeutics. The WNV protease heterocomplex, NS2B/NS3, is a prime target for antiviral therapy and has become the focus of much research. It is important to understand protease function first, in order to develop effective inhibitors. The overall goal of this thesis was to gain a better understanding into the function of the full-length NS2B/NS3 protease heterocomplex within the intracellular microenvironment. I hypothesized that there are critical residues essential for the interaction between NS2B and NS3 that affect protease activity and protein stability. The first aim of this project was to generate a cell-based fluorescent substrate assay to investigate the protease activity of the full-length NS2B/NS3 protease heterocomplex within the cell. My results demonstrate that the full-length NS2B/NS3 protease heterocomplex functions differently within the context of the cell, compared to what has been previously observed in vitro (Chapter 2). In the second aim, I investigated NS2B function on NS3 protease cis-cleavage and trans-cleavage activity. My results reveal an important dual role the NS2B protein plays in the proper function of the full-length NS2B/NS3 protease heterocomplex (Chapter 3). In the third aim, I utilized the information gathered to rationally design and test a serine protease inhibitor directed against the full-length NS2B/NS3 protease heterocomplex (Chapter 4). Taken together, my results highlight the importance of utilizing cell-based assays to assess protease activity, as this allows for the investigation of NS2B/NS3 protease function in a more physiologically relevant environment. The results presented in this thesis further our understanding of the activity of the full-length WNV NS2B/NS3 protease heterocomplex within the context of the cell. The information gathered gives insight into the regulation of viral protease function that could be utilized in the rational drug design towards the WNV NS2B/NS3 protease heterocomplex.
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Ruiz, Arroyo Víctor Manuel. "Structural and functional analysis of Zika Virus NS5 protein." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671922.
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Zika virus (ZIKV) belongs to the Flaviviridae family and constitute an important public health concern since ZIKV infection produced devastating effects in new born infants. Flaviviruses present a positive sense single stranded RNA genome flanked by highly structured untranslated regions (UTR) carrying one open reading frame that codifies for three structural proteins (C, prM, E) and five nonstructural proteins (NS1-5). At the most C-terminal end, NS5 protein carries a RNA dependent RNA polymerase (RdRP) and a methyl transferase domain (MTase) for genome copying and 5’ capping activities of the newly synthesized RNA, respectively. Given the crucial role of this enzyme for viral replication, NS5 constitutes an attractive antiviral target to inhibit viral replication. In this study, we determined the structure of the ZIKV NS5 protein using X-Ray crystallography combined with several structural biology approaches to characterize the supramolecular arrangement of the ZIKV NS5 protein. We identified the monomer-monomer and dimer-diner interactions to form fibril-like structures, and evaluated the role of oligomer formation, using in-vitro polymerization assays. We also evaluated the in-vivo effect of NS5-oligomerisation in chicken embryos, stablishing a connection between this protein and microcephaly.
One of the most important RNA structures present at the 5’UTR of flavivirus genomes is the 5SLA. This structure was identified previously to bind the NS5 protein, acting as a promoter and being essential for viral replication. We assayed and optimized the NS5-5SLA complex stability using biophysical and biochemical techniques and determined the structure of the complex by single particle cryo-EM. Comparisons between the NS5-5SLA complex and the NS5 crystallographic structure revealed for the first time in flavivirus, important conformational changes in the NS5 RdRP. We identified the residues involved in complex formation and characterized the effect of this binding on NS5 polymerization, shedding new light on the understanding of replication mechanisms in flaviviruses.El virus Zika (ZIKV) pertenece a la familia Flaviviridae y constituye una amenaza para la salud pública, especialmente debido a las malformaciones provocadas en neonatos. Los flavivirus presentan un genoma RNA de simple cadena con polaridad positiva, flanqueado por regiones no traducidas (UTR) que presentan una elevada estructura secundaria, seguido de una región codificante para una única