Introdução: O Vírus da Hepatite B (HBV) é um vírus de DNA com tropismo por hepatócitos, que apresenta um genoma circular parcialmente dupla fita. Baseado na divergência de sequência do genoma completo, dez linhagens evolutivas, denominadas “genótipos” de HBV, foram descritas (A-J), sendo F e H considerados como “indígenas” da América. Os genótipos de HBV apresentam uma forte estruturação geográfica, o que pode refletir padrões das migrações humanas. Na América do Sul, áreas de alto endemismo incluem a região amazônica, e as maiores taxas de infecção têm sido observadas em populações Nativas Americanas. Embora a forte estruturação geográfica seja indicativa de uma origem antiga, a maioria das análises visando datar a origem dos genótipos “americanos” F e H resulta em datações extremamente recentes que não condizem com eventos históricos relacionados ao HBV. Objetivo: Os objetivos desse trabalho compreendem avaliar o impacto de diferentes taxas evolutivas e da seleção purificadora sobre as estimativas de datação molecular a fim de inferir quais taxas são mais condizentes com a origem do HBV na América; caracterizar o HBV circulante em uma amostra histórica de Nativos Americanos, e discutir os processos históricos que possam ser relevantes para entender os padrões observados. Material e Métodos: Nós realizamos análise Bayesiana utilizando sequências disponíveis dos genótipos F e H e diferentes taxas evolutivas previamente reportadas, e comparamos a ocorrência de mutações sinônimas e não-sinônimas em ramos da filogenia classificados como “antigos” ou “recentes” a fim de inferir a atuação da seleção purificadora ao longo do tempo. Para caracterização do HBV presente nas populações Nativas Americanas, detecção e amplificação do DNA viral foi obtida através de PCR seguido de sequenciamento e análise filogenética. Análise Bayesiana de Skyline Plot foi realizada para comparar a dinâmica populacional do subgenótipo A1 presente na amostra de Nativos Americanos e em outras cepas isoladas no Brasil. Resultados e conclusão: Nossos resultados mostram que as estimativas de datação molecular são fortemente influenciadas pelas taxas evolutivas utilizadas na análise. Além disso, foi observado excesso de mutações não-sinônimas nos ramos recentes da filogenia, o que é compatível com a ocorrência de seleção purificadora, e pode gerar um viés sobre as estimativas, produzindo datações recentes demais. Na amostra de Nativos Americanos, nós constatamos o predomínio do subgenótipo A1, relacionado com populações africanas. Análise de Skyline Plot mostrou que a expansão populacional nas cepas isoladas de Nativos Americanos é mais recente que aquela inferida para outras cepas brasileiras, sugerindo que os processos históricos que contribuíram para a formação do subgenótipo A1 dos Nativos Americanos são relacionados com ondas migratórias mais recentes em direção à região amazônica. / Introduction: Hepatitis B virus (HBV) is a hepatotropic DNA virus that presents a partially double-stranded circular genome. Based on sequence divergence of the complete genome, ten HBV evolutionary lineages, called “genotypes” have been described (A-J), with F and H being considered as indigenous from the Americas. HBV genotypes present a remarkable geographic structure which may reflect historic patterns of human migrations. In South America, areas of high endemism include the Amazon basin, and high prevalence rates have been observed in Native American populations. Although the strong geographical structure indicates an ancient origin, most analysis trying to date the origin of the “American” genotypes F and H result in extremely recent dates that disagree with historical events related with HBV. Objective: The aims of this study comprise evaluate the impact of different evolutionary rates and of the purifying selection on molecular dating estimates in order to infer which rates are in better agreement with the origin of HBV in the Americas; to characterize the HBV circulating in a historical sample of Native Americans, and discussing the historical processes that might be relevant to understand the observed patterns. Materials and Methods: We performed a Bayesian analysis using the available sequences of F and H genotypes and different evolutionary rates previously reported, and compared the occurrence of synonymous and non-synonymous mutations in branches of phylogeny classified as “old” or “young” in order to infer the effects of purifying selection over time. For the characterization of HBV from Native American populations, detection and amplification of viral DNA were obtained through PCR followed by sequencing and phylogenetic analysis. Bayesian Skyline Plot analysis was performed to compare the population dynamics of the A1 subgenotype present in the sample of Native American and in other strains isolated from Brazil. Results and Conclusion: Our results show that molecular dating estimates are strongly influenced by the evolutionary rate assumed in the analysis. In addition, we observed an excess of non-synonymous mutations in recent branches of phylogeny, which is compatible with the occurrence of purifying selection and may create a bias on the estimates, producing too recent datings. In the sample of Native Americans, we observed a predominance of the A1 subgenotype, related with African populations. Skyline Plot analysis showed that population expansion in strains isolated from Native Americans is more recent than that inferred from other Brazilian strains, suggesting that the historical processes that contributed to the presence of A1 subgenotype A1 Native Americans are related with more recent migratory waves towards the Amazon region.

