Made available in DSpace on 2015-04-11T13:55:00Z (GMT). No. of bitstreams: 1 midia barbosa.pdf: 2406682 bytes, checksum: 16c04c78a5dd5af5cfe75dbf61b6b5e6 (MD5) Previous issue date: 2013-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hepatitis C is a disease characterized by the inflammation of the liver with HCV transmitted primarily through infected blood. It is estimated that 170 million people, about 3% of the world population, is infected with hepatitis C. Unlike other viruses that cause hepatitis, HCV does not generate adequate immune response in the body, which makes 80% of the infected to evolve into chronicity, whereas 15 to 20% of infected people heal spontaneously. In Brazil there is a significant variation in the prevalence of infection, the main cause of this epidemiological distinction is the characteristics differences between populations and geographic that each region studied has, and the limited epidemiological data about the disease. There is no vaccine against hepatitis C virus, thus early diagnosis increases the effectiveness of treatment. The aim of this study was to characterize the epidemiological, virological and immunological anti-HCV general population living in the city of Iranduba, metropolitan region of Manaus. Where were evaluated 700 volunteers and it was detected a positive case, using a rapid anti-HCV test, despite the presence of classical risk factors for infection in the seronegative volunteers. The realization of a molecular test, the polymerase chain reaction, confirmed the diagnosis of infection and by phylogenetic analysis of the NS5B region of the viral genome was observed genotype 2b. Among cytokines quantified, in the anti-HCV positive case, IL-2, IL-6, IL-10, TNF-α and IL-17A were more prevalent. The study showed serumprevalence anti-HCV of 0.14%, ranking the city as a place of low endemicity for HCV and reinforcing the heterogeneous characterization of the Amazon of hepatitis C. / A hepatite C é uma doença caracterizada pela inflamação do fígado, sendo o vírus HCV transmitido, principalmente, através de sangue contaminado. Estima-se que 170 milhões de pessoas, cerca de 3% da população mundial, sejam portadores de hepatite C. Ao contrário dos demais vírus que causam hepatite, o HCV não gera resposta imunológica adequada no organismo, o que faz com que 15 a 20% das pessoas infectadas curem espontâneamente, enquanto 80% evoluem para cronificação da doença. No Brasil ocorre grande variação na prevalência da infecção, isso se deve principalmente, as distintas características epidemiológicas entre as populações e a região geográfica estudada, além de restritas informações sobre a doença. Não existe vacina contra a hepatite C, assim o diagnóstico precoce amplia a eficácia do tratamento. O objetivo deste trabalho consistiu em caracterizar o perfil epidemiológico, virológico e imunológico anti-HCV na população geral, residente no município de Iranduba, região metropolitana de Manaus. Participaram da pesquisa 700 voluntários. Foi detectado um caso positivo no teste rápido anti-HCV, apesar da presença dos clássicos fatores de risco para a infecção nos voluntários soronegativos. A realização do teste molecular, reação em cadeia da polimerase, confirmou o diagnóstico da infecção e através da análise filogenética da região NS5B do genoma viral foi constatado o genótipo 2b. Dentres as citocinas quantificadas, no caso anti-HCV positivo, IL-2, IL-6, IL-10, TNF-α e IL-17A foram mais prevalentes. O estudo apontou soroprevalência anti-HCV de 0,14%, classificando o município de Iranduba como local de baixa endemicidade para o HCV e reforçando a caracterização heterogênea do Amazonas para a hepatite C.

