Au Chi Chuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 157-169). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.v / 論文摘要 --- p.vii / Abbreviations --- p.viii / List of Figures --- p.ix / List of Tables --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Hepatitis B virus and Hepatocellular carcinoma --- p.3 / Chapter 1.3 --- Pathogenesis of HBV related HCC --- p.6 / Chapter 1.4 --- Current screening test and tumor markers --- p.10 / Chapter 1.5 --- Expressed sequence tag (EST) sequencing --- p.13 / Chapter 1.6 --- Aim of the present study --- p.15 / Chapter 1.7 --- Characterization of KIAA0022 --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of liver HCC and normal counterpart libraries --- p.19 / Chapter 2.2 --- Plating out the human liver cDNA libraries --- p.19 / Chapter 2.3 --- PCR amplification of clones human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4 --- Cycle sequencing of cloned human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4.1 --- Dye-primer cycle sequencing (Pharmacia) --- p.21 / Chapter 2.4.1.1 --- Using Pharmacia LBKA.L.F. DNA sequencer --- p.21 / Chapter 2.4.1.2 --- Using Li-Cor 4200 Automated DNA sequencer --- p.22 / Chapter 2.4.2 --- Dye-terminator cycle sequencing (Pharmacia) --- p.22 / Chapter 2.5 --- Sequences analysis --- p.23 / Chapter 2.6 --- Cloning of full-length cDNA of KIAA0022 --- p.24 / Chapter 2.6.1 --- Amplification of KIAA0022 gene using PCR --- p.24 / Chapter 2.6.2 --- Purification of the PCR product --- p.25 / Chapter 2.6.3 --- Ligation --- p.25 / Chapter 2.6.4 --- One Shot® TOP 10 Chemical Transformation --- p.25 / Chapter 2.6.5 --- Small-scale preparation of the plasmid DNA --- p.26 / Chapter 2.6.6 --- Large-scale preparation of the plasmid DNA Table of Contents (continued) --- p.26 / Chapter 2.6.7 --- DNA sequencing of the full-length cDNA of KIAA0022 --- p.28 / Chapter 2.7 --- Northern Hybridization --- p.29 / Chapter 2.7.1 --- The Human multiple tissue Northern Blot --- p.29 / Chapter 2.7.2 --- Synthesis of the radiolabeled DNA probe --- p.29 / Chapter 2.7.3 --- Hybridization of the Northern blot --- p.30 / Chapter 2.8 --- Subcellular localization of KIAA0022 by tagging with green fluorescence protein (GFP) --- p.30 / Chapter 2.8.1 --- Amplification and purification of the KIAA0022 gene product --- p.30 / Chapter 2.8.2 --- Restriction enzymes digestion --- p.31 / Chapter 2.8.3 --- DNA ligation --- p.31 / Chapter 2.8.4 --- Preparation of the Escherichia coli competent cells for transformation --- p.31 / Chapter 2.8.5 --- Transformation of the plasmid DNA into competent Escherichia coli cells --- p.32 / Chapter 2.8.6 --- Small-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.7 --- Large-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.8 --- DNA sequencing of the cloned plasmid DNA --- p.33 / Chapter 2.8.9 --- Transfection --- p.33 / Chapter 2.8.10 --- Fluorescence microscopy examination --- p.33 / Chapter 2.9 --- Yeast two-hybrid screening assay --- p.34 / Chapter 2.9.1 --- "Cloning of the KIAA0022 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.34 / Chapter 2.9.2 --- Small-scale transformation of pGBKT7-KIAA0022 plasmid --- p.34 / Chapter 2.9.2.1 --- Preparation of yeast competent cells --- p.34 / Chapter 2.9.2.2 --- Transformation of the pGBKT7-KIAA 0022 plasmid into the yeast strain PJ69-2A --- p.35 / Chapter 2.9.3 --- Screening a pretransformed library by yeast mating --- p.35 / Chapter 2.9.4 --- β -Galactosidase analysis - colony lift filter assay --- p.36 / Chapter 2.9.5 --- Analysis of yeast plasmid inserts using PCR and DNA sequencing --- p.37 / Chapter 2.9.5.1 --- PCR --- p.37 / Chapter 2.9.5.2 --- DNA sequencing --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Results of ESTs sequencing in normal counterpart and HCC libraries --- p.38 / Chapter 3.1.1 --- The sequencing results of the normal counterpart cDNA clones --- p.38 / Chapter 3.1.2 --- Sequencing results of the human liver cancer cDNA clones --- p.41 / Chapter 3.1.3 --- The accuracy of the automated sequencing technique --- p.41 / Chapter 3.1.4 --- Catalogue of normal counterpart ESTs --- p.45 / Chapter 3.1.5 --- Catalogue of liver