poliproteína que por proteólisis dará lugar a tres proteínas estructurales (C, prM, E) y cinco proteinas no estructurales (NS1-5). En el extremo C-terminal se encuentra la proteina NS5 que presenta actividad ARN polimerasa dependiente de ARN (RdRP) y un dominio metil-transferasa (MTase) para copiar el genoma y añadir una caperuza al extremo 5’ del nuevo ARN sintetizado, respectivamente. Dado el papel crucial de este enzima en la replicación viral, la proteina NS5 constituye una diana antiviral muy atractiva para inhibir la replicación del virus. En este estudio, determinamos la estructura de la proteína NS5 de ZIKV, usando cristalografía de Rayos-X combinada con diferentes técnicas biofísicas para caracterizar la organización supramolecular de la proteína. Identificamos las interacciones monomero-monomero y dimero-dimero para caracterizar las estructuras fibrilares de la proteína y evaluamos los efectos de la dimerización en la actividad polimerasa in-vitro. También evaluamos los efectos de la oligomerización de NS5 in-vivo en embriones de pollo, estableciendo una conexión entre esta proteína y la aparición de microcefalia en fetos infectados. Una de las estructuras de ARN más importantes presentes en el 5’UTR del genoma de los flavivirus es el 5SLA. Previamente se describió que esta estructura se unía a NS5 y actuaba como un promotor, siendo ademas esencial para la replicación viral. Medimos y optimizamos la estabilidad del complejo NS5-5SLA mediante técnicas biofísicas y bioquímicas y determinamos la estructura del complejo mediante cryo-EM. Las comparaciones entre la estructura cristalográfica y cryo-EM de NS5 revelaron, por primera vez en flavivirus, cambios conformacionales importantes en el dominio RdRP. Identificamos los residuos implicados en la formación del complejo y caracterizamos el efecto de la unión de NS5 a 5SLA sobre su actividad polimerasa. Estos resultados arrojan nueva luz para entender los mecanismos de replicación en los flavivirus.
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Alves, Rúbens Prince dos Santos. "Desenvolvimento de uma vacina de subunidade contra o sorotipo 2 do vírus dengue baseada na proteína não estrutural 5 (NS5)." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-06102015-193757/.
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A dengue é uma doença que afeta milhões de pessoas e possui um número significativo de mortes. Não há nenhum tratamento vacinal legalizado para uso. As estratégias vacinais contra a dengue baseadas em proteínas não estruturais têm demonstrado serem mais seguras do que as baseadas em proteínas estruturais. A proteína não estrutural 5 (NS5) do vírus dengue é a proteína mais conservada entre os quatro sorotipos e desempenha um papel crucial na replicação viral. Neste estudo, foi gerada uma forma recombinante da NS5 expressa em E. coli com propriedades antigênicas preservados. As condições de cultura foram optimizadas, o que permitiu a expressão dessa proteína na forma solúvel. A imunização de camundongos Balb/c com a NS5 sozinha ou em combinação com um adjuvante (poli (I:C)) promoveu o aumento da sobrevida de camundongos após desafio letal com DENV2. A combinação da NS5 com poli (I:C) emulsionado em Montanide 720 levou a expansão de linfócitos T CD8+ específicos. Os resultados indicam que a proteína NS5 obtida preserva determinantes antigênicos da proteína nativa e pode ser uma ferramenta útil para estudos sobre a biologia do DENV, busca de drogas antivirais e desenvolvimento de vacinas.Dengue fever is a disease affecting millions of people worldwide and causing a significant number of deaths. There are no effective treatments or vaccine approaches capable of preventing such infection. Anti-DENV vaccine strategies based on nonstructural proteins as antigens have been shown to be safer than those based on structural proteins. The DENV nonstructural protein 5 (NS5), plays a crucial role in viral replication. In this study, we generated a recombinant form of DENV2 NS5 expressed in E. coli in high amounts and with preserved antigenic properties with regard to the native protein. Culture conditions were optimized in order to allow expression of NS5 as a soluble protein. The immunization of Balb/c mice using this protein alone or in combination with poly (I:C) led to increased survival after intracranial challenge with the DENV2 JHA1 strain. The combination of the protein with poly (I:C) emulsified in Montanide 720 led to the activation of NS5-specific CD8+ T lymphocytes. Altogether, the results indicate that the recombinant NS5 protein preserves antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV\'s biology, search for anti-viral drugs and vaccine development.
