MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p> In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p> Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p> This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.

Magistrinio darbo metu sukonstruotos mielių plazmidės, turinčios pelių poliomos viruso pagrindinį kapsidės baltymą VP1 su jame įterptais hepatito C viruso apvalkalo baltymų peptidais. Šiomis plazmidėmis buvo transformuotos S. cerevisiae mielės. Transformuotos mielės sintetina chimerinius baltymus: MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329]. Į virusus panašias daleles (VPD) renkasi tik MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329] baltymai. Kiti baltymai: MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329] nesirinko į VPD. / In the present study, plasmids for expression of major capsid proteins VP1 of murine polyomavirus with inserted sequences from Hepatitis C virus envelope proteins in yeast S. cerevisiae were constructed. The plasmids were used to transform yeast cells. The transformed yeast produced proteins: MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329]. Virus-like particles (VLPs) composed MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329]. Other proteins: MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329] did not form VLPs.

abstract: Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation. / Dissertation/Thesis / M.S. Biological Design 2011

Background Shelter employees of the Victoria Cool Aid Society (VCAS) work with clients living with or at risk of contracting, the Human Immunodeficiency Virus (HIV) and the Hepatitis C Virus (HCV). The purpose of this thesis is to assess whether the VCAS shelter staff need further HIV/AIDS and HCV education to support shelter clients. Methods A two-part (A and B) survey consisting of 70 questions asked 38 Emergency Support Workers to: A) rate their ability (expertise) to answer HIV/AIDS and HCV related questions, and B) identify which questions contain important knowledge to know for their work. Staff were recruited via Posters on bulletin boards around shelters sites as well as an email, and two follow up emails, informing staff about the survey. The survey explored the following subjects: 1) HIV/AIDS (12 questions), 2) Hep C (11 questions), 3) Health and Substance Use (3 questions), 4) Protocol (3 questions), and 5) Community Agencies (6 questions). From this format, it was possible to assess where staff felt their knowledge levels could use improvement (low and medium knowledge levels) and what topics they felt important to know for their work (high importance to know). These two parts of the survey, together, were then used to determine questions to include in a future training course i.e. questions were staff reported low or medium knowledge levels and high importance to know. Results Results for each of the five sections showing both lower levels of knowledge (expertise) and higher knowledge importance, were as follows: 1) HIV/AIDS: 8 out of 12 questions, 2) HCV: 10 out of 11 questions, 3) Health and Substance Use: 1 out of 3 questions, 4) Protocol: 3 out of 3 questions, and 5) Community Agencies: 3 out of 6 questions. Survey results were delivered via Power Point presentation to management of the Victoria Cool Aid Society using simple graphs and charts to describe easily the findings to stakeholders. The presentation emphasised that staff overall are in need of specific HIV/AIDS and HCV education. Conclusion Emergency shelter workers are in need of HIV/AIDS and HCV education. The results can inform a HIV/AIDS and HCV educational course for VCAS shelter staff. / Graduate

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-06-10T18:28:37Z No. of bitstreams: 1 Dissertação_ICS_Iury Souza.pdf: 1047267 bytes, checksum: 06bc8acdb629b7e8ae63feab201995ad (MD5) / Made available in DSpace on 2013-06-10T18:28:37Z (GMT). No. of bitstreams: 1 Dissertação_ICS_Iury Souza.pdf: 1047267 bytes, checksum: 06bc8acdb629b7e8ae63feab201995ad (MD5) / CAPES / Cerca de 33,3 milhões de pessoas apresentam infecção pelo Human Immunodeficiency Virus (HIV) no mundo; 180 milhões estão infectados pelo Hepatitis C Virus HCV e estima-se que 360 milhões apresentem infecção ativa pelo Hepatitis B Virus (HBV). Outra realidade mundial é a co-infecção entre esses vírus. Os dados mostram a importância global dessas viroses e a urgência do desenvolvimento de novos ensaios de diagnóstico sensíveis, específicos, rápidos e de baixo custo, que possam atender à demanda de entidades públicas inseridas em programas para prevenção e diagnostico dessas doenças. O presente trabalho consiste em validação relativa de um novo teste imunocromatográfico desenvolvido pela empresa canadense Medmira para detecção de anticorpos específicos contra HIV, HCV e HBV. Os resultados encontrados foram extremamente favoráveis para a detecção de anticorpos específicos para HIV, apresentando 98,6% de sensibilidade e 100% de especificidade. Para o anti-HBV a sensibilidade e especificidade encontradas foram de 90,0% e 98,6%, e de 86,3% e 100%, para anti-HCV, respectivamente. Nenhuma reatividade cruzada foi encontrada e a reprodutibilidade e repetitividade foram de 100%. O índice kappa e a acurácia global do teste foram de 0,91 (0,88-0,94) e 95,5% (93,5-97,5), respectivamente. Conclui-se que o ensaio imunocromatográfico é clinicamente útil em triagens rápidas para detecção de anticorpos anti-HIV, HCV e HBV. / Salvador