Orientadores: Sophie Françoise Mauricette Derchain, Kazue Panetta, Venancio Avancini Ferreira Alves / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-22T01:25:24Z (GMT). No. of bitstreams: 1 Martins_CeciliaMariaRoteli_M.pdf: 3183591 bytes, checksum: 579334ce540fe96c88e403f90c7612a2 (MD5) Previous issue date: 1996 / Resumo: Os objetivos deste estudo foram avaliar a prevalência de NIC 2 e 3 em mulheres sexualmente ativas, com alterações compatíveis com infecção por HPV na colpocitologia e na colposcopia, verificando a distribuição das mulheres segundo o diagnóstico histopatológico de cervicite, NIC 1 e NIC 2 e 3, com relação a cor, escolaridade, estado conjugal, número de parceiros, idade ao início da atividade sexual, uso de anticoncepcional hormonal, paridade e idade. Investigou-se, também, a associação entre o hábito de fumar, as infecções vaginais, o pH vaginal, o tipo DNA viral diagnosticado pela captura de híbridos e os resultados da colpocitologia, com as lesões cervicais determinadas pela biópsia. É um estudo observacional de corte transversal, realizado com 100 mulheres de 20 a 35 anos de idade, atendidas no Serviço de Patologia Cervical do Hospital Maternidade "Leonor Mendes de Barros" e do "Centro de Atenção Integral à Saúde da Mulher". Para análise bivariada e múltipla utilizaram-se os métodos de Qui-quadrado, o teste exato de Fisher e a regressão logística, com limite de confiança de 95%. A prevalência de NIC 2 e 3 foi de 23%. Os grupos de cervicite, NIC 1 e NIC 2 e 3 foram semelhantes em relação a cor, escolaridade, estado conjugal, número de parceiros, idade ao início da atividade sexual, uso de anticoncepcional hormonal e paridade. O aumento da idade e o hábito de fumar estiveram significativamente associados com a gravidade da lesão cery ica I. A prevalência de Gardnerella vaginalis foi significativamente maior nas pacientes com NIC 2 e 3. O Mobiluncus sp, Trichomonas vaginalis e a Chlamydia trachomatis apresentaram uma distribuição semelhantes nos três grupos. Entretanto, a Candida sp apresentou uma tendência a ser mais freqüente nas pacientes com cervicite e NIC 1. Houve uma associação significativa entre NIC 2 e 3 e a detecção de DNA-HPV de alto risco oncológico. Concluímos que, nesta amostra de mulheres com alterações compatíveis com infecção por HPV na colpocitologia e colposcopia, a idade acima de 25 anos, o I hábito de fumar, a presença de Gardnerella vaginalis e a presença de HPV de alto risco oncológico, estiveram altamente associados com NIC 2 e 3 / Abstract: The target of this study was to evaluate the NIC 2 and 3 prevalence in sexually active women, with colpocitology and colposcopy showing changes compatible with HPV infection. It was verified the distribution of women according toe histopathologic diagnostic of cervicite, NIC 1 and NIC 2 and 3, with respect to color, schooling, marital status, number of sexual partners, age of first sexual intercourse, oral contraceptive use, parity and age. Also, It was investigated the relationship between smoking habit, vaginal infections, vaginal pH, viral DNA type diagnostic by hybrid capture.. and the colpocitology results, with the cervical lesions showed by biopsy. This is a cross sectional observation study carried out on 100 women, between 20 and 35 years of age, attended in the cervical pathology department of "Hospital Leonor Mendes de Barrosl" and "Centro qe Atenção Jntegral à Saúde da Mulher - CAISM." The data analysis was done with Chi-Square, Fisher test and logistical regression, with 95% confidence interval. he NIC 2 and 3 prevalence was 23%. The cervicite, NIC 1, NIC 2 and 3 groups' were similar in respect to color, schooling, marital status, number of sexual partners, age of first sexual intercourse, oral contraceptive use and parity. However, the increase in age and the smoking habit was significantly associated with the severity of cervical lesion. The Gardnerella vaginalis prevalence was significantly higher in patients with NIC 2 and 3. The Mobiluncus sp, Trichomonas vaginalis and Chllamydia trachomatis showed similar distribution in the three groups. However, the Candida sp showed a tendency to be more frequent in patients with cervicite and NIC 1. The association between NIC 2 and 3 and the detection of high risk DNA-HPV was significant. We concluded that, in this group of 100 women with changes in colpocitology I and colposcopy compatible with HPV infection, the age above 25, the smoking I habit, the presence of Gardnerella vaginalis and the presence of HPV of high oncological risk, was strongly associated with NIC 2 and 3 / Mestrado / Tocoginecologia / Mestre em Medicina