cancer ESTs --- p.47 / Chapter 3.2 --- Identification of genes differentially expressed in HCC using in silico method --- p.115 / Chapter 3.3 --- Sequence analysis of KIAA0022 --- p.121 / Chapter 3.3.1 --- Structural analysis of KIAA0022 --- p.121 / Chapter 3.3.2 --- Homology alignment --- p.122 / Chapter 3.4 --- Tissue distribution and expression profile of KIAA0022 using Northern blot analysis --- p.132 / Chapter 3.5 --- Subcellular localization of the KIAA0022 tagging by green fluorescence protein --- p.134 / Chapter 3.6 --- Yeast two-hybrid screening assay --- p.136 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Large-scale partial cDNA sequencing --- p.138 / Chapter 4.2 --- Characterization of ESTs --- p.139 / Chapter 4.3 --- Identification of genes differentially expressed in liver cancer using Poisson probability --- p.143 / Chapter 4.4 --- Characterization of KIAA0022 --- p.154 / Reference --- p.157 / Appendix --- p.170

Le virus de l'hépatite B (VHB) reste un problème de santé majeur dans le monde malgré la disponibilité du vaccin. Le VHB n’est pas éradiqué par les thérapies actuelles et 240 millions de personnes infectées chroniquement restent à risque de développer une cirrhose du foie et un carcinome hépatocellulaire (CHC).Le VHB est un petit virus hépatotrope doté d'un génome à ADN double brin partiel (ADNrc). Après infection l'ADNrc est converti en ADN épisomal (ADNccc) qui est ensuite organisé en minichromosome viral, qui est le modèle pour la transcription et qui initie la réplication. La protéine de l'hépatite B x (HBx) est recrutée sur l'ADNccc pour initier et maintenir la transcription de l'ADN ccc. HBx cible aussi directement des gènes cellulaires impliqué dans le développement du CHC.Nous avons utilisé une approche ChIP-Seq pour identifier toutes les cibles génomiques de HBx dans les cellules qui répliquent le VHB. Les cibles HBx sont à la fois des gènes codant les protéines et des ARNnc (75 miARN et 34 lncRNA). Nous avons montré que HBx réprimait un sous-ensemble de miARNs qui réguleraient négativement la réplication virale (ex : miR-24) et des miARNs impliqués dans le développement du CHC (ex : miR-21). Parmi les lncARNs ciblés pour HBx, nous avons étudié DLEU2, qui est fortement surexprimé dans l’infection par le VHB et le CHC. Nous avons en outre montré que DLEU2 lie à la fois HBx et l’histone méthyltransférase Ezh2, la sous-unité catalytique du complexe répressif PRC2. L'interaction avec DLEU2 et HBx relie les fonctions Ezh2/PRC2 conduisant à l'activation constitutive d'un sous-ensemble de gènes cibles d'Ezh2 qui sont normalement conservés dans un état réprimé. Nous avons également montré que l’interaction de HBx avec DLEU2 se produisait sur le minichromosome de l’ADNccc où elle stimulait la transcription/réplication du virus. Enfin, nous avons caractérisé par ATAC-Seq les changements d'accessibilité de la chromatine imposés par HBV dans les hépatocytes humains primaires / Hepatitis B virus (HBV) remains a major health problem worldwide despite the availability of the vaccine. No cure is available for the 240 million peoples chronically infected with HBV that are at risk to develop liver cirrhosis and hepatocellular carcinoma (HCC). Viral suppression, achieved by long term treatment with nucleotides analogues (NUCs), impacts on liver fibrosis and prevents liver decompensation but HCC risk is not reduced in the first 5 years of treatment. HBV is a small hepatotropic virus with a partially double strand DNA (rcDNA) genome. After hepatocyte infection the rcDNA is converted into the cccDNA episome that is then organized into a viral minichromosome that is the template for all viral transcripts and initiates replication. The hepatitis B x protein (HBx) is recruited on the cccDNA and is required to launch and maintain cccDNA transcription. HBx has also been shown to directly target cellular genes and this has been related to HCC development.We used a ChIP-Seq approach to determine the full repertoire of HBx genomic targets in HBV replicating cells. HBx targets include both protein coding genes and ncRNA (75 miRNAs and 34 lncRNAs). We showed that HBx represses a subset of