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Freifrau, von Hammerstein-Gesmold Franziska [Verfasser]. "Investigating allosteric inhibition of flaviviral NS2B-NS3 proteases / Franziska Freifrau von Hammerstein-Gesmold." Mainz : Universitätsbibliothek der Johannes Gutenberg-Universität Mainz, 2020. http://d-nb.info/1223379094/34.
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Street, Andrew A. "Activation of phosphatidylinositol 3-kinase signalling by the hepatitis C virus NS5A protein." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417733.
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Kelly, Lorna Jane. "Development of tools to investigate resistance of HCV genotype 3 to NS5A inhibitors." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/19307/.
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HCV infection leads to liver failure. Genotype 3 (GT3) is known to respond poorly to newly-developed direct-acting antivirals, especially inhibitors of the multifunctional NS5A protein. This work reports the establishment of efficient transient replication of the S52 GT3 sub-genomic replicon (SGR) by further culture adaptation of S52 in the context of an optimised luciferase reporter. Also documented is the development of hepatoma cells with immune-attenuating modifications and expressing the lipid binding factor hSEC14L2 to support transient replication of S52. In parallel, stable replication of S52 in SGR-harbouring cells was used to investigate the differences between early and established replication. Differences in sensitivity to the NS5A inhibitor Daclatasvir (DCV) in both transient and stable S52 were observed compared to other genotypes, and between transient and stable replication. In addition, it is shown here that the resistance-associated substitution (RAS) Y93H conferred a significant fitness cost which is not apparent for stable S52 selected with DCV, despite this RAS being detected. This thesis explores the molecular basis of such an observation and highlights a potential mechanism which warrants further research. The role of RAS in the development of resistance is still unclear though this work reports that the presence of a RAS within a mixed population greatly influenced the development of resistance to DCV in vitro. Moreover, this work identified that a cellular metabolism-regulating factor, AMP-mediated protein kinase (AMPK), may be differentially regulated during GT3 infection compared to other GTs. This thesis presents the hypothesis that AMPK regulation by HCV may contribute to hepatic steatosis as a direct consequence of viral infection, which is unique to GT3. More insight into the propensity of GT3 to develop resistance can aid further antiviral design, and an understanding of the molecular basis of steatosis offers a rationale for treating the symptoms of HCV in addition to direct targeting of the virus.
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Oliva, Cíntia Bittar [UNESP]. "Evolução das quasiespécies da proteína NS5A do vírus da hepatite C genótipo 3a." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/102747.
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oliva_cb_dr_sjrp.pdf: 1844534 bytes, checksum: 40948c5116118b7b10a75b5b44e849cc (MD5)A Hepatite C é uma doença presente em todo o mundo. O vírus da Hepatite C (HCV), o agente etiológico dessa doença, é um vírus de RNA de fita simples positiva. Seu genoma codifica uma única poliproteína precursora que após processamento origina dez proteínas virais. A NS5A, uma das proteínas virais não estruturais, esta associada com a resposta ao tratamento baseado em Interferon, tratamento aprovado para Hepatite C no Brazil.O HCV tem uma alta taxa de mutação levando a uma alta variabilidade, fator importante para a evasão da resposta imune e a resposta ao tratamento. O objetivo deste trabalho foi analisar a evolução das quasiespécies antes, durante e após o tratamento em pacientes infectados com HCV genótipo 3a que apresentaram diferentes respostas ao tratamento. O RNA viral foi extraído, o cDNA sintetizado, a região NS5A amplificada e clonada e 15 clones de cada ponto de coleta foram seqüenciados. As sequências foram analisadas com relação a história evolutiva, diversidade genética e seleção. Nossas análises mostram que a população viral que persiste após o tratamento na maioria dos pacientes não respondedores está presente em amostras pré-tratamento sugerindo uma aptidão para evadir o tratamento. Ainda a maioria das amostras pré-tratamento de pacientes respondedores ao final do tratamento ou não apresentou a população encontrada nas amostras pós-tratamento ou apresentou em menor freqüência. As exceções ilustram a característica única do processo evolutivo e conseqüentemente o processo de resistência ao tratamento em cada paciente. A evolução do vírus da Hepatite C ao longo do tratamento aparenta ser o resultado de uma relação evolutiva única entre as cepas virais e cada hospedeiro humano, levando a persistência do vírus ou a resposta ao tratamento
Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses.Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection. Our analysis shows that the viral population that persists after treatment for most non-response patients are is present in before-treatment samples, suggesting it is fitted to evasion of treatment. Accordingly, most before-treatment samples from end-of-treatment response patients either did not show the population found after the relapse or showed it in low abundance. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient.Hepatitis C virus evolution throughout treatment appears to be the result of a unique evolutionary relationship between viral strains and each human host, leading to either persistence or clearance
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BALDACIM, SANDRO A. "Desenvolvimento, processamento e caracterizacao de compositos ceramicos Sisub(3)Nsub(4)-SiCsub(w)." reponame:Repositório Institucional do IPEN, 2000. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10877.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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FELIPPE, MONICA T. S. D. "Estudo de fluxo de oxido nitroso (Nsub(2)O) regional na bacia amazonica." reponame:Repositório Institucional do IPEN, 2010. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9547.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Oliva, Cíntia Bittar. "Evolução das quasiespécies da proteína NS5A do vírus da hepatite C genótipo 3a /." São José do Rio Preto : [s.n.], 2012. http://hdl.handle.net/11449/102747.
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Orientador: Paula RahalCoorientador: Isabel Maria Vicente Guedes de Carvalho Mello
Banca: Flora Maria de Campos Fernandes
Banca: Camila Malta Romano
Banca: Adriano Mondini
Banca: Maria Tercília Vilela de Azeredo Oliveira
Resumo: A Hepatite C é uma doença presente em todo o mundo. O vírus da Hepatite C (HCV), o agente etiológico dessa doença, é um vírus de RNA de fita simples positiva. Seu genoma codifica uma única poliproteína precursora que após processamento origina dez proteínas virais. A NS5A, uma das proteínas virais não estruturais, esta associada com a resposta ao tratamento baseado em Interferon, tratamento aprovado para Hepatite C no Brazil.O HCV tem uma alta taxa de mutação levando a uma alta variabilidade, fator importante para a evasão da resposta imune e a resposta ao tratamento. O objetivo deste trabalho foi analisar a evolução das quasiespécies antes, durante e após o tratamento em pacientes infectados com HCV genótipo 3a que apresentaram diferentes respostas ao tratamento. O RNA viral foi extraído, o cDNA sintetizado, a região NS5A amplificada e clonada e 15 clones de cada ponto de coleta foram seqüenciados. As sequências foram analisadas com relação a história evolutiva, diversidade genética e seleção. Nossas análises mostram que a população viral que persiste após o tratamento na maioria dos pacientes não respondedores está presente em amostras pré-tratamento sugerindo uma aptidão para evadir o tratamento. Ainda a maioria das amostras pré-tratamento de pacientes respondedores ao final do tratamento ou não apresentou a população encontrada nas amostras pós-tratamento ou apresentou em menor freqüência. As exceções ilustram a característica única do processo evolutivo e conseqüentemente o processo de resistência ao tratamento em cada paciente. A evolução do vírus da Hepatite C ao longo do tratamento aparenta ser o resultado de uma relação evolutiva única entre as cepas virais e cada hospedeiro humano, levando a persistência do vírus ou a resposta ao tratamento
Abstract: Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses.Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection. Our analysis shows that the viral population that persists after treatment for most non-response patients are is present in before-treatment samples, suggesting it is fitted to evasion of treatment. Accordingly, most before-treatment samples from end-of-treatment response patients either did not show the population found after the relapse or showed it in low abundance. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient.Hepatitis C virus evolution throughout treatment appears to be the result of a unique evolutionary relationship between viral strains and each human host, leading to either persistence or clearance
Doutor
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Maqbool, Muhammad Ahmad. "Etude de l’impact de la variabilité génétique de la protéine NS5A du virus de l’hépatite C dans la pathogenèse et la réplication virale." Thesis, Paris Est, 2012. http://www.theses.fr/2012PEST0026/document.