Objective: Evaluate The Ottawa Hospital Viral Hepatitis Program (TOHVHP) telemedicine (TM) program for patient retention, treatment initiation and sustained virologic response (SVR) rates. Methods: Retrospective analysis of TOHVHP cohort data for patients entering HCV care between 2012 and 2016. Logistic regression modeling was used to assess characteristics associated with patient retention, treatment initiation, and achieving SVR. TM outcomes were compared to the standard outpatient clinic and mixed delivery outcomes. Results: Treatment initiation rates were comparable between TM and the outpatient clinic. TM delivered Direct Acting Antiviral treatments achieved high SVR outcomes across all patient populations. Patient retention was lower among TM patients. Conclusion: TOHVHP TM program engaged patients facing barriers to traditional HCV care models. Efforts to improve TM retention are needed.

Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.

Orientador: Márjorie de Assis Golim / Resumo: Vírus da Hepatite C (VHC) é uma das principais causas de doença inflamatória crônica do fígado, com progressão variável para a fibrose e cirrose hepática. Cerca de 30 a 40% dos pacientes com hepatite C crônica tem manifestações extra-hepáticas, sendo uma variedade destas descritas como associadas ao VHC. Quando a produção da medula é incapaz de manter as necessidades do organismo, em reação secundária a doença mieloproliferativa (policitemia vera, leucemia granulocítica crônica), ou até mesmo de origem idiopática, pode ocorrer hematopoese extramedular (HEM), presente mais comumente no fígado, baço e gânglios linfáticos, os quais passam a exercer função hematopoiética. Com o objetivo de avaliar a existência de hematopoese extramedular hepática em pacientes com hepatite C crônica e sua influência na gênese da fibrose hepática, foram avaliados 69 pacientes VHC positivos, os quais foram submetidos à biópsia hepática percutânea, e estratificados em grupos conforme a classificação METAVIR, conforme descrição abaixo: G1 – n = 19: pacientes no estágio F1 (fibrose portal sem septos); G2 – n = 16: pacientes no estágio F2 (septos poucos); G3 – n = 17: pacientes em estágio F3 (septos numerosos sem cirrose); G4 – n = 17: pacientes em estágio F4 (cirrose); G5 - n = 15: indivíduos saudáveis (grupo controle). Foram realizadas quantificações plasmáticas de quimiocinas (CXCL8, CCL5, CXCL9, CCL2 e CXCL10) e fatores de crescimento (TGF-beta, VEGF, FGF, PDGF) e investigada a presença de HEM em co... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hepatitis C Virus (HCV) is a major cause of chronic liver inflammatory disease with variable progression for fibrosis and cirrhosis. About 30 to 40% of patients with chronic hepatitis C has extrahepatic manifestations and a variety of these described as an association with HCV. When the production of the bone marrow is not able to maintain the homeostasis due a secondary reaction to myeloproliferative disease (polycythemia vera, chronic granulocytic leukemia), or idiopathic causes, it may occur an extramedullary hematopoiesis (EMH), most commonly in the liver, spleen and lymph nodes, which can start the development of hematopoietic function. The purpose of this study is the evaluation of existence of liver extramedullary hematopoiesis in patients with chronic hepatitis C, and the influence in the pathogenesis of liver fibrosis. It was evaluated 69 liver biopsy from HCV positive patients and stratified into groups according to METAVIR rating as described below: G1 - n = 19: patients in the F1 stage (portal fibrosis without septa); G2 - n = 16: patients in stage F2 (few septa); G3 - n = 17: patients with stage F3 (numerous septa without cirrhosis); G4 - n = 17: patients in stage F4 (cirrhosis); G5 - n = 15: healthy individuals (control group). It was quantifed the chemokines (CXCL8, CCL5, CXCL9, CCL2 and CXCL10) and growth factors (TGF-, VEGF, FGF, PDGF) from plasma and investigated the presence of EMH in the liver tissue sections by immunohistochemistry use of CD61 / CD34... (Complete abstract click electronic access below) / Mestre

