Le virus de la grippe A est un virus à ARN négatif appartenant à la famille des Orthomyxoviriadea dont la réplication se produit dans le noyau des cellules infectées. L'organisation du génome est segmentée en huit segments d'ARNv de polarité négative, codant pour un minimum de 16 protéines virales différentes. Ces ARN viraux (ARNv) sont en complexe avec de nombreuses copies de nucléoprotéines et liés par leurs extrémités 5' et 3' au complexe hétérotrimérique de l'ARN-polymérase ARN-dépendante composé des sous unités PA, PB1 et PB2. Cet assemblage macromoléculaire (ARNv / polymérase / NP) nommée Ribonucléoprotéine (RNP) constitue une entité génomique indépendante. Dans le contexte de la RNP, l'ARN-polymérase assure à la fois la transcription et la réplication du génome ARNv. En assurant ces deux fonctions, l'ARN-polymérase joue un rôle majeur dans la réplication virale et constitue une cible antivirale privilégiée. Les travaux de recherche présentés dans cette thèse se concentrent sur les éléments structuraux participants à l'assemblage de l'ARN polymérase et son interaction avec les avec les ARNv. Pour atteindre ces objectifs, notre laboratoire, en collaboration avec d'autres groupes, a mis en place un système d'expression en polyprotéines permettant d'exprimer la polymérase. Plus encore, cette méthode a aussi permis de reconstituer des complexes entre l'ARN-polymérase et des partenaires cellulaires, notamment RanBP5 qui appartient à la famille des importines-β. / Influenza A virus is a negative-strand RNA virus belonging to the Orthomyxoviriadea family whose replication occurs in the nucleus of infected cells. The genome organisation of influenza virus is segmented in eight vRNA segments of negative polarity coding for at least 16 different viral proteins. Each vRNA is bound to multiple copies of nucleoprotein (NP) and to the heterotrimeric RNA-dependent RNA-polymerase complex (PA, PB1 and PB2) through its 5' and 3' extremities. This macromolecular assembly (vRNA/polymerase/NP) forms the ribonucleoprotein (RNP) particle, which acts as a separate genomic entity within the virion. The RNP complex is at the core of viral replication and in the context of RNPs, the polymerase performs both transcription and replication of the vRNA genome. As such, the polymerase constitutes a major antiviral drug target. The research work presented within this thesis focuses on the underlying determinants of the RNA polymerase assembly process and its interaction with its vRNA genome. To fulfill these goals, our lab, in collaboration with other groups, has set up a novel polyprotein expression system to express the polymerase but also to reconstitute polymerase and cellular partner complexes, notably RanBP5, which belongs to the importin-β family.

Orientador: Tereza Cristina Cardoso Silva / Banca: Roberto Gameiro de Carvalho / Banca: Andréa Fontes Garcia / Banca: Ana Carolina Borsanelli / Banca: Jamila Cristina Baptistella / Resumo: O herpes vírus bovino 5 (HVB-5) é um agente infeccioso pertencente a família Herpesviridae, sub-família Alphaherpesvirinae e gênero Varicellovirus. É um vírus neurotrópico que causa doenças neurológicas principalmente em animais jovens em todo mundo, especialmente nos países Sul-americanos, resultado em significantes perdas econômicas. A infecção pelo herpesvírus bovino 5 em gado jovemestá associado a doença neurológica que é, usualmente, fatal, sendo um ótimo modelo para estudar a patogênese da meningoencefalite induzida por vírus. O HVB5 replica na mucosa nasal, e invadesistema nervoso central (SNC), principalmente por meio do sistema olfatório. A resposta imune inata desencadeada pelo hospedeiro frente à replicação do vírus por meio da via olfativa não é totalmente entendida. Estudos verificaram as variações nos níveis de expressão de receptores Toll-Like (TLRs) em diferentes regiões do sistema nervoso central bovino durante a infecção aguda e reativação de bovinos infectados por HVB-1 e HVB-5-.Uma nova perspectiva para entender a relação do patógeno e o hospedeirorelacionando os microRNAs(miRNAs) que interagem com a resposta imune inatadurante infecções virais neurotrópicas. / Abstract: Bovine herpes virus 5 (BHV-5) is an infectious agent belonging to the family Herpesviridae, subfamily Alphaherpesvirinae and genus Varicellovirus. It is a neurotropic virus that causes neurological diseases mainly in young animals worldwide, especially in the South American countries, resulting in significant economic