Made available in DSpace on 2015-04-20T12:31:42Z (GMT). No. of bitstreams: 1 PRE-TEXTUAL.pdf: 394027 bytes, checksum: d5e64d94f3ade196d4079ab1173940e8 (MD5) Previous issue date: 2009-06-30 / Fundação de Amparo à Pesquisa do Estado do Amazonas / The global epidemic of AIDS began in 1981, the United States, and after the introduction of antiretroviral drugs to patients infected with HIV / AIDS have been given a higher survival emerged a new challenge, co-infection with the hepatitis C virus (HCV), which is already the leading cause of death in these individuals. The HCV was discovered in 1989 and its main route of transmission, parenteral, is common to HIV, thus increasing the prevalence of co-infection HIV / HCV and are currently more than 30% of those infected by HIV also infected by HCV. There are few studies on co-infection HIV / HCV in the Amazon and this study provides an opportunity to evaluate the association of these viruses. The work is a description of a number of cases and is intended to study the clinical implications and immunobiologicals of co-infection HIV / HCV in a population of patients in the Tropical Medicine Foundation of Amazonas (FMTAM), in the period 2000 to 2007. This study found a prevalence of patients co-infected HIV / HCV of 4.42% (n = 70), with an average annual growth of 3.6%. Of these 72.9% were male, 47.1% were aged 30 | - 40. Of these 80% had completed the first grade, 50% received up to a minimum wage and 55.7% were natural in the city of Manaus where 94.3% from the capital too. Of the patients studied, 68.6% were heterosexuals and 84.3% of patients there was sexual promiscuity as a risk factor. During the study period 34.3% of the patients died. Co-infected individuals, only 25.7% (n = 18/70) to attend the clinic for viral hepatitis FMTAM for collection of biological material. In these patients (n = 18) the mean AST was 61.5 ± 61 U / L, ALT of 62.2 ± 37 U / L and the AST / ALT was 0.88 ± 0.33 U / L. As for lipids 38.9% had total cholesterol> 200mg/dl, 83.3% had HDL ≤ 40 mg / dL, 77.8% had triglycerides> 150 mg / dL and 33.3% had glucose> 110 mg / dL . Included 83.3% of patients used HAART scheme whereby 100% of these were using the protease inhibitor regimen. In applying the FIB-4 score in predicting fibrosis found that 77.8% with a cutoff point was <1.45 and 22.2% with a cutoff> 3.25. As for CD4 + T cells 72.2% had <500 cls/mm3 with a median of 271 cls/mm3, on the T CD8 + 88.9% had ≥ 215 cls/mm3 with a median of 794.5 cls/mm3. The ratio CD4 + / CD8 + was 0.32 cls/mm3. As the viral load of HIV and HCV there was a median of 16,911 copies / mL and 543,209 copies / mL, respectively. In this population 88.9% had the genotype 1 of HCV and 94.4% had a sub-type B HIV. Of these, 83.3% had Child-Pugh A and 61.1% who had normal liver on ultrasound. When the dose cytokines IL4, IL6, IL8, IL10, IL12 and IFN-γ in these patients found that only the IL6 (p = <0.001) showed statistical significance especially when correlated to the logarithm of the HCV viral load (0.031). The results found in this study, despite the low prevalence, have annual growth of co-infection due to improvement in the research of hepatitis C in patients with HIV. These results contribute to a better understanding of the clinical, epidemiological and immunological profile of patients co-infected in the north, because these data may lead to greater understanding of the interaction of these two viruses resulted in early diagnosis and consequent reduction of deaths. / A epidemia mundial da AIDS teve início em 1981, nos Estados Unidos, e pós a introdução dos anti-retrovirais os pacientes infectados com HIV/AIDS passaram a ter uma sobrevida maior surgindo um novo desafio, a co-infecção com o vírus da hepatite C (HCV), a qual já é a principal causa de morte nestes indivíduos. O HCV foi descoberto em 1989 e sua principal via de transmissão, a parenteral, é comum ao HIV, com isso aumentando a prevalência da co-infecção HIV/HCV e estando atualmente, mais de 30% dos infectados pelo HIV também infectados pelo HCV. Existem poucos estudos sobre co-infecção HIV/HCV no Amazonas e o presente estudo oferece uma oportunidade de avaliar a associação destes vírus. O trabalho é do tipo descritivo de uma série de casos e tem o objetivo de estudar as implicações