Estima-se que aproximadamente 400 milhões de indivíduos sejam portadores crônicos do vírus da hepatite B no mundo. Quando incide em adultos, a doença apresenta elevada proporção de evolução para a cura, ao passo que na ocorrência de transmissão vertical, o risco de cronificação chega a 90%, aumentando muito a possibilidade de graves conseqüências para a criança, entre as quais cirrose e hepatocarcinoma primário. A possibilidade de triagem para a identificação das gestantes portadoras do vírus da hepatite B e a conseqüente adoção de medidas profiláticas – imunização ativa e passiva – permite a prevenção segura da transmissão vertical. Em 1999, o Programa do Pré-Natal da Secretaria Municipal da Saúde de Ribeirão Preto introduziu em sua rotina o screening para o HBsAg, com a finalidade de identificar as gestantes portadoras do vírus. Este trabalho objetiva estudar alguns aspectos referentes à hepatite B entre as gestantes atendidas pela Rede Municipal de Saúde, bem como realizar uma avaliação sistematizada do Programa do Pré-Natal. Para este trabalho foram criados dois grupos de gestantes. O primeiro formado pelas pacientes com primeira avaliação sorológica do Pré-Natal para aquela gestação, realizada no período de 01 de novembro de 2001 a 31 de outubro de 2002, com a finalidade de estimar a prevalência do HBsAg e também verificar a proporção de recém-nascidos, filhos de mães portadoras deste marcador, que receberam os cuidados preconizados para tal situação. Já o segundo grupo foi formado pelas pacientes, na mesma condição, avaliadas no período de 01 de julho de 2002 a 30 de junho de 2003, com a finalidade de também estimar a prevalência do HBsAg, bem como a proporção de portadoras do HbeAg e anti-Hbe. Os valores de prevalência do HBsAg encontrados foram os seguintes: para o primeiro grupo 0,5 (IC 95% : 0,3 – 0,7) e, 0,4 (IC 95%: 0,2 – 0,6) para o segundo. Verificou-se que em 25,0% dos 24 partos realizados no município não foram realizados os procedimentos de profilaxia preconizados como ideais, no que diz respeito à rapidez da solicitação de vacina e imunoglobulina. Isso foi devido, parcialmente, a deficiências na qualidade de registro das informações em diferentes instâncias. As proporções de portadoras do HBeAg e anti-HBe foram respectivamente 5,9% (IC 95%: 0 – 17,1) e 90,5% (IC 95%: 77,9 – 100). Este trabalho procura apresentar informações que sirvam de base para reflexões a respeito do fluxo de procedimentos do Programa do Pré-Natal, visando elevar sua efetividade e superar os obstáculos encontrados. / It is estimated that about 400 million persons are chronic carriers of the hepatitis B virus worldwide. When it occurs in adults, the disease shows high proportions of benign evolution, meanwhile in vertical transmission the risk of becoming a chronic carrier approaches 90%, elevating the possibilities of serious consequences to the child, including cirrhosis and primary hepatocellular carcinoma. The possibility of performing screening tests for the identification of pregnant women carriers of the hepatitis B virus and the consequent adoption of prophylactic measures – active and passive immunization – allow safe prevention of the vertical transmission. In 1999, the Pre-Natal Program of the Municipal Secretary of Health of Ribeirão Preto, SP, Brasil, included in its routine services the screening for the HBsAg, towards the identification of pregnant carriers of the virus. This study aims to look into some aspects referring to hepatitis B among the pregnants being attended by the Municipal Health Network, as well as perform a careful systematic evaluation of the Pre-Natal Program. In order to perform this study, two groups of pregnants were selected. The first one formed by those patients having had their first pre-natal serological evaluation done for that pregnancy during the period of November 1, 2001 until October 31, 2002, with the purpose of estimating the HBsAg prevalence and also to verify the proportion of the new-borns of HBsAg carriers that received the recommended care for that situation. The second group was formed by those patients on the same condition, evaluated during the period of July 1, 2002 until June 30, 2003, with the purpose of also estimating the HBsAg prevalence, as well as estimating the proportion of HBeAg and anti-HBe carriers. The HBsAg prevalences were the following: for the first group of pregnants 0,5 (CI 95% : 0,3 – 0,7) and 0,4 (CI 95%: 0,2 – 0,6) for the second one. It was verified that in 25,0% of the 24 births that happened in the city, the recommended care were not taken, when it comes to the prompt request of the specific vaccine and immunoglobulin. This fact was, partially, due to deficiency in the data files quality of the different institutions. The proportions of HBeAg and anti-HBe carriers found were, respectively, 5,9% (CI 95%: 0 – 17,1) and 90,5% (CI 95%: 77,9 – 100). This study intends to present data that can be the starting point towards reflections on the established procedures of the Pre-Natal Program, in order to increase its effectiveness and surpass the found obstacles.