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Le virus de l’Hépatite C (VHC), de la famille Flaviviridae, est à l’origine d’une pandémiemondiale. L’infection par le VHC provoque le dévelopment d’hépatites chroniques, decirrhoses et de carcinomes hépatocellulaires (CHC). Les fonctions de la majorité des protéinesvirales sont connues, mis à part pour NS5A dont la seule fonction directe attribuée à ce jour,équivaut à celle d’un facteur d'activation transcriptionnelle. Notre laboratoire a montréprécédemment que les variants de quasiespèce de NS5A isolés à partir du sérum d’un mêmepatient présentaient des différences significatives dans leurs propriétés intrinsèques detransactivation. Fort de ces résultats, nous avons analysé des variants de NS5A isolés à partirde tissu hépatique d’un patient chroniquement infecté par le VHC de génotype 1b. Cesanalyses ont révélé une compartimentation génétique et fonctionnelle des variants de NS5Aentre le tissu tumoral et le tissu non-tumoral adjacent. Nous avons donc émis l’hypothèse queles propriétés transactivatrices de NS5A pourraient jouer un rôle dans la pathogenèse ainsique dans la réplication virale, et que la variabilité naturelle de NS5A pourrait influencer sespropriétés transactivatrices. L’objectif de ce travail de thèse était d’analyser le rôle despropriétés transactivatrices de NS5A dans la pathogenèse hépatique viro-induite ainsi quedans la réplication virale. Pour étudier le rôle des propriétés de transactivation de NS5A dans la pathogenèse hépatique,nous avons développé des vecteurs lentiviraux pour exprimer dans les hépatocytes primaireshumains les variants choisis de NS5A portants différents potentiels de transactivation. Enutilisant la technologie RNA-Seq d’Illumina, l’analyse des transcriptomes d’hépatocytestransduits exprimant les variants transactivateurs fort et faible de NS5A, sera utiliser pouridentifier les voies cellulaires ciblées par les propriétés transactivatrices de NS5A. Pour lesétudes in vivo, nous avons lancé le développement des souris transgénique permettantl’activation conditionnelle de l’expression des variants de NS5A avec fort et faible potentielde transactivation, spécifiquement dans le foie. Ces souris transgéniques seront utilisées pourétudier le rôle potentiel des propriétés transactivatrices dans la pathogenèse VHC induite etplus particulièrement dans le développement des cancers. Pour étudier le rôle des propriétés de transactivation de NS5A dans la réplication virale, nousavons utilisé le système de réplicon subgénomique de VHC exprimant les variants de NS5Aprécédemment caractérisés. Pour exercer ses propriétés transactivatrices, NS5A doit êtrelocalisée au moins partiellement dans le noyau. Nous avons démontré qu’une partie de NS5Ase retrouve dans noyau et est recruté sur des promoteurs cellulaires, modulant ainsidirectement l’expression de gènes cellulaires essentiels pour la réplication de l’ARN viral.Nous avons observé que les variants de NS5A avec différents potentiels de transactivation,confèrent différentes capacités de réplication au réplicon subgénomique, et corrèlent avec lepotentiel de transactivation de variant correspondant. En accord avec ces observations,l’inhibition de translocation nucléaire de NS5A entraine une inhibition de la réplication virale,suggerant un rôle potentiel des propriétés transactivatrices de NS5A dans la réplication l’ARNvirale. En conclusion, nous avons démontré que l’activation transcriptionnelle des gènes cellulairespar la NS5A est essentielle pour la réplication de l’ARN du VHC. Cette modulation des gènescellulaires pourrait également être impliquée dans les mécanismes de la pathogenèse viroinduite.Nous confirmerons cette hypothèse grâce aux souris NS5A. Par ailleurs, ces résultatspourraient contribuer au développement de nouvelles thérapies anti-VHC, basées surl’inhibition de translocation nucléaire de NS5AHepatitis C virus (HCV) causes a chronic infection in the majority of infected patients,ultimately leading to liver cirrhosis and hepatocellular carcinoma (HCC). Although the rolesof the HCV proteins in the viral life cycle are increasingly understood, the precise function ofthe HCV NS5A protein has yet to be elucidated. To date, the only putative direct functionattributed to NS5A is its transcriptional transactivation properties. Our