clínicas e imunobiológicas da co-infecção HIV/HCV em uma população de pacientes atendidos na Fundação de Medicina Tropical do Amazonas (FMTAM), no período de 2000 a 2007. Este estudo encontrou uma freqüência de pacientes co-infectados HIV/HCV de 4,4% (n=70), com uma média de crescimento anual de 3,6% dos casos. Destes 72,9% eram do sexo masculino, 47,1% tinham entre 30|- 40 anos. Destes 80% possuíam o primeiro grau completo, 50% recebiam até um salário mínimo e 55,7% eram naturais da cidade de Manaus sendo que 94,3% também procedentes da capital. Dos pacientes estudados, 68,6% eram heterossexuais e em 84,3% dos pacientes encontrou-se a promiscuidade sexual como fator de risco. No período do estudo 34,3% dos pacientes foram à óbito. Dos indivíduos co-infectados, apenas 25,7% (n=18/70) comparecerem ao ambulatório de hepatites virais da FMTAM para coleta de material biológico. Nestes pacientes (n=18) a média da AST foi 61,5±61 U/L, da ALT 62,2±37 U/L e a relação AST/ALT foi 0,88±0,33 U/L. Quanto aos lipídeos 38,9% apresentaram colesterol total >200mg/dL, 83,3% apresentaram HDL ≤ 40 mg/dL, 77,8% tinham triglicerídeos >150 mg/dL e 33,3% tinham glicemia >110 mg/dL. Dos pacientes incluídos 83,3% usavam esquema HAART sendo que 100% destes faziam uso de inibidor da protease no esquema. Ao aplicar o escore FIB-4 para predizer fibrose verificou-se que 77,8% ficou com ponto de corte <1,45 e 22,2% com ponto de corte >3,25. Quanto às células T CD4+ 72,2% tinham <500 cls/mm3 com uma mediana de 271 cls/mm3, quanto ao T CD8+ 88,9% tinham ≥215 cls/mm3 com uma mediana de 794,5 cls/mm3. A razão CD4+ /CD8+ foi 0,32 cls/mm3. Quanto à carga viral do HIV e do HCV verificou-se uma mediana de 16.911 cópias /mL e de 543.209 cópias/mL, respectivamente. Nesta população 88,9% apresentaram o genótipo 1 do HCV e 94,4% apresentaram o sub-tipo B do HIV. Destes, 83,3% apresentavam Child-Pugh A sendo que 61,1% apresentavam fígado normal na ultra-sonografia. Ao dosar as citocinas IL4, IL6, IL8, IL10, IL12 e IFN-γ nestes pacientes verificou-se que apenas a IL6 (p= <0,001) apresentou significância estatística principalmente quando correlacionada ao logaritmo da carga viral do HCV (0,031). Os resultados encontrados neste estudo, apesar da baixa prevalência, apresentam crescimento anual da co-infecção provavelmente devido à melhora na investigação da hepatite C nos pacientes com HIV. Estes resultados contribuem para um melhor conhecimento sobre os dados clínicos, epidemiológicos, bem como perfil imunológico dos pacientes co-infectados da região Norte, uma vez que estes dados podem levar à uma maior compreensão da interação destes dois vírus resultando em diagnóstico precoce, e conseqüente redução dos óbitos.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-27T10:31:50Z No. of bitstreams: 2 Dissertação - Rachel de Paula Santos Ribeiro - 2017.pdf: 12713551 bytes, checksum: c361c2531a99b684a3f4f3633f77b96c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-27T10:32:23Z (GMT) No. of bitstreams: 2 Dissertação - Rachel de Paula Santos Ribeiro - 2017.pdf: 12713551 bytes, checksum: c361c2531a99b684a3f4f3633f77b96c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-27T10:32:24Z (GMT). No. of bitstreams: 2 Dissertação - Rachel de Paula Santos Ribeiro - 2017.pdf: 12713551 bytes, checksum: c361c2531a99b684a3f4f3633f77b96c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-26 / Autoimmune diseases can be triggered by viruses, bacteria and parasites. However, the participation of these infectious agents in the etiology of vitiligo, it is a current research topic. In this study, the serum of 51 participants with vitiligo and 51 control subjects was analyzed for the presence of anti-Toxoplasma gondii (T.gondii) IgG, anti-herpes simplex (HSV) 1/2 IgG, anti-cytomegalovirus (CMV) IgG and anti-hepatitis C (HCV) IgG. / As doenças automimunes podem ser desencadeadas por vírus, bactérias ou parasitas. E o envolvimento destes agentes infecciosos na etiologia do vitiligo, é tema de intensa investigação atual. Neste estudo, o soro de 51 participantes com vitiligo e de 51 pessoas controle pareados por sexo e idade foi analisado para presença de imunoglobulinas IgG anti- Toxoplasma gondii (T.gondii), anti-herpes simples (HSV) 1/2, anti citomegalovírus (CMV) e anti-hepatite C (HCV).