Orientador: Marcos A. Machado / (Tese doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-22T21:50:22Z (GMT). No. of bitstreams: 1 Targon_MariaLuisaPenteadoNatividade_D.pdf: 6263544 bytes, checksum: fc6bcef3ec00b19dbb7a60841e7d7717 (MD5) Previous issue date: 1997 / Doutorado / Genetica Vegetal / Doutor em Ciências Biológicas

Orientador : Jose Vassallo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-07-29T01:36:43Z (GMT). No. of bitstreams: 1 Engelman_DianaEliciSader_D.pdf: 58580585 bytes, checksum: 71ae6bf64ac3e567e7b34853950e1f5a (MD5) Previous issue date: 2001 / Resumo: o carcinomada vulva é ainda hoje uma doençade etiologiaindefinida.Estudosrecentesapontam para uma origem multifatorial onde os papilomavírushumanos (HPV) seriam os agentes responsáveis pelo desenvolvimentoda neoplasia vulvar em grupos etários mais jovens, enquanto que nas pacientes mais idosas estaria provavelmente relacionada à presença de lesões distróficas, de mutações genéticas, além de outros fatores ainda pouco conhecidos. Tivemos como objetivo avaliar a presença do HPV e da proteína p53 e correlaciona-Ias com outros parâmetros clínico-patológicos nos grupos de pacientes com neoplasia intraepitelial vulvar (NN m - 38 casos), carcinoma superficialmente invasivo (nove casos) e carcinoma escamoso invasivo da vulva (55 casos). A detecção do HPV foi realizada através de exame imuno-histoquímico com o anticorpo anti-HPV policlonal e da hibridização molecular in situ com sondas biotiniladas de amplo espectro e para os tipos 6/11, 16/18 e 31/35/51. A proteína p53 foi identificada através de exame imuno-histoquímico com o anticorpo monoclonal DO-7. No grupo com NN m a idade média das pacientes foi de 47,5 anos; 45,7% tinham lesão de condiloma associado, o HPV estava presente em 57,9% dos casos e a proteína p53 em 21,1%. As pacientes com carcinoma superficialmente invasivo tinham idade média de 57,9 anos, lesão de condiloma em 22,2%, detecção do HPV em 33,3% e da proteína p53 em 66,7% dos casos. No carcinoma escamoso invasivo a idade média foi de 67,8 anos e apenas 7,3% das pacientes tinham lesão de condiloma associado; lesões distróficas estavam presentes em 60% dos casos e NN m em 25,5%; o HPV foi identificado em 7,3% e a proteína p53 em 58,2%. A detecção do HPV foi mais freqüente no grupo com lesão de NN m e estava relacionada à idade mais jovem das pacientes. No grupo com carcinoma invasivo, a variante histológica mais freqüente foi a queratinizante usual e estava associada à presença de lesões distróficas e da proteína p53. Entretanto, uma pequena subpopulação de pacientes mais jovens continham a variante basalóide do carcinoma invasivo, freqüentemente associada à lesão de NN m e à infecção pelo HPV. Os nossos resultados são semelhantes aos da literatura, onde um grupo de lesões está relacionado com a infecção pelo HPV e outro não. O HPV 16/18 foi o tipo predominanteem todos os grupos de lesões. Índices mais baixos de detecção da p53 nas lesões de NN m e maiores e com proporções semelhantes no carcinoma superficialmente invasivo e invasivo, além da ausência de relação entre a presença da p53 e do HPV, indicam que a imunodetecção da p53 é secundária à invasão do estroma pela neoplasia e é independente da presença de infecção viral. A detecção da proteína p53 estava associada apenas com a profundidade de invasão tumoral e não mostrou relação com neoplasia recidivante ou metástases ganglionares e, portanto, não tem valor como marcador para lesões recidivantes ou metastáticas, pelo menos para o grupo de pacientes incluídas neste trabalho / Abstract: Vulvar squamous cell carcinoma still is a disease of unknown etiology. Recent studies have pointed out to a multifactorial origin, with HPV as the causal agent in the developrnentof vulvar neoplasia in younger age groups, whereas in older patients it would be possibly related to dystrophic lesions,genetic mutations, and other factors still unknown. Our purpose was to analyze the presence