losses. Bovine herpesvirus 5 infection in young cattle is associated with neurological disease, which is usually fatal and is a good model for studying the pathogenesis of virus-induced meningoencephalitis. The BHV5 replicates in the nasal mucosa, and invades the central nervous system (CNS), mainly through the olfactory system. The innate immune response triggered by the host against virus replication via the olfactory route is not fully understood. Studies have found variations in the levels of Toll-Like receptor (TLR) expression in different regions of the bovine central nervous system during acute infection and reactivation of BoHV-1 and BoHV-5 infected bovines. A new perspective to understand the relationship of the pathogen and the host relating the microRNAs (miRNAs) that interact with the innate immune response during neurotropic viral infections. / Doutor

3C-like protease (3CLpro) of severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for autoprocessing of the polyproteins 1a and 1ab, and is a potential target for treating coronaviral infection. To obtain a thorough understanding of its substrate preference, we created a substrate library of 19 x 8 variants by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer (FRET). The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high beta-sheet propensity P4 prefers small hydrophobic residues: P2 prefers hydrophobic residues without beta-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. / Inhibition of SARS-CoV 3CLpro proteolytic activity suppresses virion replication and virus-induced cytopathic effects. Peptidomimetic inhibitors with nitrile warheads, which inhibit Cys protease activity, have been applied for clinical therapy. To investigate whether the nitrile group can target 3CLpro, a series of nitrile-based peptidomimetic inhibitors with various protective groups, peptide length and peptide sequences were synthesized. Inhibitor potency in terms of IC50 and Ki values was determined by FRET assay. Most of these nitrile-based inhibitors in micromolar range can significantly reduce 3CLpro activity. The most potent inhibitor is the tetrapeptidomimetie inhibitor linked with carbobenzyloxy (cbz) group 'cbz-AVLQ-CN' with IC50 and Ki values of 5.9 +/- 0.6 muM and 0.62 +/- 0.11 muM respectively. Crystal structures of 3CLpro-inhibitor complexes demonstrated that nitrite warhead covalently bonded to Cys145, while P1 -- P4 residues interacted with 3CLpro as substrate bound. The cbz group in 'cbz-AVLQ-CN' flipped into a cavity of Gu166 -- Pro168, providing an extra binding force to enhance inhibitor potency. In conclusion, the nitrile-based peptidomimetic inhibitor with cbz group is a convincing model for drug development. / Substrate specificities of various 3CLpro were further investigated by using the substrate library of SARS-CoV 3CLpro. Among various viral strains, the proteases of HCoV-NL63, HCoV-OC43 and infectious bronchitis virus (IBV) were selected from group I, IIa and III respectively for specificity profiling. Their proteolytic rates against 19 x 8 variants were obtained by FRET assay, and correlated with structural properties of substituting residues. Like SARS-CoV 3CLpro in group IIb, these 3CLpro consistently prefer small hydrophobic P4 residues, positively charged P3 residues, hydrophobic P2 residues without beta-branch, P1-Gln and small P1' residues. These proteases also tend to accommodate P5 and P3' residues with positive charge, and P2' residues with small size. In contrast, their preferences on secondary structure are diverse. Correlation was found between IBV 3Clpro activity and beta-sheet propensity at P5 position, while no strong correlation with secondary structure propensities was observed in HCoV-NL63 and HCoV-0C43. Collectively, all 3CLpro share universal preferences on charge, side chain volume and hydrophobicity, but not secondary structure. Their relative activities against universal and specific super-active substrates were elevated to 1.4 -- 4.3, showing synergetic effects by combining preferred residues. These substrates were examined by group I HCoV-229E and group IIa HCoV-HKU1 in parallel. Their activities were highly comparable to those of other group members. / Chuck, Chi Pang. / Adviser: Chi-Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [179]-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lam, Ka Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 77-82). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Content --- p.vii / List of Abbreviations and Symbols --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Severity of influenza --- p.1 / Chapter 1.2 --- Introduction of influenza viruses --- p.3 / Chapter 1.2.1 --- Virion and genome structure --- p.4 / Chapter 1.2.2 --- The replication cycle of influenza viruses --- p.5 / Chapter 1.3 --- Influenza virus NP --- p.8 / Chapter 1.3.1 --- The importance of NP in RNP structure maintenance --- p.9 / Chapter 1.3.2 --- NP self oligomerization --- p.10 / Chapter 1.3.3 --- NP-RNA interaction --- p.12 / Chapter 1.3.4 --- NP and other interacting partners --- p.13 / Chapter 1.4 --- Aim of the project --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Biological materials --- p.18 / Chapter 2.2 --- Construction of NP mutants --- p.19 / Chapter 2.3 --- Luciferase assay --- p.22 / Chapter 2.4 --- Western blot --- p.23 / Chapter 2.5 --- Protein expression and purification --- p.23 / Chapter 2.6 --- Circular dichroism spectroscopy --- p.24 / Chapter 2.7 --- Static Light scattering --- p.24 / Chapter 2.8 --- Surface plasmon resonance --- p.25 / Chapter 2.9 --- Co-immunoprecipitation (co-IP) --- p.26 / Chapter Chapter 3 --- Identification of residues crucial for NPB oligomerization and ribonucleoprotein activity / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Result --- p.31 / Chapter 3.2.1 --- NPB mutants showed deficiency in overall transcription and replication activity --- p.31 / Chapter 3.2.2 --- Expression and purification of NP mutants with low RNP activity --- p.37 / Chapter 3.2.2.1 --- Expression of MBP-tagged NP variants --- p.37 / Chapter 3.2.2.2 --- Purification of MBP-tagged NP variants --- p.38 / Chapter 3.2.3 --- Secondary structures of NP variants were comparable t o wild type NP --- p.41 / Chapter 3.2.4 --- NP variants with low RNP activity were abnormal in oligomerization in vitro --- p.42 / Chapter 3.2.5 --- NP variants with low RNP activity were impaired in homo-oligomer formation in vivo --- p.45 / Chapter 3.2.6 --- Discussion --- p.47 / Chapter Chapter 4 --- Identification of residues crucial for NP 一 RNA interaction and ribonucleoprotein activity / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Result --- p.58 / Chapter 4.2.1 --- NPB mutants showed deficiency in overall transcription and replication activity --- p.58 / Chapter 4.2.2 --- Expression and purification of NP variants with low RNP activity --- p.62 / Chapter 4.2.3 --- Secondary structures of NP variants were comparable t o wild type NP --- p.63 / Chapter 4.2.4 --- NP variants with low RNP activity were abnormal in RNA binding --- p.64 / Chapter 4.3 --- Discussion --- p.68 / Chapter Chapter 5 --- Conclusion and future prospect --- p.73 / Copyright --- p.76 / References --- p.77

Submitted by Suzana Dias (suzana.dias@famerp.br) on 2018-10-19T20:56:33Z No. of bitstreams: 1 JaquelineCalçaAssis_dissert.pdf: 1656909 bytes, checksum: 0f9e5425c7d3ffa51b86c71d5b4b9687 (MD5) / Made available in DSpace on 2018-10-19T20:56:33Z (GMT). No. of bitstreams: 1 JaquelineCalçaAssis_dissert.pdf: 1656909 bytes, checksum: 0f9e5425c7d3ffa51b86c71d5b4b9687 (MD5) Previous issue date: 2016-11-07 / The presence of anti-HBc alone can have several meanings: false positive, healing immune window, delayed immunity or occult hepatitis B virus infection (OBI). In clinical practice, it is important and necessary to clarify the diagnosis to prevent transmission to the risk population such as hemodialysis patients, blood donors, transplant recipients and co-infected individuals with HIV and/or HCV. Objectives: The aim of the study was to determine the prevalence of anti-HBc alone and occult hepatitis B, respectively, in blood samples from Adolfo Lutz Institute - Regional Laboratory Center X - São José do Rio Preto (IAL - CLR X - SJRP) and patients from Municipal Ambulatory of Viral Hepatitis (AMHV) both from São José do Rio Preto city in the period from January 1st, 2009 to December 31st, 2014. Methods: The study population of IAL - CLR X - SJRP is from the region served by the 15th Health Regional Division (DRS), and the AMHV is a population screened for clarification, monitoring and treatment of viral hepatitis in the city. In this population with anti-HBc alone, patients were immunized against hepatitis B and the individuals without vaccine response were selected for the performance of HBV-DNA research for the diagnosis of occult hepatitis B. Results: During the study period, 6805 samples were evaluated