Os exames laboratoriais são fundamentais como ferramentas para elucidar o diagnóstico e avaliar prognóstico na prática médica. Novos exames são constantemente adicionados ao elenco já existente na clínica, o que pode resultar em elevação dos custos, nem sempre com o retorno esperado. A padronização da solicitação de exames tem sido estimulada, tanto na saúde pública quanto na saúde suplementar considerando os impactos clínicos, financeiros e ambientais de solicitações desnecessárias. O Hospital das Clínicas de Ribeirão Preto (HCRP) tem revisto protocolos e criado uma hierarquização para solicitação de testes complementares. Este estudo justifica-se por avaliar uma ação que visou adequar a solicitação de marcadores sorológicos de hepatite B, ajustando-os às normas preconizadas por resolução da Secretaria da Saúde do Estado de São Paulo. Objetivo: Avaliar a adequação das requisições médicas dos marcadores sorológicos para hepatite B enviadas ao laboratório de sorologia do HCRP e o impacto da triagem (\"reflective testing\"/\"reflex testing\") instituída pelo laboratório. Métodos: Estudo retrospectivo, de caráter descritivo, com dados coletados através de 24.649 solicitações médicas de marcadores de hepatite B recebidas em dois períodos: Fase 1 - sete meses entre 2012 e 2013; Fase 2 - nove meses entre 2014 e 2015. Avaliamos os marcadores solicitados e a hipótese diagnóstica informada pelo médico. Foram consideradas adequadas as solicitações de rastreio para HBV com os marcadores HBsAg e Anti-HBcAg Ig-total isolados ou em conjunto. A rotina de adequação incluiu a exclusão de marcadores inadequados ou a complementação dos marcadores sorológicos indicados, sempre que o HBsAg era positivo, independente da solicitação médica. Uma análise de custos foi realizada, considerando os exames solicitados e aqueles que foram efetivamente realizados. Resultados: Fase 1 - das 11.167 solicitações avaliadas, 1.143 (10,23%) foram consideradas inadequadas. Fase 2 - das 13.482 solicitações analisadas, 251 (1,86%) foram consideradas inadequadas. Com o processo de triagem realizado pelo laboratório foi possível economizar respectivamente R$ 19.617,25 e R$ 1.683,80 nos dois períodos analisados. Estas informações servirão de base para estruturação de uma proposta de orientação para solicitação dos marcadores sorológicos da hepatite B, junto às equipes onde identificamos maior frequencia de inadequação. Conclusão: Os processos de triagem manual (\"reflective testing\") e eletrônico (\"reflex testing\") realizados pelo laboratório demonstraram ser custo-efetivos e aceleram a liberação de resultados relevantes para a tomada de decisão clínica / Laboratory tests are critical as tools to elucidate the diagnosis and assess prognosis in medical practice. New tests are constantly added to the list already exists in the clinic, which can result in higher costs, not always with the expected return. The standardization of test ordering has been stimulated both in public health and in health insurance considering the clinical, financial and environmental impacts of unnecessary requests. The Hospital of the Faculty of Medicine of Ribeirão Preto (HCRP) has revised protocols and created a hierarchy to request additional tests. This study is justified by assessing an action that aimed to suit the request of serological markers of hepatitis B, adjusting them to the standards established by resolution of the São Paulo State Health Department. Objective: To evaluate the adequacy of medical requests of serological markers for hepatitis B sent to the serology lab HCRP and the impact of screening (\"reflective testing\" / \"reflex testing\") established by the laboratory. Methods: Retrospective study of a descriptive nature, with data collected through 24,649 medical claims of hepatitis B markers received in two