miRNAs that would negatively regulate viral replication (i.e. miR-24) and miRNAs involved in HCC development (i.e. miR-21). Among the HBx targeted lncRNAs we focused DLEU2, which is strongly upregulated in HBV infection and HCC. We further showed that DLEU2 binds both HBx the Ezh2 histone methyltransferase, the catalytic subunit of the repressive PRC2 complex. The interaction with DLEU2 and HBx re-wires Ezh2/PRC2 functions leading to the constitutive activation of a subset of Ezh2 target genes that are normally kept in a repressed state. We also showed that HBx interaction with DLEU2 occurs on the cccDNA minichromosome where it boosts HBV transcription/replication. Finally, we characterized by ATAC-Seq HBV imposed changes of chromatin accessibility in primary human hepatocytes

INTRODUCTION: Mortality associated with hepatitis C virus (HCV) infection is increasing, yet only a small percentage of HCV-infected individuals are aware of their infections, complete treatment, and achieve a cure, defined as a sustained virologic response. In March 2015, the Section of General Internal Medicine at Boston Medical Center (BMC), New England’s largest safety-net hospital, implemented the Adult Primary Care HCV Treatment and Triage Program to increase access to treatment. We are unaware of prior studies that have explored a pharmacist-centered primary care-based HCV treatment model in the era of newer direct-acting antiviral (DAA) medications. OBJECTIVES: To gain a deeper understanding of the roles of each program staff member, as well as an understanding of how primary care providers (PCPs) who refer patients to the program perceive and interact with the program. Such an understanding will help promote implementation and dissemination of the program. METHODS: We conducted in-depth semi-structured interviews with six staff members and with five PCPs in the Section of General Internal Medicine at BMC who refer patients to the program. We asked staff members about their roles and their perception of the program’s impact on patient linkage to HCV treatment. We probed PCPs about their experiences with HCV screening, referral, and follow-up processes, and differences in accessing HCV treatment for their patients prior to and following the implementation of the program. We audiotaped and transcribed interviews, and identified major themes through qualitative analysis. RESULTS: We identified five major themes that characterize how the HCV treatment program delivers care: 1) efficiency (“So here I feel like…they get evaluated…they get treated. Boom, it’s done”); 2) clear and open communication (“…one of the strengths of our program is that we have…a lot of direct contact with patients…”); 3) personalized medicine (“…I've set up the pill box for them [patients]…we tailor it to whatever they need”); 4) high patient engagement (“So if I get a referral for a patient…I call the patient three times. If I haven’t heard from the patient…I send them a letter and I tell the PCP”); 5) patient empowerment through education (“I think patient education is the best thing…if the patient is involved then… they’ll do what they need to do”). Additionally, the public health social worker and the pharmacist play key roles in the program. The social worker supports patients throughout treatment and addresses psychosocial barriers to treatment engagement (“I had a patient…who stopped taking his medication because his apartment was infested with bed bugs…[Social worker] got the patient furniture for free and got an exterminator…”). The pharmacist provides medication management during face-to-face patient visits (“…I go over everything imaginable...proper adherence…adverse effects, interactions…”). CONCLUSIONS: The HCV treatment program at BMC is a promising model to deliver HCV treatment to urban, underserved patient populations. Our findings suggest that public health social workers and pharmacists may be one approach to increasing access to HCV treatment in primary care settings in the era of DAA medications. Further study of the program’s efficacy in improving HCV outcomes is warranted.

Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.