without duplication in IAL - CLR X - SJRP, of these 624 samples had anti-HBc positive, and the prevalence of anti-HBc alone was 17.63% (110/624). In the AMHV, 940 patients anti-HBc isolated were evaluated, from these 816 (86.81%) were vaccinated and after the criterion of disregarding the vaccinated patients who did not have anti-HBs evaluated after vaccination (85 - 10.42%), 731 (89.58%) patients were considered for analysis of the vaccine response, and 568 (77.70%) presented seroconversion with anti-HBs positive and 163 (22.30%) non-seroconverted patients. The research of HBV-DNA was performed in 25.77% (42/163) patients without a vaccine response, finding a prevalence of occult hepatitis B (OBI) of 47.62% (20/42).The presence of antibodies to HIV and HCV was 25.40% and 13.25% in the blood samples IAL - CLR X - SJRP and in AMHV was 1.80% and 0.33%, respectively. Conclusion: The results show the occurrence of antiHBc alone in IAL - CLR X - SJRP and the need of monitoring this population. In AMHV, the vaccination was effective for most cases, which demonstrates the need of vaccine introduction as a routine in anti- HBc alone patients in the overall population. The occult hepatitis B was found in almost half of patients assessed without vaccine response. / A presença do anti-HBc isolado pode ter vários significados: falso positivo, janela imunológica de cura, imunidade tardia ou infecção oculta pelo vírus da hepatite B (IOB). Na prática clínica é importante e necessário o esclarecimento diagnóstico para evitar transmissão em populações de risco como pacientes hemodialisados, doadores de sangue, transplantados e indivíduos coinfectados com HIV e/ou HCV. Objetivo: O objetivo do estudo foi determinar a prevalência de anti-HBc isolado e hepatite B oculta, respectivamente, em amostras de sangue do Instituto Adolfo Lutz - Centro de Laboratório Regional X - São José do Rio Preto (IAL - CLR X - SJRP) e pacientes do Ambulatório Municipal de Hepatites Virais (AMHV) ambos da cidade de São José do Rio Preto, no período de 01 de janeiro de 2009 a 31 de dezembro de 2014. Casuística e Métodos: A população estudada do IAL - CLR X - SJRP é proveniente da região atendida pela Divisão Regional de Saúde (DRS) XV e a do AMHV é uma população triada para esclarecimento, acompanhamento e tratamento das hepatites virais do município. Nesta população com anti-HBc isolado os pacientes foram imunizados contra hepatite B e os indivíduos sem resposta vacinal foram selecionados para realização da pesquisa de HBV-DNA para diagnóstico da hepatite B oculta. Resultados: Durante o período de estudo, foram avaliadas 6805 amostras, sem duplicação, no IAL - CLR X - SJRP, destas, 624 amostras apresentavam anti-HBc reagente, sendo a prevalência de anti-HBc isolado 17,63% (110/624). No AMHV foram analisados 940 pacientes com anti-HBc total isolado destes 816 (86,81%) foram vacinados e depois de aplicado o critério de desconsiderar os pacientes vacinados que não tiveram o anti-HBs avaliado após a vacinação (85 - 10,42%), 731 (89,58%) pacientes foram considerados para análise da resposta vacinal, sendo que 568 (77,70%) apresentaram soroconversão com anti-HBs positivo e 163 (22,30%) pacientes não soroconverteram. A pesquisa do HBV-DNA foi realizada em 25,77% (42/163) dos pacientes sem resposta vacinal, encontrando uma prevalência de hepatite B oculta (IOB) de 47,62% (20/42). A presença de anticorpos contra HIV e HCV foi de 25,40%, 13,25% nas amostras do IAL - CLR X - SJRP e no AMHV foi de 1,80%, 0,33%, respectivamente. Conclusão: Os resultados obtidos demonstram a ocorrência de anti-HBc isolado nas amostras do IAL - CLRX - SJRP e a necessidade de acompanhamento dessa população. No AMHV a vacinação esclareceu a maioria dos casos, o que demonstra a necessidade da introdução da vacina como rotina em pacientes anti-HBc isolado na população geral. A hepatite B oculta foi encontrada em quase metade dos pacientes não respondedores vacinais avaliados.