periods: Phase 1 - seven months between 2012 and 2013; Phase 2 - nine months between 2014 and 2015. We evaluated the requested markers and the diagnosis reported by the physician. Tracing requests for HBV with HBsAg and anti-HBcAg alone or together were considered adequate. The adjustment routine includes the exclusion of inappropriate markers or serological markers of the indicated complementation, where the HBsAg was positive, whether for medical application. A cost analysis was performed, considering the requested examinations and those who have been realized. Results: Phase 1 - 11,167 of assessed requests, 1,143 (10.23%) were considered inadequate. Phase 2 - the 13,482 requests analyzed, 251 (1.86%) were considered inadequate. With the screening process carried out by the laboratory was possible to save respectively R $ 19,617.25 and R $ 1,683.80 in both periods analyzed. This information will form the basis for structuring a proposal to request guidance of serological markers of hepatitis B, with teams where we identify higher frequency of inadequacy. Conclusion: The manual sorting processes (\"reflective testing\") and electronic (\"reflex testing\") performed by the laboratory have shown to be cost-effective and accelerate the release of relevant results for clinical decision making

A imunização por via nasal pode representar uma alternativa as imunizações intramusculares, pois a aplicação por essa rota não é invasiva e há fácil acesso da vacina à mucosa. Além disso, a mucosa nasal apresenta diversas características que podem favorecer a imunização, por exemplo, há uma grande área superficial altamente vascularizado disponível para a absorção dos antígenos. Outra característica fundamental é a capacidade da mucosa de responder a antígenos, através das células imunocompetentes presentes, como as células M e dendríticas. Apesar disso, outros métodos podem ser empregados para melhorar absorção e disponibilidade dos antígenos pela mucosa, como a utilização de polímeros biodegradáveis. Entre estes, a quitosana é um biopolímero, derivado da desacetilação da quitina, que tem como principal característica, a possibilidade de estrutura-los em nanopartículas. Outra característica importante é sua propriedade catiônica a qual possibilita a sua ligação a proteínas e também à mucosa, que provoca maiores taxas de retenção de antígenos pela mucosa. Assim, neste trabalho, foi avaliado a imunogenicidade da inoculação do antígeno de superfície da hepatite B (HBsAg), via mucosa nasal em camundongos, os quais produziram anticorpos IgG contra HBsAg, apresentaram aumento da secreção de IgA pela mucosa nasal, e também ao avaliar a resposta de citocinas em células RAW 264.7, houve secreção de TNF-α . / The nasal vaccination is not invasive since its do not require needles for your application and your administration for is easy, thus the immunization via nasal route could be an alternative to intramuscular immunizations. Furthermore, the nasal mucosa has several characteristics like highly vascularized surface area available for absorption of antigen that could elicited the mucosal immune response caused by the competents cell available on the mucosal tissue. Nevertheless, other methods can be employed to improve absorption and availability of antigens to the mucosa, such as the use of nanoparticles of chitosan. Chitosan is a biopolymer product of deacetylation reaction of chitin, that has as main characteristic, the moldability, which enables the production of nanoparticles and its cationic property which allows its binding to proteins and also to the mucosa, which would lead to higher rates of absorption of antigens through the nasal mucosa. Thus, this work investigated the immunogenicity of the administration nanoparticles of chitosan with the surface anti-gen of hepatitis B (HBsAg) via the nasal mucosa in mice, which show levels of IgA in nasal lavages and serum IgG, as well as cytokines such as TNF-α released by RAW 264.7 cells of mice.