Ce travail concerne le développement d’un système de vectorisation nanoparticulaire pour l’interférence ARN, constituant une nouvelle proposition thérapeutique applicable à des pathologies virales ou tumorales, et possiblement complémentaire aux traitements existants. La vectorisation de siRNA est ici basée sur l’enrobage multicouche de nanoparticules de phosphate de calcium, la multicouche étant constituée de dépôts alternés de PEI modifié et de siRNA. Ce système permet d’obtenir une efficacité de transfection des cellulaires cibles supérieure à celle des procédés conventionnels et une rémanence fonctionnelle in vitro jusqu’à neuf jours. Les résultats d’interférence ARN obtenus ont permis notamment d’inhiber l’infection par le virus de l’hépatite C jusqu’à 99,95%, l’inhibition de l’expression d’une protéine intrinsèque jusqu’à90,5%, et le ralentissement de la croissance cellulaire dans un modèle 3D mimant une tumeur hépatique jusqu’à 46,5%. Ces nanoparticules pourraient présenter un intérêt majeur, en offrant une action à long terme et en résolvant la plupart des difficultés rencontrées en utilisant des siRNA en thérapie. / This work concerns the development of a nanoparticle vector system for RNA interference,constituting a new therapeutic option applicable to viral and tumor pathologies,and possibly complementary to existing treatments.siRNA vectorisation is here based on multi layer coating of alcium phosphate nanoparticles,the multi layer being constituted of alternate coatings of modified PEI and siRNA.This system triggers a better transfection efficiency of target cells than classic techniques,as well as a functional persistence up to 9 days in vitro.RNA interference results using CPnp allowed inhibition of hepatitis C virus infection up to 99.95%,of intrinsic protein expression up to 90.5%,and of cell growth in a 3 D model mimicking an hepatic tumor up to 46.5%.These nanoparticles could be of major interest,by offering a long term action,and resolving most of the issues found in the use of siRNA in therapy.

L'hépatite C chronique est une des étiologies principales du carcinome hépatocellulaire. En revanche, les mécanismes de tumorigenèse sont peu connus. Récemment, plusieurs modifications du métabolisme du glucose ont été décrites dans les cellules infectées par le HCV. Celles-ci évoquent les reprogrammations métaboliques mises en place dans les cellules cancéreuses. L'effet Warburg, ou glycolyse aérobie, est une des caractéristiques principales des cellules tumorales. Ce phénomène permet d'assurer une production énergétique ainsi qu'un stock en précurseurs de macromolécules suffisants pour permettre la prolifération. Par ailleurs, en complément de l'utilisation de glucose, les cellules tumorales deviennent dépendantes de la métabolisation de la glutamine pour alimenter leur métabolisme énergétique et les différentes voies de biosynthèse. Mes travaux de thèse ont porté sur l'étude des changements métaboliques caractéristiques des cellules cancéreuses, et plus précisément sur le métabolisme de la glutamine, dans les cellules infectées par le HCV. Dans le modèle de culture cellulaire HCVcc, une activation de l'utilisation de la glutamine par le virus a pu être mise en évidence. L'infection par HCV entraine une augmentation du facteur de transcription MYC, de plusieurs transporteurs de glutamine ainsi que de la glutaminase, l'enzyme limitante de la glutaminolyse. De façon intéressante, ces changements semblent survenir également chez les personnes chroniquement infectés par le virus, comme le suggère l'analyse des biopsies de patients. Ces altérations métaboliques pourraient participer à la mise en place d'un environnement favorable au développement tumoral / Chronic infection with hepatitis C virus (HCV) is one of the main etiologies of hepatocellular carcinoma (HCC). However, mechanisms of HCV-related tumorigenesis are ill-defined. Recent literature data suggest that HCV infection may reprogram glucose metabolism in a cancerlike fashion. The Warburg