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-22T16:21:30Z No. of bitstreams: 1 Veronica Arns da Silva.pdf: 500226 bytes, checksum: 2a21789fca9964130708e123521a1f6c (MD5) / Made available in DSpace on 2017-02-22T16:21:30Z (GMT). No. of bitstreams: 1 Veronica Arns da Silva.pdf: 500226 bytes, checksum: 2a21789fca9964130708e123521a1f6c (MD5) Previous issue date: 2007-02-22 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Infectious Myonecrosis Viral (IMNV), restricted action to the Brazilian northeast up to 2006, is disease of bigger negative impact in the shrimp cultured of the region. Having the objective to verify occurrence and evolution of the IMNV in Litopenaeus vannamei cultured on the coast of Pernambuco, Brazil, histopathological examination in 60 samples of shrimps from four farms in two cycles of aquaculture was proceeded. The histopatological findings had been correlated the data of the wet mount and the inquiry. Suggestive injuries of IMNV (coagulation necrosis, muscular hemocitic infiltration, spheroid of the ectopic lynphoid organ) were found in samples of the four studied farms with bigger occurrences in the first cycle. However, in the rainy period it had reduction of the occurrence, being associated the handling change. The raise time and the density were variables which influenced significantly (P<0,05) in the manifestation of the IMNV. / A Mionecrose Infecciosa Viral (IMNV), de ação restrita ao nordeste brasileiro até 2006, é a doença de maior impacto negativo na carcinicultura da região. Com objetivo de verificar ocorrência e evolução da IMNV em Litopenaeus vannamei, cultivado no litoral de Pernambuco, Brasil, procedeu-se ao exame histopatológico em 60 amostras, provenientes de quatro fazendas, em dois períodos (estio e chuvoso). Os resultados histopatológicos foram relacionados com os do exame a fresco e do inquérito. Em amostras das quatro fazendas foram identificadas lesões sugestivas de IMNV (necrose de coagulação, infiltração hemocítica na musculatura, esferóides ectópicos do órgão linfóide), havendo maior ocorrência de lesões no período de estio. Entretanto, no período chuvoso houve redução da ocorrência, sendo associada a mudança de manejo. O tempo de cultivo e a densidade de estocagem, foram variáveis que influenciaram significativamente (P<0,05) na manifestação da IMNV.

Introdução: As infecções virais respiratórias agudas são as doenças mais comuns em humanos e estão associadas a grande morbidade e mortalidade em crianças, principalmente menores de 2 anos de idade, sobretudo nos países em desenvolvimento e em idosos nos países desenvolvidos. As crianças que apresentam cardiopatias congênitas estão mais susceptíveis a adquirir infecção viral devido à sua mecânica pulmonar alterada, o que pode gerar diversas complicações tanto no período pré-operatório quanto no período pós-operatório, tais como aumento no tempo de internação hospitalar, maior tempo de ventilação mecânica e maiores taxas de mortalidade. Este estudo teve como objetivo identificar a epidemiologia das infecções virais respiratória em crianças com cardiopatia congênita e comparar os desfechos: tempo de internação, tempo de ventilação mecânica e mortalidade, na presença ou não de infecção viral respiratório e determinar qual o momento que essas crianças adquirem a infecção viral. Trata-se de estudo longitudinal, observacional, do tipo coorte. Foram coletadas amostras de secreção nasofaringe no período pré e pós operatório de todos os pacientes submetidos à cirurgia cardíaca e analisados os dados gerais dos pacientes durante o tempo de internação no centro de terapia intensiva pediátrica, por meio de prontuário médico, entre maio de 2013 a maio de 2014. Resultados: Foram analisados 43 pacientes. Foi encontrada elevada prevalência de vírus respiratórios (39%) em crianças com cardiopatia congênita. No presente estudo não houve diferença estatisticamente dos desfechos em relação a infecção viral respiratória no modelo estatístico bivariável, por motivo de interferência de variáveis confundidoras, idade e RACHS-1. A seguir, foram ajustados modelos de regressão multivariável, para analisar os desfechos com a variáveis idade, RACHS-1 e infecção viral. A variável infecção viral respiratória apresentou efeito estatisticamente significativo no desfecho diferença arteriovenosa de oxigênio, enquanto as covariáveis idade e RACHS-1 tiveram efeito significativamente em todos os desfechos pesquisados no estudo. Conclusão: A prevalência de infecção viral respiratória em crianças submetidas a cirurgia cardíaca é alta. A infecção viral respiratória não apresentou efeito sobre os principais desfechos, apenas na diferença arteriovenosa de oxigênio / Introduction: Acute respiratory viral infections are the most common diseases in humans and are associated with high morbidity and mortality in children, especially those under two years of age, particularly in developing countries, and in the elderly from developed countries. Children with congenital