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T19:01:30Z No. of bitstreams: 1 MarinildeSouza.pdf: 1783855 bytes, checksum: bc20000b025261af153ffc4f6418fad7 (MD5) / Made available in DSpace on 2017-06-14T19:01:30Z (GMT). No. of bitstreams: 1 MarinildeSouza.pdf: 1783855 bytes, checksum: bc20000b025261af153ffc4f6418fad7 (MD5) Previous issue date: 2016-05-20 / Introduction: The hepatitis B virus (HBV) present diversity of its genome, which is to be classified in different genotypes and subgenotypes (A-J). It has been demonstrated that different genotypes are related to the natural history of infection. The maintenance of viral replication could be one of the factors related to genotypes. Objectives: To identify the viral replication status of HBV carriers among the subgenótipos A1 and D4. Materials and methods: HBV carriers identified have been studied in two studies involving individuals from the state of Maranhão, northeast,Brazil, which had genotyping and subgenotypes, serology for HBeAg and anti-HBe and certain viral loads. Serological tests were performed by ELISA, HBV – DNA quantification real time PCR and genotyping performed by sequencing. Results: We identified 146 patients. Among these, 136 were subgenotype A1 or D4. It is 85 - A1 (62.5%) and 51 - D4 (37.5%). No difference was found between groups when age was evaluated (42 ± 12 vs 38 ± 17 p=0.11) or gender (male 48.5% vs 51.5% p=00.18). Among the D4 subgenotype carriers had more patients with HBeAg positive (23.5% vs 9.4%, p=0.02) and a higher proportion of patients with viral loads above 20.000 IU / ml (43.1% vs 12.9 % p <0.0001), even when only those with negative HBeAg (25.6% vs 6.5%, p=0.007) when compared with the A1 subgenotype. Conclusion: HBV carriers, subgenotype D4, compared to A1 subgenotype have delayed HBeAg seroconversion and higher levels of HBV – DNA, suggesting that this subgenotype is possibly related to / Introdução: O vírus da hepatite B (VHB) apresenta diversidade do seu genoma, o que o faz ser classificado em diferentes genótipos e subgenótipos (A-J). Tem sido demonstrado que os diversos genótipos estão relacionados com a história natural da infecção. A manutenção da replicação viral pode ser um dos fatores relacionado aos genótipos. Objetivos: Identificar o estado de replicação viral do VHB entre portadores dos subgenótipos A1 e D4. Materiais e métodos: Foram estudados portadores do VHB identificados em dois estudos que envolveram indivíduos provenientes do estado do Maranhão, nordeste do Brasil, que tinham determinação de genótipos e subgenótipos, sorologias para o HBeAg e anti-HBe e cargas virais determinadas. Sorologias foram realizadas por ELISA, VHB–DNA quantificado por PCR em tempo real e genotipagem realizada por sequenciamento. Resultados: Foram identificados 146 portadores. Dentre estes, 136 eram subgenótipo A1 ou D4. Sendo 85 - A1 (62,5%) e 51 - D4 (37,5%). Não houve diferença entre os grupos quando foi avaliado idade (42±12 vs 38±17 p=0,11) ou gênero (masculino 48,5% vs 51,5% p=0,18). Entre os portadores do subgenótipo D4 havia mais indivíduos com HBeAg positivo (23,5% vs 9,4%, p=0.02) e maior proporção de portadores de cargas virais acima de 20.000 UI/ml (43,1% vs 12,9% p<0,0001), mesmo quando avaliados apenas aqueles com HBeAg negativos (25,6% vs 6,5% p=0,007), quando comparados com os de subgenótipo A1. Conclusão: Portadores do VHB, subgenótipo D4, quando comparados com subgenótipo A1 apresentam soroconversão mais tardia do HBeAg e maiores níveis de VHB–DNA, sugerindo que esse subgenótipo possivelmente está relacionado com potencial para doença mais grave e maior facilidade de transmissão da infecção.