mormaeche@dge.gob.pe / Objective: To assess the seroprevalence of hepatitis B virus (HBV), syphilis, and HIV and associated risk factors in pregnant women and their male partners from six indigenous populations of the Peruvian Amazon Basin. Methods: A cross-sectional study was performed in six indigenous populations from the Peruvian Amazon Basin. Blood samples were obtained and tested for HBV (antibodies to the hepatitis B core antigen (anti-HBc) and hepatitis B surface antigen (HBsAg)), for syphilis (rapid plasma reagin and microhemagglutination assay for Treponema pallidum antibodies), and for HIV (ELISA and indirect immunofluorescence test). A survey was also performed to identify associated risk factors. Results: One thousand two hundred and fifty-one pregnant women and 778 male partners were enrolled in the study. The seroprevalence of anti-HBc in pregnant women was 42.06% (95% confidence interval (CI) 39.28–44.85%) and in their male partners was 54.09% (95% CI 50.32–57.86%). The seroprevalence of HBsAg in pregnant women was 2.11% (95% CI 0.78–3.44%) and in their male partners was 3.98% (95% CI 1.87–6.08%). The seroprevalence of syphilis in pregnant women was 1.60% (95% CI 0.86–2.33%) and in their male partners was 2.44% (95% CI 1.22–3.66%). HIV seroprevalence in pregnant women was 0.16% (95% CI 0.02–0.58%) and in their male partners was 0.29% (95% CI 0.04–1.03%). Sexual risk factors were strongly related to blood markers of syphilis and HBV. Conclusions: Hepatitis B was found to be hyperendemic and strongly related to sexual factors, suggesting an important sexual component in the transmission of the disease in the populations studied. Syphilis was found to have an endemicity in pregnant women above the national level and this may be indicative of high mother-to-child transmission. HIV has started to show its presence in indigenous populations of the Amazon Basin and the results suggest the epidemic is concentrated. / Revisión por pares

Malaria is a major health problem and an effective vaccine is essential for the eradication of the disease. Despite extensive efforts, a malaria vaccine remains elusive due to the parasite's complex life cycle, diverse morphology, and immune system evasion mechanisms. Antibodies against C terminal domain of merozoite surface protein 1 (MSP1-19), a highly conserved protein and the main vaccine candidate for blood-stage malaria, can inhibit erythrocyte invasion by the parasite and alleviate the disease symptoms. However, MSP1-19 is poorly immunogenic and classic protein-in-adjuvant MSP1-19-based vaccine formulations failed to induce strong immune responses due to low immunogenicity and generation of ineffective antibodies. The aim of this study was to use hepatitis B virus core (HBc) particles to increase the immunogenicity of MSP1-19. HBc forms particles with protruding spikes and induces a strong and specific immune response against foreign epitopes inserted at the tips of the spikes. In addition, positioning of MSP1-19 on the particle can influence the accessibility of certain antibody binding sites, possibly altering elicited antibody fine specificity and vaccine efficiency. MSP1-19 domain was inserted into the middle of the HBc sequence so that it is displayed at the tips of the HBc particle. Two HBc-MSP1-19 constructs, having different insert flanking linkers, displayed soluble particle formation after bacterial expression and lysis optimization. The particles were purified and the suitability of these two constructs as malaria vaccine candidates was assessed. Firstly, binding of the conformational anti-MSP1-19 antibodies indicated that MSP1-19 domain in the chimeric proteins has the correct disulphide bond pattern which is crucial for the protective properties of an MSP1-19-based vaccine. Furthermore, electron microscopy imaging and determination of initial 3D structures confirmed that both HBc MSP1-19 constructs form particles resembling the wild-type HBc particles, meaning the insertion of MSP1-19 did not heavily distort the overall HBc particle structure. In addition, it was shown that MSP1-19 domains are displayed at the tips of the particle spikes. Particle formation and foreign epitope display are important for the epitope's immunogenicity improvement. The immunogenicity of the chimeric particles was then assessed in mice. Both constructs elicited similar high antibody titres without the use of additional adjuvants, but no difference was observed between the particulate constructs and a non-particulate control (an MSP1-19-based protein). Interestingly, although both HBc-MSP1-19 and non-particulate MSP1-19-elicited antibodies recognized native malarial parasite, only the particulate construct antibodies demonstrated a moderate parasite growth inhibition while the antibodies from the control group did not show parasite inhibition above the background levels. In conclusion, it was shown that MSP1-19 can be expressed in bacteria as a soluble correctly folded protein fused to HBc. More importantly, the fusion protein is capable of forming immunogenic particles which generate antibodies that recognize native MSP1 and inhibit parasite growth more effectively than the protein without the HBc. Therefore, this work lays grounds and supports further chimeric HBc-MSP1-19 research and development.