effect, or aerobic glycolysis, is a hallmark of cancer. Activation of this pathway allows tumor cells to sustain high rates of energy production and provide sufficient biosynthetic precursors for proliferation. Likewise, the induction of similar metabolic alterations may favor HCV multiplication through the rapid production of nucleotides, amino acids and lipids. To complement aerobic glycolysis, tumor cells become frequently dependent on glutamine. The partial oxidation of glutamine through the glutaminolytic pathway can fuel their energy metabolism and several anabolic pathways. However, the role of glutamine metabolism in HCV life cycle has not been documented so far. I focused my PhD research project on the characterization of metabolic alterations triggered by HCV. In particular, I evaluated the occurrence of distinctive features of tumor cell metabolism in HCVinfected cells, with a specific attention on glutamine utilization. In the HCVcc cell culture model, I report the induction of a metabolic reprogramming towards higher rates of glutaminolysis upon HCV infection. HCV-induced transcriptional activation of MYC, along with several glutamine transporters and glutaminase, is likely to be responsible for this metabolic shift. Interestingly, increases in transcript levels of these factors in liver biopsies of patients with chronic hepatitis C suggest that this metabolic reprogramming may be relevant in vivo. Moreover, these metabolic changes may expose new drug targets against HCV as suggested by the inhibition of the virus replication upon suppression of glutaminolysis via different strategies. Altogether, these findings uncover a potential link between chronic hepatitis C and HCC through the installation of a favorable metabolic environment for tumor development

Les septines sont une famille de protéines GTPases qui peuvent former des structures d'ordre supérieur, comme les filaments et les anneaux, et capables de se lier avec les membranes cellulaires par leur interaction avec les phosphoinositides (PIs) via un domaine polybasique en N-terminal de leur domaine de liaison au GTP. Nous avons montré par une analyse transcriptomique réalisée en utilisant les données GSE14323 que la septine 9 est significativement surexprimée dans la cirrhose induite par le virus de l'hepatite C (VHC). Nos résultats montrent, ainsi, que la septine 9 induit l’augmentation en taille des gouttelettes lipidiques (GLs) par un mécanisme dépendant le phosphatidylinositol-5-phosphate et des microtubules. Nous avons montré, également, que cette voie de régulation des GLs est exploité par le VHC. De plus, nous avons montré que la septine 9 est impliquée dans la régulation de la morphologie de l’appareil Golgi et la mise en place de la polarité cellulaire par son interaction avec les phosphoinositides via deux domaines polybasiques. Ces résultats apportent une nouvelle compréhension du mécanisme moléculaire de l’interaction des septines avec les phosphoinositides et montrent pour la première fois l’importance de cette interaction dans des fonctions cellulaires de la septine 9. / Septins are a GTPases proteins family that can form high order structures such as filaments and rings, and able to bind cell membranes by interacting with phosphoinositides via a polybasic domain located at the N-terminal of their GTP binding domain. Here, We show by the transcriptomic analysis performed using the GSE14323 dataset that septin 9 is significantly upregulated in hepatitis C virus induced cirrhosis. Our findings show that septin 9 induce the lipid droplet growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV. In addition, we have shown that the septin 9 is involved in Golgi apparatus morphology regulation and cell polarity installation by interacting with phosphoinositides via two polybasic domains. These results provide new understanding of the molecular mechanism of septins interaction with the phosphoinositides and show its importance in septin 9 cellular functions shown for the first time.