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-11-13T14:21:15Z No. of bitstreams: 1 Fábio Gomes Teixeira.pdf: 415617 bytes, checksum: 372db933a02a007f513b9a2d77a17362 (MD5) / Made available in DSpace on 2017-11-13T14:21:15Z (GMT). No. of bitstreams: 1 Fábio Gomes Teixeira.pdf: 415617 bytes, checksum: 372db933a02a007f513b9a2d77a17362 (MD5) Previous issue date: 2011-10-18 / Cohort study using retrospective data to evaluate the effectiveness of the treatment program for chronic hepatitis C in users of Farmácia Estadual de Medicamentos Excepcionais do Maranhão (FEME) to determine the rate of sustained virologic response (SVR) and correlate it with demographic, clinical, laboratory, histological and virological features of patients and to identify the rates of discontinuation of treatment. We analyzed data from 256 patients treated for chronic hepatitis C in FEME for the period January 2005 to July 2009, being an SVR by intention to treat 57%. Males predominated (66%). The mean age was 52.5 years, with a predominance of non-white individuals in relation to whites. Genotype 1 was the most common (77%) and 150 (58.6%) patients had viral load above 400,000 UI/ml. With respect to the treatment regimen, pegylated interferon associated with ribavirin was used by 80.5% of patients, the rate of discontinuation of treatment of 13,3%. They were identified as factors independently associated with SVR: white, non-cirrhotic, have not a genotype 1 and viral load below 400,000 IU / ml. These findings demonstrate the effectiveness of treatment provided by FEME, which makes it possible to cure most patients, preventing progression to end-stage liver disease and its disastrous consequences. The factors associated with SVR have seen in other studies, leads us to believe that the results are reliable and that the program is fulfilling the role it has set itself. / Estudo de coorte com dados retrospectivos com o objetivo de avaliar a efetividade do programa de tratamento da hepatite C crônica em usuários da Farmácia Estadual de Medicamentos Excepcionais do Maranhão (FEME), determinar a taxa de resposta virológica sustentada (RVS) e correlacioná-la com características demográficas, clínicas, laboratoriais, histológicas e virológicas dos pacientes, além de identificar as taxas de interrupção do tratamento. Foram analisados os dados de 256 pacientes, tratados para hepatite C crônica na FEME no período de Janeiro de 2005 a Julho de 2009, encontrando-se uma RVS por intenção de tratar de 57%. O sexo masculino foi predominante (66%). A média de idade encontrada foi de 52,5 anos, havendo predomínio de indivíduos não brancos em relação aos brancos. O genótipo 1 foi o mais comum (77%) e 150 (58,6%) pacientes apresentaram carga viral superior a 400.000 UI/ml. Com relação ao esquema de tratamento, o interferon peguilado associado ribavirina foi utilizado por 80,5% dos pacientes, sendo a taxa de interrupção do tratamento de 13,3%. Foram identificados como fatores independentemente associados à RVS : cor branca, não cirróticos, ter genótipo não 1 e carga viral abaixo de 400.000 UI/ml. Estes achados demonstram a efetividade do tratamento fornecido pela FEME, que possibilita a cura da maioria dos pacientes, prevenindo a progressão para doença hepática terminal e suas consequências desastrosas. A RVS associada a fatores já vistos em outros estudos, nos faz acreditar que os resultados são confiáveis e que o programa está cumprindo o papel a que se propôs.

Respondent-driven sampling (RDS) is very useful in collecting data from individuals in hidden populations, where a sampling frame does not exist. It starts with researchers choosing initial respondents from a group which may be involved in taboo or illegal activities, after which they recruit other peers who belong to the same group. Analysis results in unbiased estimates of population proportions though with strong assumptions about the underlying social network and RDS recruitment process. These assumptions bear little resemblance to reality, and thus compromise the estimation of any means, population proportions or variances inferred from studies. The topology of the contact network, denoted by the number of links each person has, provides insight into the processes of infectious disease spread. The overall objective of the thesis is to identify the topology of an injection drug use network, and critically review the methods developed to produce estimates. The topology of people who inject drugs (PWID) collected by RDS in Ottawa, 2006 was compared with a Poisson distribution, an exponential distribution, a power-law distribution, and a lognormal distribution. The contact distribution was then evaluated against a small-world network characterized by high clustering and low average distances between individuals. Last a systematic review of the methods used to produce RDS mean and variance estimates was conducted. The Poisson distribution, a type of random distribution, was not an appropriate fit for PWID network. However, the PWID network can be classified as a small world network organised with many connections and short distances between people. Prevention of transmission in such networks should be focussed on the most active people (clustered individuals and hubs) as intervention with any others is less effective. The systematic review contained 32 articles which included the development and evaluation of 12 RDS mean and 6 variance estimators. Overall, the majority of estimators perform roughly the same, with the exception of RDSIEGO which outperformed the 6 other RDS mean estimators. The Tree bootstrap variance estimate does not rely on modelling RDS as a first order Markov (FOM) process, which seems to be the main limitation of the other existing estimators. The lack of FOM as an assumption and the flexible application of this variance estimator to any RDS point estimate make the Tree bootstrapping estimator a more efficient choice.