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-08-04T14:12:52Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação - Tássia Brena da Costa.pdf: 2477280 bytes, checksum: c34cd8503224fb8886f2d07b5e20b69a (MD5) / Made available in DSpace on 2017-08-04T14:12:52Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação - Tássia Brena da Costa.pdf: 2477280 bytes, checksum: c34cd8503224fb8886f2d07b5e20b69a (MD5) Previous issue date: 2016-03-29 / CNPQ / FACEPE / A metabonômica pode ser definida como um conjunto de ferramentas, analíticas e de estatística multivariada, utilizadas para identificar mudanças de concentração dos metabólitos em um dado biofluido, associando-as à perturbação sofrida pelo organismo. Sendo assim, ela seria capaz de identificar qualquer doença no organismo, desde que seja empregado o biofluido adequado e as informações sejam corretamente extraídas. Para isso, a ferramenta mais empregada é a Espectroscopia de Ressonância Magnética Nuclear de Hidrogênio-1 (RMN de ¹H), e é necessário o uso de técnicas quimiométricas para extrair as informações do espectro. Neste trabalho, foram construídos modelos metabonômicos para: (1) identificar pacientes portadores de esteatose, e dos vírus da hepatite B (HBV) e da hepatite C (HCV), utilizando amostras de urina; e (2) classificar o grau de fibrose hepática em pacientes com hepatites crônicas por HBV ou HCV, utilizando amostras de soro sanguíneo. O modelo para classificação de pacientes com esteatose, obteve 100% de sensibilidade e de valor preditivo positivo. Para identificar esteatose independentemente de ser um portador de HBV ou HCV, o modelo construído obteve 97,9% de exatidão. Para classificar portadores de HBV e HCV, os modelos apresentaram sensibilidade de 100% e 92,6%, respectivamente. O modelo construído para diferenciar pacientes com diferentes lesões no fígado: esteaose e hepatites virais B ou C, obteve 94% de exatidão. Para classificar pacientes com fibrose significativa; fibrose avançada; e cirrose, alcançamos 98,4; 100; e 96,8% de exatidão, respectivamente. Através da combinação dos resultados dos modelos de fibrose significativa e fibrose avançada, foi possível determinar os pacientes com grau F2, no METAVIR, com percentual de acerto de 96,8%. Nas análises de fibrose, a exatidão observada para os modelos metabonômicos foram superiores aos observados para os métodos não-invasivos normalmente utilizados na prática clínica, APRI (do inglês, Aspartate aminotransferase Platelet Ratio Index) e FIB-4. A estratégia metabonômica demonstrou capacidade de avaliar a presença de diferentes doenças hepáticas em uma única análise, não invasiva, e determinar o grau de fibrose hepática, de forma minimamente invasiva. / The metabonomics can be defined as a set of analytics and multivariate statistics tools, used to identify the metabolite concentration changes in a certain biofluid, associating them to the disturbance suffered. Therefore, it would be able to identify any disease in the body, if employed the appropriate biofluid and correctly extract the information. The most commonly used tool is Nuclear Magnetic Resonance Spectroscopy for hydrogen-1 (¹H NMR), and chemometrics techniques are used to extract the information of the spectrum. In this work we built metabonomics models to: (1) identify patients with steatosis, hepatitis B (HBV) and hepatitis C (HCV), using urine samples; and (2) classify the degree of liver fibrosis in patients with chronic hepatitis, HBV or HCV, using blood serum samples. The classification model for patients with steatosis obtained 100% to sensitivity and positive predictive value. To identify steatosis, without regard the presence of HBV or HCV, the constructed model achieved 97.9% accuracy. To classify carriers of HBV and HCV, the models showed 100 and 92.6% of sensitivity, respectively. The constructed model to differentiate patients with different liver damage: steatosis and viral hepatitis B or C, achieved 94% accuracy. To classify patients with significant fibrosis; advanced fibrosis; and cirrhosis, the models reached 98.4; 100; and 96.8% accuracy, respectively. By combining the results of significant fibrosis models and advanced fibrosis, it determined the patients with F2 in the METAVIR, with 96.8% of accuracy. In fibrosis analysis, the accuracy observed for metabonomics models were higher than those observed for the non-invasive methods commonly used in clinical practice, APRI (Aspartate aminotransferase Platelet Ratio Index) and FIB-4. The metabonomics strategy demonstrated ability to assess the presence of different liver diseases in a single non-invasive analysis and determine the degree of liver fibrosis, in a minimally invasive way.

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