group has previouslyshown that quasispecies variants of NS5A isolated from the serum samples of the samepatient bear different transactivating properties according to their amino acid sequence. Basedon these observations, we performed preliminary phylogenetic and functional analysis ofNS5A variants isolated from liver tissue of individuals infected with HCV of genotype 1b.This analysis revealed genetic and functional compartmentation of NS5A variants in tumoraland adjacent non-tumoral tissue. We hypothesized that the natural variability of NS5A mayimpact its proposed transactivation properties. We also hypothesized that NS5A’s putativetransactivation properties could play a role in HCV replication and in liver pathogenesis. Theaim of the study presented in this thesis was to investigate the role of NS5A transactivationproperties in the development of HCV-induced liver pathogenesis as well as in viralreplication. To study the role of NS5A transcriptional activation properties in liver pathogenesis, wedeveloped lentiviral vectors for the expression of selected NS5A variants bearing differenttransactivation potentials in cultured primary human hepatocytes. We now intend to extendthese preparations using RNAseq technology to analyse the, transcriptome of primaryhepatocytes transduced with lentiviral vectors encoding strongly and weakly transactivatingNS5A variants to identify the cellular pathways targeted by NS5A, allowing us to decipherthe role of NS5A mediated host gene regulation in development of HCV inducedpathogenesis. For in vivo studies, we have begun the development of transgenic mice allowingliver-specific conditional expression of NS5A variants with high and low transactivationpotentials. These transgenic mice will be used to study the possible role of NS5Atransactivation properties in development of HCC. To study the role of NS5A transcriptional activation properties in HCV RNA replication, weused the sub-genomic replicon system expressing previously characterized NS5A sequences..Using this system, we have demonstrated that a subset of NS5A protein can translocate to thenucleus and is recruited to cellular promoters of host cell genes known to be required forefficient replication of HCV replicon RNA as well as those implicated in pathogenesis.Moreover, we have shown that NS5A directly regulate the expression of these genes.Consequently, it was observed that replicons encoding NS5A variants with differenttransactivation potentials exhibited different replication capacities, and that this correlatedwith the transactivation potential of the corresponding NS5A variant. In agreement with theseobservations, inhibition of nuclear translocation of NS5A resulted in the inhibition ofreplication of the HCV subgenomic replicon, further confirming the role of NS5Atransactivation properties in viral RNA replication. In conclusion, we have demonstrated that NS5A-mediated transcriptional regulation ofcellular genes is required for HCV replication. Such NS5A-mediated modulation of cellulargenes may also constitute one of the mechanisms involved in HCV-related liver pathogenesisand development of HCC, an aspect which is currently under investigation using the toolsdeveloped during this project. This study will contribute towards deciphering the role ofNS5A in viral replication as well as providing insight into its role in HCV-induced liverpathogenesis
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François, Catherine. "Étude des relations entre la protéine NS5A du VHC, l'apoptose et le système interféron." Paris 6, 2002. http://www.theses.fr/2002PA066147.
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Taube, Stefan. "Charakterisierung des Hepatitis-Virus NS5A-Proteins als funktionalen Inhibitor der Interferon induzierten antiviralen Immunantwort." [S.l.] : [s.n.], 2005. http://www.diss.fu-berlin.de/2006/75/index.html.
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Shelton, Holly Ann. "Hepatitis C virus NS5A poly-proline motif interactions with cellular proteins containing SH3 domains." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435924.
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Yin, Chunhong. "Functional analysis of domain I of the hepatitis C virus non-structural NS5A protein." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/20605/.