Soňa Fraňková: Role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C Abstract Hepatitis C virus infection represents a leading cause of liver disease in western countries. The primary goal of HCV therapy is elimination of the virus, i.e. sustained virological response (SVR) achievement. Genetic factors have long been suspected of playing a crucial role in determining response to IFN-α-based therapies, but pretreatment predictors of response were only poorly defined and did not allow personalization of therapy. The aim of the thesis is to describe the role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C in specific groups of patients. First, we focused on the role of the IFNG -764G/C promoter variant in SVR achievement. We did not prove that this variant predicted SVR in Czech HCV-infected individuals. Next, we focused on the role of IL28B and IFNL4 in HCV-infected patients: we confirmed that the IL28B rs12979860 CC genotype slows down the progression of liver fibrosis in chronic HCV infection and that IFNL4 ss469415590 TT|ΔG genotyping does not bring a better prediction of treatment success than IL28B rs12979860 in the Czech population. Third, we assessed prediction of treatment response in HCV positive liver...

Unravelling the regulators of translation and replication of Hepatitis C virus Hepatitis C virus (HCV) is a positive sense, single stranded RNA virus belonging to the genus Hepacivirus and the family Flaviviridae. It infects human liver cells predominantly. Although, the treatment with α interferon and ribavirin can control HCV in some cases, they fail to achieve sustained virological response in others, thus emphasizing the need of novel therapeutic targets. The viral genome is 9.6 kb long consisting of a 5’ untranslated region (5’UTR), a long open reading frame (ORF) that encodes the viral proteins and the 3’ untranslated region (3’UTR). The 5’UTR contains a cis acting element, the internal ribosome entry site (IRES) that mediates the internal initiation of translation. The HCV 5’UTR is highly structured and consists of four major stem-loops (SL) and a pseudoknot structure. HCV proteins are synthesized by the IRES mediated translation of the viral RNA, which is the initial obligatory step after infection. The viral proteins are synthesized in the form of a long continuous chain of proteins, the polyprotein, which is then processed by the host cell and the viral proteases. Once viral proteins are synthesized sufficiently, the viral RNA is replicated. However the mechanism of switch from translation to viral RNA replication is not well understood. Several host proteins as well as the viral proteins help in the completion of various steps in the HCV life cycle. In this thesis, the role of two such factors in HCV RNA translation and replication has been characterized and exploited to develop anti-HCV peptides. The HCV proteins are categorized into two major classes based on the functions broadly: the non structural and the structural proteins. HCV NS3 protein (one of the viral non structural proteins) plays a central role in viral polyprotein processing and RNA replication. In the first part of the thesis, it has been demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding owing to the participation of the catalytic triad residue (Ser 139) in this RNA protein interaction. More importantly, NS3pro binding to the SLIV region hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, thus resulting in the inhibition of HCV-IRES activity. Moreover excess La protein could rescue the inhibition caused by the NS3 protease. Additionally it was observed that the NS3 protease and human La protein could out-compete each other for binding to the HCV SL IV region indicating that these two proteins share the binding region near the initiator AUG which was further confirmed using RNase T1 foot printing assay. Although an over expression of NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation in the cells, replication of HCV RNA was enhanced significantly. These observations suggested that the NS3pro binding to HCV IRES reduces translation in favour of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might contribute in the regulation of the molecular switch from translation to replication of HCV. In the second part the interaction of NS3 protease and HCV IRES has been elucidated in detail and the insights obtained were used to target HCV RNA function. Computational approach was used to predict the putative amino acid residues