Le Virus de l’Hépatite C (HCV) est un problème majeur de santé publique qui touche plus de 170 millions de personnes dans le monde. Le HCV cible essentiellement les hépatocytes où il effectue son cycle de réplication qui peut-être divisé en trois étapes majeures : l’entrée de la particule virale qui aboutit à la libération de l’ARN viral dans le cytoplasme, la traduction/réplication du génome viral et l’assemblage/sécrétion des particules néosynthétisées. Durant ma thèse, nous nous sommes intéressés à l’étape d’entrée qui est un processus complexe multiséquentiel faisant intervenir de nombreux facteurs cellulaires. Le virus se lie d’abord à la surface cellulaire via des facteurs d’attachement puis interagit avec des facteurs d’entrée spécifiques tels que la tétraspanine CD81, le récepteur scavenger B1 et les protéines de jonctions serrées CLDN-1, -6, -9 et OCLN. La particule virale est ensuite internalisée via une endocytose dépendante de la clathrine, puis sous l’action du pH acide des endosomes les enveloppes virale et cellulaire fusionnent permettant la libération de la capside virale dans le cytoplasme de la cellule infectée. Parmi les facteurs cellulaires impliqués dans l’entrée du HCV, deux protéines de la famille des tétraspanines ont été identifiées, CD81 et CD63. Les tétraspanines sont des protéines transmembranaires capables de former des microdomaines enrichis en tétraspanines en interagissant entre elles ainsi qu’avec des protéines dites partenaires. Nous avons testé l’implication dans l’entrée virale des tétraspanines exprimées dans les hépatocytes. Ainsi, nous avons montré que CD151 semble également important pour l’entrée du HCV. CD151 aurait un rôle indirect sur l’entrée en jouant sur l’organisation membranaire, en empêchant CD81 de former des clusters qui restreignent l’entrée du virus. Nous avons aussi étudié le rôle que pouvait jouer les polymorphismes de facteurs d’entrée sur l’entrée virale. Sur une cohorte de patients toxicomanes infectés par le HIV mais non infectés par le HCV, deux mutations ont été identifiées, R209Q dans CLDN-6 et P24A dans OCLN, qui n’étaient pas présentes dans les populations contrôles ou coinfectées par le HCV. Chez les patients toxicomanes, il est très rare que les patients infectés par le HIV ne le soient pas par le HCV, suggérant une résistance naturelle de ces patients à l’infection. Nous avons émis l’hypothèse que ces deux mutations pouvaient bloquer l’entrée du HCV. Cependant, la caractérisation de ces mutations a montré qu’elles n’avaient pas d’effet fonctionnel in vitro. Enfin, nous avons exploré l’effet de la Monensine, un ionophore polyéther, sur l’infection par le HCV. Cette molécule augmente le pH des endosomes via ses capacités de transfert d’ions à travers les membranes cellulaires. Nous avons montré qu’elle inhibe l’entrée du HCV en bloquant la fusion. De manière intéressante, la transmission cellule-cellule, un mécanisme d’entrée du virus encore mal caractérisé, était également bloquée par la Monensine, suggérant une étape de fusion dépendante du pH pour cette voie. Nous avons généré des mutants de résistance à la Monensine, notamment le clone FL-8 qui portait deux mutations dans les glycoprotéines d’enveloppe, Y297H dans E1 et I399T dans E2. Les particules virales portant ces deux mutations infectaient les cellules de manière indépendante du pH, présentaient des propriétés physicochimiques différentes et ne se transmettaient plus de cellule à cellule. En conclusion ces études soulignent l’importance de l’organisation membranaire pour l’entrée du virus via certains facteurs cellulaires tels que CD151. Par