of HPV and p53 overexpression and correlate them with other clinical and pathological parameters in patients with vulvar intraepithelial neoplasia grade m (VIN m - 38 cases), superficially invasive carcinoma (nine cases), and invasive squamous cell carcinoma of the vulva (55 cases). HPV infection was determined by immunoperoxidase reaction with a policlonal antibody against HPV and by in situ hybridization with biotinilated probes for wide spectrum and HPV types 6/11, 16/18 and 31/35/51. P53 overexpression was detected by immunoperoxidasereaction with the monoclonal antibody DO-7. In the VIN m group, the mean age was 47.5 years; 45.7% had associated condylomatous lesions, HPV infection was detected in 57.9% ofthe cases, and p53 overexpression in 21.1%. The patients with superficially invasive carcinoma had mean age of 57.9 years, condylomatous lesions in 22.2%, HPV infection in 33.3%, and p53 overexpression in 66.7% ofthe cases. In the invasive squamous carcinoma group the mean age was 67.8 years; 7.3% ofthe patients had associated condylornatous lesions, 60% presented with dystrophic lesions and 25.5% with VIN m; HPV were identified in 7.3%, and p53 overexpression in 58.2% of the cases. Our findings revealed that HPV detection occurred mainly in the VIN m group and was related to younger patients. In the invasive squamous carcinoma group, the most common histological variant was the usual type of keratinizing squamous cell carcinoma, and it was associated to dystrophic lesions and p53 overexpression. However, a small subpopulation of younger patients contained the basaloid type of invasive carcinoma, and it was :&equentlyassociated to VIN m lesions and HPV infection. Our results are similar to other reports, where two separate entities of the disease exist, one associated with HPV infection, and other unrelated to it. HPV 16/18 was the predominant type in all the three groups of lesions. The weakness or absence of p53 overexpression in VIN m lesions, and its higher detection, with stronger positivity and similar distribution in superficially invasive and invasive carcinoma, together with the lack of relationship between p53 overexpression and HPV infection, indicates that p53 overexpression is related to neoplastic stromal invasion and it is independent of HPV infection. P53 detection was associated only to the depth of stromal invasion and unrelated to tumor recurrence or lymph node metastasis and therefore, it had no significance as a prognostic marker, at least in the group of patients included in this study / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas

Orientador: Alvaro Santos Costa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-15T00:06:19Z (GMT). No. of bitstreams: 1 Figueira_AntoniadosReis_D.pdf: 5723352 bytes, checksum: 05d1b0ff6cd856f8737ab8693ebb4e0c (MD5) Previous issue date: 1983 / Resumo: Foram detectados dois fortes inibidores da transmissão mecânica do vírus do mosaico do mamoeiro em uma triagem efetuada entre diferentes substâncias químicas e sucos de diferentes espécies de plantas. Foram eles o detergente comercial ODD, utilizado na limpeza doméstica, e o suco de Turnera ulmifolia L. Foi estudado o mecanismo de ação desses inibidores sobre a transmissão do vírus do mosaico do mamoeiro e do fumo, porém alguns outros testes realizados mostraram que eles foram capazes de inibir a transmissão mecânica de outros vírus como o do mosaico da alfafa, Y de Piracicaba e acro-necrose. A inibição pela aplicação previa do suco de T. ulmifolia em mamoeiro mostrou efeito positivo quando feita um dia antes, mas não 4 ou mais dias antes da inoculação mecânica. O ODD mostrou boa inibição quando aplicado 8 dias antes (76,6%) e mesmo aplicado 12 dias antes induziu inibição de 40%. Com o vírus do mosaico do fumo (TMV) o inibidor de T. ulmifolia exerceu algum efeito positivo aplicado até 8 dias antes da inoculação (35,9%); o ODD teve efeito inibidor razoável mesmo quando aplicado 12 dias antes (60,7%). Quando esses inibidores foram misturados com o inoculo ou aplicados imediatamente após a inoculação do TMV em plandas de fumo TNN, houve bom efeito inibidor. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Exploratory screening of a number of chemical substances and of plant juices from many species permitted the detection of two effective plant virus inhibitors; ODD, a comercial household detergent sold in Brasil, and the leaf juice of the Turneraceae, Turnera ulmifolia L. Both inhibitors were tried against tobacco mosaic and papaya ring spot viruses in the majority of tests, but they were also active against two potyviruses that infect bean and alfafa mosaic virus. Seedlings of papaya (Carica papaya L.), Turkish necrotic tobacco (Nicotiana tabacum L.), and bean (Phaseolus vulgaris L.) were used as test plants. The inhibitory present in the leaf juice of T.ulmifolia, as well as that in ODD, are inhibitors of virus infections and not of virus replication. Both were effective when applied prior or immediaately after inoculation, although ODD was still effective when treatment was 4 hr afterwards. These results were interpreted as indicating the leaf juice inhibitor was an impediment in the initial interaction virus-host cell, whereas that in the detergent could act likewise or after it had occurred. The virus inhibitory effect of ODD is attributed to its main constituint, sodium alkylbenzenesulfonate, a component of most commercial brands of detergents. ...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Doutor em Ciências Biológicas

Puesta a punto PCR Multiplex para el diagnóstico de 14 virus respiratorios

The role of the non-structural protein NS3 in the viral life cycle of African horsesickness virus (AHSV) is an active area of research. It has been shown to be involved in the lytic and nonlytic mechanisms of viral release. How the NS3 protein acts in the lytic release of viral progeny has not yet been clarified. When expressed in a baculovirus system in insect cells the protein causes cell lysis. This phenomenon could be related to the lytic activity of the NS3 protein that facilitates viral release. It has been proposed that the AHSV NS3 protein may function as a viroporin at the plasma membrane causing damage and in this way facilitates viral release. Several residues of unknown function had been identified previously that are highly conserved in the NS3 protein of AHSV and the cognate proteins of other closely related viruses. The nature and characteristics of these residues and the motifs, which they resemble, were predicted to be important in protein folding and protein-protein interaction. Thus, they may be required for the function of NS3 and possibly for the viroporin-like activity of NS3. This study set out to characterise the functions of these residues by mutational studies. Targets for mutation were selected and mutations were designed that involved either the hydrophobic domain 1 (HD1), hydrophobic domain 2 (HD2) or the intervening spacer region (ISR). Eight mutant constructs were produced during the course of this project and three constructs, produced previously, were included in the various assays. These mutant versions of the NS3 proteins were expressed using the baculovirus expression system and assayed for membrane association, sucellular localisation, intracellular trafficking and their effects on cell viability. The mutations had various effects on membrane association of the proteins, as well as on their subcellular localisation, intracellular trafficking. There were intrinsic differences in the reponses of the AHSV-2 NS3 and AHSV-3 NS3 proteins to the introduced mutations. The tendency of the AHSV-2 NS3 mutant proteins to retain their nuclear localisation may be due to the presence of a nuclear localisation signal which is not present in AHSV-3 NS3. The intrinsic characteristics of the proteins can influence possible responses of proteins to modifications, thus generalised conclusions relating to mutagenesis could not be made. The modifications had varying effects on the cytotoxic effect on insect cells. Mutation of the HD domains seemed to interfere with plasma membrane trafficking, but this did not always correlate with a loss of cytotoxic ability. There were however intranuclear bodies observed, of which the functions still remain unclear. When the formation of the intranuclear bodies were lost there was a significant reduction in cell death. This may indicate that the intranuclear bodies have a role in AHSV NS3 induced cell death. / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

No description available.