within the protease that might be involved in the interaction with the HCV IRES. Based on the predictions a 30-mer peptide (NS3proC-30) was designed from the RNA binding region. This peptide retained the RNA binding ability and also inhibited IRES mediated translation. The NS3proC-30 peptide was further shortened to 15-mer length (NS3proC-C15) and demonstrated ex vivo its ability to inhibit translation as well as replication. Additionally, its activity was tested in vivo in a mice model by encapsulating the peptide in Sendai virus based virosome followed by preferential delivery in mice liver. This virosome derived from Sendai virus F protein has terminal galactose moiety that interacts with the asialoglycoprotein receptor on the hepatocytes leading to membrane fusion and release of contents inside the cell. Results suggested that this peptide can be used as a potent anti-HCV agent. It has been shown earlier from our laboratory, that La protein interacts with HCVIRES near initiator AUG at GCAC motif by its central RNA recognition motif, the RRM2 (residues 112-184). A 24 mer peptide derived from this RRM2 of La (LaR2C) retained RNA binding ability and inhibited HCV RNA translation. NMR spectroscopy of the HCV-IRES bound peptide complex revealed putative contact points, mutations at which showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM2 structure. A 7-mer peptide (LaR2C-N7) comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred NOE (Nuclear Overhauser Effect) experiments suggested it to be a βturn. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition HCV-IRES function. More importantly, the peptide significantly inhibited replication of HCVRNA. Smaller forms of this peptide however failed to show significant inhibition of HCV RNA functions suggesting that the 7-mer peptide as the smallest but efficient anti-HCV peptide from the second RNA recognition motif of the human La protein. Further, combinations of the LaR2C-N7 and NS3proC-C15 peptide showed better inhibitory activity. Both the peptides were found to be interacting at similar regions of SLIV around the initiator AUG. The two approaches have the potential to block the HCV RNA-directed translation by targeting the host factor and a viral protein, and thus can be tried in combination as a multi drug approach to combat HCV infection. Taken together, the study reveals important insights about the complex regulation of the HCV RNA translation and replication by the host protein La and viral NS3 protein. The interaction of the NS3 protein with the SLIV of HCV IRES leads to dislodging of the human La protein to inhibit the translation in favour of the RNA replication. These two proteins thus act as the regulators of the translation and the replication of viral RNA. The peptides derived from these regulators in turn regulate the functions of these proteins and inhibit the HCV RNA functions.

Hepatitis C infection is a global disease that causes an estimated 399,000 deaths per year. Treatment has improved dramatically in recent years through the development of direct acting antivirals that target specific regions of the Hepatitis C virus (HCV). Unfortunately the virus can have a preexisting resistance or become resistant to these drugs by mutations in the genes that code for the target proteins. These mutations are called resistance-associated substitutions (RASs). Since RASs can cause treatment failure for patients, resistance detection is performed in clinical practice to select the ideal regimen. Currently RASs are detected by using Sanger sequencing and a partly manual workflow that can discriminate the presence of a RAS if it is present in 15-20% of viruses in a patients blood. A new method with the capacity to detect lower ratios of RASs in HCV sequences was developed, which utilizes Pacific Biosciences’ (PacBio’s) sequencing and a bioinformatics analysis software called CLAMP. To validate this new approach, 123 HCV patient samples were sequenced with both methods and then analyzed. The RASs detected with the new method were congruent to what was found with the Sanger-based workflow. The new approach was also shown to correctly genotype the virus samples, identify any co-existing mutations on the same sequences, and detect if there were any mixed genotype infections in the samples. The new procedure was found to be a valid replacement for the Sanger based workflow, with the possibility to perform additional analyses and perform automated and time efficient RAS detection.