ailleurs, nous avons mis en évidence que la transmission cellule-cellule était dépendante du pH et que des mutations ponctuelles dans E1 et E2 permettent une fusion indépendante du pH. En revanche, des polymorphismes naturels de CLDN-6 et OCLN n’affectent pas leur capacité à supporter l’entrée virale. / Hepatitis C Virus (HCV) is a global health problem with over 170 million infected people worldwide. HCV targets mainly the hepatocytes and its lifecycle is divided into three steps. Viral particle entry leads to the release of viral genomic RNA into the cytoplasm where translation and replication take place. Then, new viral particles are assembled and secreted. During my thesis, we focused on the entry step. Indeed, HCV entry is a complex multistep process that requires numerous cellular factors. First, the virus interacts with attachment factors and then interacts with specific cellular factors including the tetraspanin CD81, the scavenger receptor B1 and the tight junction proteins CLDN-1, -6, -9 and OCLN. The viral particle is next internalized through a clathrin-dependent endocytosis. Finally, fusion at low pH occurs between viral and endosomal membranes leading to the release of the capsid into the cytoplasm. Among cellular factors involved in HCV entry, two members of the tetraspanin superfamily were identified, CD81 and CD63. Tetraspanins are transmembrane proteins able to organize themselves in tetraspanin-enriched microdomains through tetraspanin-tetraspanin interactions and interactions with partner proteins. We tested the involvement in HCV entry of tetraspanins expressed in hepatocytes. Interestingly, we showed that CD151 is also involved in HCV entry. CD151 seems to play an indirect role in HCV entry through regulating membrane organization, especially by preventing CD81 clustering, which is unfavourable to HCV entry. We also studied the effect of some entry factors polymorphisms on viral entry. In a cohort of drug users infected by HIV but not by HCV, we identified two mutations, R209Q in CLDN-6 and P24A in OCLN, not found in control or coinfected patients. It is very rare for drug users patients infected by HIV not to be infected by HCV, suggesting a natural resistance of these patients to HCV infection. We hypothesized that the two mutations identified might impair HCV entry. However, the characterization of these mutations showed that they did not have a functional effect in vitro. Finally, we investigated the effect of Monensin, a polyether ionophore, on viral entry. This molecule increases endosomal pH through its ions transfer properties across cell membranes. We showed that Monensin inhibits HCV entry through impairing the fusion step. Interestingly, HCV cell-to-cell transmission, another poorly characterized entry pathway, was also blocked by Monensin, suggesting that a pH-dependent fusion step is also required for this transmission route. We generated resistant mutants to Monensin, notably the FL-8 mutant carrying two mutations in envelope glycoproteins, namely Y297H in E1 and I399T in E2. Viral particles expressing these two mutations infected cells in a pH-independent manner, displayed different biophysical properties and were not cell-to-cell transmitted. To conclude, these studies highlight the importance of membrane organization for viral entry through cell factors like CD151. We also pointed out that cell-to-cell transmission is a pH-dependent process. In addition, point mutations in E1 and E2 are sufficient to enable HCV to be pH-independent for its entry. However, polymorphisms in CLDN-6 and OCLN seem to have no effect on viral entry.