heart disease are more likely to get viral infections due to their altered lung mechanics, which can lead to several complications in both the preoperative and postoperative period, such as increased hospital stay, longer mechanical ventilation and higher mortality rates. This study aimed to identify the epidemiology of respiratory viral infections in children with congenital heart disease, to compare the outcomes: hospital stay, duration of mechanical ventilation and mortality, in the presence or absence of respiratory viral infection, and determine the time when these children acquire viral infection. This is a longitudinal, observational cohort study. Nasopharyngeal secretion samples were collected pre- and postoperatively for all patients undergoing cardiac surgery. General data of patients were obtained during hospital stay from medical records, from May 2013 to May 2014. Results: We enrolled 43 patients. We found a high prevalence of respiratory viruses (39%) in children with congenital heart disease. In this study there was no statistically significant difference in outcomes in relation to respiratory viral infection in bivariate statistical model, because of interference from confounding variables, age and RACHS-1. We then used multivariate regression models to analyze outcomes with respect to independent variables age, RACHS-1 and viral infection. Respiratory viral infection showed a statistically significant effect on the outcome arteriovenous oxygen difference, while the covariables age and RACHS-1 showed significant effects on all outcomes investigated in the study. Conclusion: The prevalence of respiratory viral infection in children undergoing cardiac surgery is high. Respiratory viral infection did not affect the outcome, just in arteriovenous oxygen difference

Made available in DSpace on 2017-07-10T14:17:13Z (GMT). No. of bitstreams: 1 Dissertacao -16.pdf: 1891470 bytes, checksum: 1b28975beba2d1e4f43d3a0f9f79a9aa (MD5) Previous issue date: 2014-02-11 / Bombyx mori is an insect of the order Lepidoptera that is only found only in germplasm banks; it is used in scientific research and for commercial purposes. In the latter case, the silk cocoon, which is produced at the end of the 5th larval instar, is used in the production of various yarns and fabrics. This branch of Brazilian agribusiness, known as sericulture, is well developed in the state of Paraná, where it is a form of small-scale family farming. Several factors impact negatively on Brazilian sericulture, such as diseases during rearing, and B. mori is susceptible to a virus from the Baculoviridae family, namely, Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), genus Alphabaculovirus (AlphaBV), which infects the larvae and jeopardises commercial production of the cocoon, causing losses to farmers and industry. Studies have proved that BmMNPV is polyorganotropic and there are several target organs, such as the tracheal system; however, details of its cytopathology are not known. The tracheal system is responsible for the aeration of the tissues of the insect. Thus, the present study aimed to describe the cytopathology of the tracheas of hybrid larvae of B. Mori, infected experimentally with BmMNPV, and isolated geographically in the state of Paraná. Fifth instar hybrid larvae were divided into two groups; one control, and the other inoculated. After ingestion, and on different days post-inoculation (dpi), from the 2nd to the 9th dpi, the larvae were anesthetized and dissected. Segments of organs such as the integument, muscle and silk gland, containing branches of the trachea, were collected and fixed in Karnovsky modified for transmission electron microscopy. On the 2st dpi, fresh hemolymph analysis was conducted in order to determine the susceptibility of the hemocytes. The results revealed that the hemocytes were infected from the 2nd dpi and the epithelial cells of the trachea were infected from the 4th dpi. The cytopathology of the tracheal cells showed hypertrophic nucleus, containing the viroplasm, the site of the synthesis of the nucleocapsids. Subsequently, the formation and development of the polyhedra occured, accentuating the nuclear hypertrophy and culminating in cell lysis. Virions were also observed, immersed in the basal lamina of the trachea, which appeared to be disorganized. Thus, the cytopathology of the trachea was consistent with the infection caused by AlphaBV, and the data that was obtained provides a better