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)

L'infection par le virus de l'hépatite B est un problème de santé publique avec 250 millions de porteurs chroniques et cela malgré l'existence d'un vaccin préventif. Les traitements actuellement utilisés sont les analogues de nucléos(t)ide et/ou l'interféron a. Bien qu'ils permettent une diminution de la charge virale, ils ne permettent pas d'éradiquer la maladie du fait de la persistance de l'ADNccc, le minichromosome de l'hépatite B. Cet ADN sert de matrice à la transcription virale, et la présence d'une seule copie permet la réactivation de l'infection. En prenant en compte la longue demi-vie des hépatocytes et de la stabilité de l'ADNccc dans leur noyau, un modèle mathématique suggère que de nombreuses années de traitement seraient nécessaires pour éliminer complètement cet ADN du foie des patients infectés chroniquement. Les techniques utilisées en routine pour la quantification de l'ADNccc ne sont pas assez sensibles pour pouvoir détecter des faibles concentrations de cet ADN, notamment dans des biopsies de patients infectés chroniquement et traités à long terme. Il est nécessaire de développer de nouvelles stratégies permettant de cibler directement l'ADNccc afin d'éliminer le virus. Ainsi les travaux de cette thèse reposent sur le développement d'une nouvelle technologie : la Droplet Digital PCR (ddPCR) pour permettre la quantification de l'ADNccc dans les biopsies de patient. Cette technique permet un gain de 2 log au niveau de la sensibilité par rapport à la qPCR, technique utilisée actuellement en routine. Elle nous a ainsi permis de constater la présence de cet ADN chez des patients traités à long terme par des analogues de nucléos(t)ides et même en présence d'interféron. La présence d'ARNpg et les expériences de ChIP ont également confirmé que l'ADNccc était encore transcriptionnellement actif. Ces résultats confirment d'autant plus la nécessité d'élaborer de nouvelles thérapeutiques pour permettre l'inactivation voire l'élimination de l'ADNccc. L'une des stratégies envisagées est le système CRISPR/Cas 9. Ainsi le dernier axe de cette thèse a été de développer ce système dans des modèles d'infection du virus de l'hépatite B. Pour vérifier l'efficacité de ce système sur le VHB, nous avons testé 8 ARN guide différents incorporer via des ribonucléoprotéines dans des cellules HepG2-NTCP. Les résultats préliminaires ont ainsi démontré que ce système pouvait réduire le pool d'ADNccc dans ces cellules et ouvre des perspectives intéressantes pour le développement de nouveaux traitements / Hepatitis B virus {HBV) is a major health problem with 250 million chronic carriers, despite the existence of a preventive vaccine. Currently the treatments used are nucleos{t)ide analogues and / or interferon a. Although they efficiently reach a decrease of the viral load, they do not allow the eradication the disease due to the persistence of the cccDNA, the minichromosome of the hepatitis B. This DNA serves as a template for the viral transcription and only a single copy suffice for the infection rebound. However, the techniques used routinely for the quantification of the cccDNA are not sensitive enough to be able to detect low concentrations of this DNA, in particular in biopsies of patients chronically infected and long term treated. ln addition, it is necessary to develop new strategies to target the cccDNA in order to clear the infection. Thus, my thesis work is based on the development of a new technology: the Droplet Digital PCR {ddPCR) to allow the quantification of cccDNA in patient biopsies. This technique allows a gain of 2 log in sensitivity compared to the qPCR technique currently used in routine. lt allowed us to see the presence of this DNA in long-term treated patients even in the presence of interferon. The presence of pgRNA and ChlP experiments also confirmed that the cccDNA was still transcriptionally active.These results confirm the requirement to develop new therapeutics to allow the inactivation or even the elimination of the cccDNA. One of the strategies envisaged is the CRlSPR / Case 9 system. Thus, the following part of this thesis was to develop this system in hepatitis B virus infection models. To reduce off-target effect we tested 8 different guide RNAs incorporated via ribonucleoproteins into HepG2- NTCP cells. Preliminary results have shown that this system can reduce the pool of cccDNA in these cells and open up the possibilities to test this model on PHH and opens interesting perspectives for the development of new treatments