O vÃrus da hepatite C (VHC) e a esteatose sÃo importantes causas de doenÃa hepÃtica crÃnica no mundo. Apesar de comum, a fisiopatologia da esteatose, e seu papel na progressÃo da fibrose em pacientes com VHC, permanece desconhecida. O objetivo deste trabalho foi quantificar esteatose macrovesicular e microvesicular e correlacionÃ-las com dados clÃnicos e histopatolÃgicos. O estudo analisou biÃpsias hepÃticas de pacientes portadores do VHC sem tratamento prÃvio. A fibrose e atividade necroinflamatÃria foram avaliadas segundo os escores de METAVIR e Ishak; as classificaÃÃes de Kleiner e Brunt foram utilizadas como suporte para o diagnÃstico de esteatohepatite realizado pelo patologista. Ademais, o nÃmero de hepatÃcitos com esteatose macrovesicular e esteatose microvesicular foram quantificados a partir do nÃmero total de hepatÃcitos. A fibrose e atividade necroinflamatÃria foram classificadas e semi-quantificadas. Foi encontrada associaÃÃo significante da fibrose avaliada pelo sistema de Ishak entre a esteatose macrogoticular e microgoticular (p= 0,017 e p= 0,0113, respectivamente). A atividade inflamatÃria global (classificaÃÃo Metavir ) apresentou correlaÃÃo linear com a piora da fibrose (< 0,001). A fibrose avaliada pelo sistema de Metavir se correlacionou com o IMC. A presenÃa de VHC associado à esteatohepatite apresentou correlaÃÃo significante com as mÃdias das AST/ALT, e com a fibrose de Ishak e Metavir, quando comparado aos dados dos pacientes com VHC, sem esteatohepatite (p = 0,006; p = 0,012; p = 0,0098 e p = 0,014, respectivamente). Em nosso trabalho, concluÃmos que a fibrose de Ishak esteve associado a esteatose macrogoticular e microgoticular. Igualmente, se demonstrou que a presenÃa da esteatohepatite esteve fortemente associado a fibrose. / The hepatitis C virus (HCV) and steatosis are important causes of chronic hepatic disease in the world. Although common, the pathofisiology of steatosis and its role in the progression of fibrosis in patients with HCV is uncertain. Our objective was to quantify the macrovacuolar and microvesicular steatosis and to correlate them with clinical and histophatologic data. The study included needle biopsy of the liver of patients with HCV without previous treatment. The fibrosis and necroinflammatory activity of hepatic damage by HCV were evaluated by METAVIR and Ishakâs scores; Kleinerâs and Bruntâs classification were used as a support for diagnosis of the steatohepatitis by the pathologist. Furthermore, the number of hepatocytes with the macrovacuolar and microvesicular steatosis was quantified in a total number of hepatocytes. Fibrosis and necroinflammatory activity were categorized and semi quantified. A significant association of the fibrosis was found and it was evaluated by the Ishak system between the macrogoticular and the microgoticular steatosis (p=0,017 e p= 0,0113, respectively). The global inflammatory activity (Metavir classification) has presented a linear correlation with the worsening of the fibrosis (< 0,001). The fibrosis which was evaluated by the Metavir system has been correlated with the BMI. The presence of HCV associated with the steatohepatitis has presented a significant correlation with the average of AST/ALT and with the Ishak and Metavir fibrosis, when compared to the data of the patients with HCV, without steatohepatitis (p= 0,006; p= 0,012; p= 0,0098 and p= 0,014, respectively) In our research we conclude that the Ishak fibrosis has been associated with the macrogoticular and microgoticular steatosis. Equally, it was demonstrated that the presence of the steatohepatitis was strongly associated to the fibrosis.

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-05-28T19:39:54Z No. of bitstreams: 1 Marina Brandão Braz Albuquerque.pdf: 605500 bytes, checksum: 1405379cffb1283fb04eb721d76cc92a (MD5) / Made available in DSpace on 2018-05-28T19:39:54Z (GMT). No. of bitstreams: 1 Marina Brandão Braz Albuquerque.pdf: 605500 bytes, checksum: 1405379cffb1283fb04eb721d76cc92a (MD5) Previous issue date: 2018-03-27 / The use of hepatic biopsy to assess the stay of the level of fibrosis continues to be gold standard but may present some risks during the procedure and high cost. Given that the WHO suggested the use of APRI and FIB-4 indexes to evaluate hepatic fibrosis in patients with chronic hepatitis C, because they are low-cost exams that can replace the use of hepatic biopsy. Objectives: Assess the level of hepatic fibrosis before and after treatment of hepatitis C by means of APRI and FIB 4 methods. Methods: This is a cross-descriptive study, carried out by reviewing medical records of patients attending the STD/AIDS and Viral Hepatitis clinics in the period from March 2016 to December 2017. The level of fibrosis was defined according to PCDT 2015 in significant fibrosis values of APRI > 1.5 and FIB-4 > 