understanding of the infectious cycle of BmMNPV in the body of the insect. The time of infection, later for the hemocytes, and the presence of virions in the basal lamina of the trachea, indicated that this system is a secondary target for infection, and also that the hemolymph is an important dispersant of viral infection / Bombyx mori é um inseto da ordem Lepidoptera encontrado somente em bancos de germoplasma, sendo utilizado em pesquisas científicas e para fins comerciais. Neste caso, seu casulo de seda, construído ao final do 5º instar larval, é usado na produção de diversos fios e tecidos. Este ramo da agroindústria brasileira, conhecido como sericicultura, se apresenta bem desenvolvido no Estado do Paraná, estando incluído no programa de agricultura familiar. Vários são os fatores que exercem influência na sericicultura nacional, como as doenças, e B. mori é susceptível a um vírus da família Baculoviridae, o Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), gênero Alphabaculovirus (AlphaBV). Ao infectar as lagartas o vírus compromete a produção comercial do casulo, causando prejuízos aos produtores rurais e a indústria. Estudos comprovam que o BmMNPV é poliorganotrófico e vários são os órgãos-alvos, como o sistema traqueal; entretanto, detalhes de sua citopatologia não são conhecidos. O sistema traqueal é responsável pela aeração dos tecidos do inseto e o presente estudo objetivou descrever a citopatologia das traqueias de lagartas híbridas de B. mori infectadas experimentalmente pelo BmMNPV, isolado geográfico do Paraná. Para tanto, lagartas híbridas de 5º instar foram divididas em dois grupos, controle e inoculado. Neste, o inóculo viral foi fornecido na alimentação e em diferentes dias pós-inoculação (dpi), do 2º ao 9º dpi, as lagartas foram anestesiadas e dissecadas; segmentos do tegumento, músculo e glândula da seda, contendo ramos da traqueia, foram coletados e fixados em Karnovsky modificado para a microscopia eletrônica de transmissão. No 2º dpi foi efetuada análise a fresco da hemolinfa, para averiguar a susceptibilidade dos hemócitos. Os resultados revelaram que os hemócitos se apresentaram infectados a partir do 2º dpi e as células epiteliais da traqueia a partir do 4° dpi. A citopatologia das células traqueais revelou núcleo hipertrófico, contendo o viroplasma, que é o local de síntese dos nucleocapsídeos. Posteriormente, houve a formação e o desenvolvimento dos poliedros, acentuando-se a hipertrofia nuclear e culminando com a citólise. Vírions também foram visualizados na lâmina basal da traqueia, que se apresentou desorganizada. Assim, a citopatologia da traqueia condiz com a infecção causada por AlphaBV, e as informações obtidas permitem um melhor entendimento do ciclo infeccioso do BmMNPV no corpo do inseto. O tempo de infecção, posterior ao dos hemócitos, e a presença de vírions na lâmina basal da traqueia, indicam que este sistema é alvo secundário e, ainda, que a hemolinfa se apresenta como um importante dispersor da infecção viral.

Chan, Wing Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 129-152). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Table of Contents --- p.7 / List of Abbreviations and symbols --- p.13 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemics and pandemics of influenza virus --- p.17 / Chapter 1.2 --- Biology of influenza virus --- p.19 / Chapter 1.2.1 --- Types of influenza virus --- p.19 / Chapter 1.2.2 --- Viral structure and viral proteins --- p.20 / Chapter 1.2.3 --- Life cycle of influenza virus --- p.21 / Chapter 1.2.4 --- An ever-changing virus --- p.22 / Chapter 1.3 --- Pathogenesis and immunology of influenza virus --- p.24 / Chapter 1.3.1 --- Diseases and symptoms caused by influenza virus infection --- p.24 / Chapter 1.3.2 --- Production of cytokines during influenza virus infection --- p.25 / Chapter 1.3.3 --- Immune responses in the hosts --- p.27 / Chapter 1.4 --- Non-structural protein 1 (NS1) --- p.28 / Chapter 1.4.1 --- Overview of NS1 --- p.28 / Chapter 1.4.2 --- Roles of NS1 in influenza virus infection --- p.29 / Chapter 1.5 --- Aims of study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Chemical reagents --- p.34 / Chapter 2.1.2 --- Buffers --- p.37 / Chapter 2.1.2.1 --- Preparation of buffers --- p.37 / Chapter 2.1.2.2 --- Commonly used buffers --- p.37 / Chapter 2.1.3 --- Strains and plasmids --- p.40 / Chapter 2.1.4 --- Primer list --- p.40 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Preparation of competent cells --- p.42 / Chapter 2.2.2 --- Molecular cloning --- p.43 / Chapter 2.2.2.1 --- Amplification of the target genes by PCR --- p.43 / Chapter 2.2.