A imunização por via nasal pode representar uma alternativa as imunizações intramusculares, pois a aplicação por essa rota não é invasiva e há fácil acesso da vacina à mucosa. Além disso, a mucosa nasal apresenta diversas características que podem favorecer a imunização, por exemplo, há uma grande área superficial altamente vascularizado disponível para a absorção dos antígenos. Outra característica fundamental é a capacidade da mucosa de responder a antígenos, através das células imunocompetentes presentes, como as células M e dendríticas. Apesar disso, outros métodos podem ser empregados para melhorar absorção e disponibilidade dos antígenos pela mucosa, como a utilização de polímeros biodegradáveis. Entre estes, a quitosana é um biopolímero, derivado da desacetilação da quitina, que tem como principal característica, a possibilidade de estrutura-los em nanopartículas. Outra característica importante é sua propriedade catiônica a qual possibilita a sua ligação a proteínas e também à mucosa, que provoca maiores taxas de retenção de antígenos pela mucosa. Assim, neste trabalho, foi avaliado a imunogenicidade da inoculação do antígeno de superfície da hepatite B (HBsAg), via mucosa nasal em camundongos, os quais produziram anticorpos IgG contra HBsAg, apresentaram aumento da secreção de IgA pela mucosa nasal, e também ao avaliar a resposta de citocinas em células RAW 264.7, houve secreção de TNF-&#945; . / The nasal vaccination is not invasive since its do not require needles for your application and your administration for is easy, thus the immunization via nasal route could be an alternative to intramuscular immunizations. Furthermore, the nasal mucosa has several characteristics like highly vascularized surface area available for absorption of antigen that could elicited the mucosal immune response caused by the competents cell available on the mucosal tissue. Nevertheless, other methods can be employed to improve absorption and availability of antigens to the mucosa, such as the use of nanoparticles of chitosan. Chitosan is a biopolymer product of deacetylation reaction of chitin, that has as main characteristic, the moldability, which enables the production of nanoparticles and its cationic property which allows its binding to proteins and also to the mucosa, which would lead to higher rates of absorption of antigens through the nasal mucosa. Thus, this work investigated the immunogenicity of the administration nanoparticles of chitosan with the surface anti-gen of hepatitis B (HBsAg) via the nasal mucosa in mice, which show levels of IgA in nasal lavages and serum IgG, as well as cytokines such as TNF-&#945; released by RAW 264.7 cells of mice.

Student Number : 9903144J - MSc (Med) dissertation - Faculty of Health Sciences / Since the introduction of vaccination against hepatitis B virus (HBV) infection in South Africa, at least one case of infection despite vaccination has occurred. The purpose of this study was to determine whether this infection was the result of mutations within the region of the surface (S) gene encoding the a determinant epitopes of the hepatitis B surface antigen, which permitted viral vaccine-escape. HBV DNA was extracted from the serum and liver tissue of the patient and amplified within the complete 3 215 bp genome and S gene, respectively. Following cloning, sequencing revealed a minor population displaying unique or uncommon S gene mutations that resulted in C138R, C139R, K141R, P142L, T143A, N146D, and T148A amino acid substitutions in the clones from the serum, and C139Y and D144N in the clones from the liver. Such isolates may represent South African HBV vaccine-escape mutants that caused chronic infection in the host prior to their reversion to wild-type.