3.25; and absence of fibrosis APRI ≤ 0.5 and FIB-4 ≤ 1.5. Patients who did not fit within the triage were defined as indeterminate. The correlation between the improvement of fibrosis and the variables was performed: gender, age and genotype. Results: 45 selected patients were assisted in the clinic, 23 of them (51.1%) were male. The average age of the patients was 57.13. The infection by genotype 1a was more prevalent n = 19 (42.2%). Among the 45 patients, 28 were within the classifications between absence and advanced by the APRI before the treatment and 17 were defined as indeterminate. In the FIB-4, 26 patients were classified between absence and advanced fibrosis and 19 defined as indeterminate. After the treatment 25 patients were classified by APRI and 20 by FIB- 4. Only the FIB-4 index had significant correlation (P < 0.05) with age and improvement in fibrosis. Conclusions: Both tests can be used to verify the staging of hepatic fibrosis and assist in daily practice, but some results may be in the indeterminate zone. In these cases, it is necessary to carry out complementary tests to better define the degree of fibrosis. / O uso da biópsia hepática para avaliar o estadiamento do grau de fibrose continua sendo padrão-ouro, contudo pode apresentar alguns riscos durante o procedimento, além de elevado custo. Diante disso, a OMS sugeriu o uso dos índices APRI e FIB-4 para avaliar a fibrose hepática em pacientes portadores de hepatite C crônica, visto que os exames têm baixo custo e podem substituir a biópsia hepática. Objetivos: Avaliar o grau de fibrose hepática pelos métodos APRI e FIB 4 antes e após tratamento de hepatite C. Métodos: Trata-se de estudo descritivo transversal, realizado por meio da revisão de prontuários médicos de pacientes atendidos no Ambulatório de DST/AIDS e Hepatites Virais em Aparecida de Goiânia no período de março de 2016 a dezembro de 2017. O grau de fibrose foi assim definido conforme PCDT 2015: fibrose significativa com valores de APRI >1,5 e FIB-4 >3,25; ausência de fibrose com APRI ≤0,5 e FIB-4 ≤1,5. Os pacientes que não se enquadravam na classificação foram definidos como indeterminados. Foi realizada a correlação entre a melhora da fibrose e as variáveis: gênero, idade e genótipo. Resultados: Foram selecionados para o estudo 45 pacientes atendidos na unidade, dos quais 23 (51,1%) eram do sexo masculino. A média de idade dos pacientes foi de 57,13 anos. A infecção pelo genótipo 1a foi mais prevalente, n= 19 (42,2%). Dos 45 pacientes, 28 estavam inseridos nas classificações entre fibrose ausente e avançada pelo APRI antes do tratamento e 17 foram definidos como indeterminados. No FIB-4, 26 pacientes foram classificados entre ausência e fibrose avançada e 19 foram definidos como indeterminados. Após o tratamento, 25 pacientes foram classificados pelo APRI e 20 pelo FIB-4. Apenas o índice FIB-4 mostrou correlação significativa (p<0,05) entre idade e melhora na fibrose. Conclusões: Ambos os testes podem ser utilizados para verificar o estadiamento da fibrose hepática e auxiliar na prática diária, porém alguns resultados podem estar na zona indeterminada. Neste caso é necessária a realização de testes complementares para definir melhor o grau de fibrose.

Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte / Converging evidences suggest that the Hepatitis B virus (HBV) core protein, beside its well-known structural role to form nucleocapsids in the cytoplasm, could have important regulatory functions in the nucleus of infected hepatocytes. Indeed, nuclear Core was shown to associate with the cccDNA and to the promoters of some cellular genes, suggesting that Core may control viral and/or cellular gene expression. In addition, Core has the capacity to bind RNA, and may thus regulate HBV RNA metabolism. To elucidate these functions, we performed a proteomic analysis of the cellular factors interacting with nuclear Core in human hepatocytes. This interactome revealed a majority of highly interconnected RNA-binding proteins (RBPs), which participate in several steps of mRNA metabolism, including transcription, splicing and nuclear egress. We focused on two major Core-interacting factors, SRSF10 and RBMX that were previously involved in splicing and DNA repair. Functional analyses performed by a siRNA approach indicated that RBMX and SRSF10 were able to differentially regulate the levels of all viral RNAs most likely by acting at different steps of the viral life-cycle. Similarly, a small compound, affecting the activity of selected RBPs, severely impaired HBV replication by strongly reducing viral RNA accumulation. Altogether, these results strongly suggest that Core interacts with some selected RBPs to control the fate of viral and/or cellular RNAs and provide new critical information for the development of novel host-targeting antiviral agents (HTA)