Oncolytic viruses (OVs) have begun to show their promise in the clinical setting, however these results have been predominantly associated with loco-regional administration of virus. The treatment of metastatic disease necessitates a systemic approach to virus delivery. The circulatory system, though, is a hostile environment for viruses and the advantages associated with intravenous (IV) delivery come at a heavy cost that must be understood and brokered. Pre-existing immunity, specifically through the function of antibody and complement, poses a significant hurdle to the IV delivery of infectious virus to dispersed tumor beds. This is of particular importance for therapeutic vaccinia viruses as a majority of today’s cancer patients were vaccinated during the smallpox eradication campaign. In vitro neutralization assays of oncolytic vaccinia virus demonstrated that the antibodies elicited from smallpox vaccination, and also the anamnestic response in patients undergoing Pexa-Vec treatment, was minimally neutralizing in the absence of functional complement. Accordingly, in a Fischer rat model, complement depletion stabilized virus in the blood of pre-immunized hosts and correlated with improved delivery to mammary adenocarcinoma tumors. Complement depletion additionally enhanced infection of tumors following direct intratumoral injection of virus. The feasibility and safety of using a complement inhibitor, CP40, was tested in a cynomolgus macaque model. Immune animals saw an average 10-fold increase in infectious virus titer at an early point after the infusion, and a prolongation of the time during which infectious virus was still detectable in the blood. We have also demonstrated that vaccinia virus engages in promiscuous interactions with cells in the blood and that these interactions may be partially complement-dependent. Additionally, we have translated this complement inhibition approach to other OV candidates and found that reovirus, measles virus and a virus pseudo typed with the LCMV glycoprotein all elicit antibodies, that to some degree, are dependent on complement activation to neutralize their target viruses. We show here that capitalizing on the complement dependence of anti-viral antibody with adjunct complement inhibitors may increase the effective dose to enable successful delivery of multiple rounds of OV in immune hosts.

In systems biology and genomics, epistasis characters the impact that a substitution at a particular location in a genome can have on a substitution at another location. This phenomenon is often implicated in the evolution of drug resistance or to explain why particular ‘disease-causing’ mutations do not have the same outcome in all in- dividuals. Hence, uncovering these mutations and their locations in a genome is a central question in biology. However, epistasis is notoriously difficult to uncover, es- pecially in fast-evolving organisms. Here, we present a novel statistical approach that takes inspiration from a model developed in ecology and that we adapt to analyze genetic data in a typically fast-evolving system: the influenza A virus. We validate the approach using experimentally-validated data: known interactions are recovered. We further evaluate the ability of our approach to detect epistasis during antigenic shifts or at the emergence of drug resistance. We show that in all cases, epistasis is prevalent in influenza A viruses, involving many pairs of sites linked together in chains, a hallmark of historical contingency. Strikingly, interacting sites are sepa- rated by large physical distances, which entail either long-range structural effects or functional tradeoffs, for which we find support with the emergence of drug resistance. Our work paves a new way for the unbiased detection of epistasis in a wide range of organisms by performing whole-genome scans.