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The research on hepatitis C virus protein NS5A has developed rapidly over the decades, primarily with the advent of the JFH1 cell culture infectious clone which allowed the study of all aspects of the virus lifecycle from entry to exit. As the important target of DAAs, the NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, a mutagenic study of 12 absolutely conserved and surface-exposed residues were conducted within the context of a JFH1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. Whilst P35A exhibited a modest reduction in infectivity, V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high-resolution confocal microscopy, it was demonstrated that V67A and P145A disrupted the recruitment of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken collectively, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. In parallel, this study also set out to investigate the interactions of NS5A domain I with cellular proteins by the approach of quantitative proteomic analysis. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.
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Potisopon, Supanee. "Insights into the RNA polymerase activity of the dengue virus NS5." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM5019.
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Le virus de la dengue cause une maladie de type grippal qui peut dans certains cas évoluer vers des fièvreshémorragiques mortelles. Mon projet de thèse porte sur la réplication de ce virus. Je focalise sur la compréhension du mécanisme d'action de la protéine NS5 de ce virus. La protéine contient 2 domaines : 1) domaine méthyltransférase, essentiel pour la traduction des protéines virales, 2) domaine polymérase, synthétisant le génome ARN du virus. Premièrement, nous avons démontré que la polymérase joue un rôle principal dans la conservation de l'extrémité 3' et 5' du génome et de l'anti-génome. Puis, j'ai caractérisé l'influence du domaine méthyltransférase sur l'activité polymérase de la protéine NS5. J'ai développé un système d'études mécanistiques en utilisant des techniques biochimiques de cinétique pré-stationnaire pour la protéine NS5, et obtenu des paramètres cinétiques et thermodynamiques de cette protéine envers ses substrats. Avec ce même système, j'ai pu tester des activités de la polymérase NS5 avec des ARN coiffés et triphosphates de différente longueur, mimant les séquences à l'extrémité 5' du génome du virus de la dengue. L'activité polymérase de NS5 est influencée par la présence de la coiffe de l'ARN, ce qui m'a permis de proposer une distance physique correspondant à environ 13 nucléotides entre les sites actifs domaines méthyltransférase et polymérase. Mes travaux ouvrent la voie à la détermination de la structure 3D de NS5 avec ses ARN et des nucléotides 5'-triphosphate.Elucider son mécanisme d'action, c'est être capable d'inhiber son action et donc de pouvoir proposer des molécules capables d'arrêter la prolifération virale lors d'une infectionDengue virus causes dengue fever, which may evolve towards life-threatening hemorrhagic fever. My research projectfocuses on dengue replication, and more precisely on the mechanism of NS5 at the molecular/atomic level. NS5 is a bifunctionalenzyme containing two domains: 1) a methyltransferase domain essential for translation of viral proteins, 2) apolymerase domain synthesizing the viral RNA genome. First, we demonstrated the main role of the polymerase in theconservation of 5' and 3' ends of dengue genome and anti-genome RNAs. Next, I showed the influence of themethyltransferase domain on the activity of the polymerase domain. I also developed a system allowing mechanistic studiesusing pre-steady state kinetics to characterize NS5 in depth. I have made use of this system to determine the catalyticparameters of NS5 towards its substrates. Using the same pre-steady state system, I was able to test the polymerase activityof NS5 with capped and uncapped 5'-triphosphate RNAs of different lengths corresponding to the 5'-end of the dengue RNAgenome. The polymerase activity of NS5 is significantly affected by the presence of the 5'-cap, which allowed me to designan experimental set-up pointing to a minimal physical distance of around 13 nucleotides between the methyltransferase andpolymerase active sites. My work will be useful to characterize the biophysics of NS5 in complex with its RNA and NTPsubstrates, and then to determine the crystal structure of such complex at play during viral RNA synthesis. Knowing thedetailed NS5 mechanism paves the way to inhibit its action and thus design drugs aiming at stopping a viral infection
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Paterson, Morris. "Inhibition of the cellular responses to interferon alpha by the hepatitis C virus NS5A protein." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325537.
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Foster, Toshana Lauria. "Structural and functional characterisation of the hepatitis C virus proteins p7, Ns2-3 and Ns5A." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531627.
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McKechnie, Victoria Margaret. "Variation in the NS5A gene of Hepatitis C Virus in response to interferon alpha therapy." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301364.
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