A doença hepática crônica causada pelo vírus da hepatite C (HCV) tornou-se, nos últimos anos, uma das principais comorbidades dos pacientes portadores do vírus da imunodeficiência humana (HIV) nos países desenvolvidos. Os pacientes coinfectados com HIV/HCV apresentam uma progressão mais rápida para a cirrose e as suas complicações que os pacientes monoinfectados com HCV. Embora os mecanismos responsáveis por esta evolução não estejam totalmente esclarecidos, a expressão da molécula de HLA-G, um HLA de classe Ib não clássico, que tem propriedades bem reconhecidas na regulação negativa da resposta imune, pode estar relacionada à progressão da doença hepática. Os objetivos deste trabalho foram analisar o perfil de expressão de HLA-G em tecido hepático de pacientes coinfectados HIV/HCV e identificar possíveis variáveis do hospedeiro, do HCV e do HIV que possam estar relacionadas com a expressão de HLA-G na biópsia hepática. Para isso, 57 amostras de biópsia hepática de pacientes coinfectados com HIV/HCV, nas quais a imuno-histoquímica para HLA-G foi realizada, foram analisadas retrospectivamente quanto à expressão desta molécula no tecido hepático. Avaliaram-se também outras características histopatológicas da biópsia como grau de fibrose, atividade inflamatória, deposição de ferro e gordura. Determinou-se o polimorfismo de inserção ou deleção de 14 pares de bases da região 3` não traduzida do exon 8 do gene do HLA-G, que está relacionada com a produção de RNA-mensageiro, em 43 destes pacientes, além do polimorfismo de IL-28B, relacionado com a resposta ao tratamento do HCV, em 44 deles. Características bioquímicas e virológicas, tanto do HIV quanto do HCV também foram avaliadas. O genótipo 1 do HCV foi o mais prevalente (87,75%), especialmente o subgenótipo 1a (60%). A expressão do HLA-G foi observada em 38 (66,7%) amostras de fígado, e foi mais frequente em estágios moderados e severos de fibrose do que em estágios mais leves (94,1% x 55%, P < 0,01). Não houve relação entre a expressão do HLA-G e os outros parâmetros estudados. Embora a progressão para a cirrose no contexto da coinfecção por HIV/ HCV seja um processo complexo, modulado por muitos factores, a associação da intensidade de fibrose com a expressão do HLA-G pode indicar que a expressão desta proteína desempenha um importante papel nos mecanismos que contribuem para a progressão da doença, por meio da regulação negativa da resposta imune contra o HCV na coinfecção pelo HIV. / Chronic liver disease induced by hepatitis C virus (HCV) infection has recently become one of the most common comorbidities in patients who are infected with the human immunodeficiency virus (HIV) in developed countries. HIV/HCV coinfected patients show faster progression to cirrhosis and its complications than the HCV monoinfected patients. Even though the responsible mechanisms for this evolution have not been entirely clarified yet, the expression of the HLA-G molecule, a HLA from the non-classic Ib class, with well-known properties of negatively regulating the immune response, may be related to the liver disease progression. The aims of the present work were to analyze the HLA-G expression profile in the liver micro ambience of HIV/HCV coinfected patients and to identify possible host factors, HIV or HCV, that may be related to the HLA-G expression on the liver biopsy. For this purpose, 57 liver biopsies of HIV/HCV coinfect patients, in which immunohistochemistry for HLA-G had been performed, were retrospectively analyzed according the HLA-G expression on the hepatic tissue. Other histopathological features in the liver biopsies, such as fibrosis degree, inflammatory activity, iron deposition and fat were also evaluated. The polymorphism of insertion or deletion in 14-base pairs of the 3`non-translated region of exon 8 of the HLA-G gene, which is related to the production of HLA-G messenger RNA, was evaluated in 43 of the patients. Also, the polymorphism of IL-28B, related to the response to HCV treatment, was evaluated in 44 of them. Biochemical and virological features of HIV and HCV were also evaluated. The HCV genotype 1 was the most prevalent (87.75%), especially the subgenotype 1a (60%). The expression of HLA-G was observed in 38 (66.7%) samples of the liver biopsies, and it was most frequent in moderate and severe stages of fibrosis than in the mild stages (94.1% x 55%, P < 0.01). There was no established relationship between HLA-G and other parameters studied. Although the progression to cirrhosis in the context of HIV/HCV coinfection is a complex process modulated by many factors, the association of HLA-G expression with the intensity of the liver fibrosis may indicate the protein expression play an important role in the mechanisms that contribute to the progression of the disease, through the negative regulation of the immune response against HCV setting of a coinfection with HIV.