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gels --- p.44 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.45 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.45 / Chapter 2.2.2.6 --- Transformation and plating out transformants --- p.46 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.46 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.47 / Chapter 2.2.2.9 --- Confirmation of insertion in the miniprep DNA by restriction digestion --- p.48 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.48 / Chapter 2.2.3 --- Cell culture --- p.53 / Chapter 2.2.3.1 --- Cultivation of human lung epithelial NCI-H292 cells --- p.53 / Chapter 2.2.3.2 --- Transfection of cell culture --- p.53 / Chapter 2.2.4 --- Western blot analysis --- p.54 / Chapter 2.2.4.1 --- Protein extraction --- p.54 / Chapter 2.2.4.2 --- Determination of protein concentration --- p.54 / Chapter 2.2.4.3 --- Protein Blotting --- p.55 / Chapter 2.2.4.4 --- Membrane blocking and antibody incubations --- p.56 / Chapter 2.2.4.5 --- Detection of proteins --- p.57 / Chapter 2.2.5 --- Total RNA extraction --- p.58 / Chapter 2.2.5.1 --- Preparation of cell culture for total RNA extraction --- p.58 / Chapter 2.2.5.2 --- Spectrophotometric analysis of total RNA --- p.58 / Chapter 2.2.5.3 --- Agarose gel electrophoresis of total RNA --- p.59 / Chapter 2.2.6 --- DNA Microarray --- p.60 / Chapter 2.2.6.1 --- Preparation of biotin-labeled antisense cRNA --- p.60 / Chapter 2.2.6.2 --- "Hybridization, washing and scanning of DNA microarray chips" --- p.60 / Chapter 2.2.6.3 --- Data processing and analysis --- p.61 / Chapter 2.2.7 --- Quantitative real-time PCR (QRT-PCR) --- p.62 / Chapter 2.2.7.1 --- Preparation of cDNA --- p.62 / Chapter 2.2.7.2 --- Analysis of mRNA gene expression by QRT-PCR --- p.62 / Chapter Chapter 3 --- Roles of NS1A and NS1B on cellular gene expression / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.67 / Chapter 3.2.1 --- NS1 protein expression in transfected NCI-H292 cells --- p.67 / Chapter 3.2.2 --- Purity and integrity of total RNA extracted --- p.67 / Chapter 3.2.3 --- Microarray data processing and analysis --- p.70 / Chapter 3.2.3.1 --- Genes perturbed by NS1A --- p.88 / Chapter 3.2.3.1.1 --- Effect of NS1A on antiviral gene expression --- p.91 / Chapter 3.2.3.1.2 --- Regulation of JAK-STAT pathway by NS1A --- p.92 / Chapter 3.2.3.2 --- Genes perturbed by NS1B --- p.93 / Chapter 3.2.3.2.1 --- Effects of NS1B on IFN-stimulated gene expression --- p.96 / Chapter 3.2.3.3 --- Genes perturbed by both NS1A and NS1B --- p.96 / Chapter 3.2.4 --- Verification of differentially expressed genes --- p.98 / Chapter 3.3 --- Discussion --- p.100 / Chapter 3.3.1 --- Human lung epithelial cell line as a model --- p.100 / Chapter 3.3.2 --- DNA microarray technology --- p.101 / Chapter 3.3.3 --- Different actions of NS1A and NS1B on host cell gene expression --- p.102 / Chapter 3.3.4 --- Novel roles of NSIA --- p.103 / Chapter 3.3.5 --- Novel role of NSIB --- p.107 / Chapter 3.3.6 --- Implications --- p.108 / Chapter Chapter 4 --- "Roles of NSIA, NS1B and NS1C on cytokine expression" / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Results --- p.113 / Chapter 4.2.1 --- NS1 protein expression in transfected NCI-H292 cells --- p.113 / Chapter 4.2.2 --- Purity and integrity of total RNA extracted --- p.113 / Chapter 4.2.3 --- QRT-PCR --- p.116 / Chapter 4.3 --- Discussion --- p.119 / Chapter 4.3.1 --- Human lung epithelial cell line as a model for cytokine study --- p.119 / Chapter 4.3.2 --- Implications of different cytokine patterns induced by different NS1 proteins --- p.120 / Chapter Chapter 5 --- General Discussion and Future Perspectives --- p.125 / References --- p.129

Orientador: Angelo José Magro / Resumo: A Hepatite C é uma doença hepática de etiologia viral cujo agente é o Vírus da Hepatite C (VHC). A infecção pelo vírus, na grande maioria dos casos evolui para a cronicidade, sendo, portanto, um problema de saúde pública devido ao seu grande potencial de evolução para cirrose e hepatocarcinoma. Portanto, atualmente é fundamental a utilização de testes moleculares como diagnóstico complementar e para o acompanhamento da doença no paciente infectado, assim como para a detecção cada vez mais precoce da infecção pelo VHC. Neste sentido, testes para a detecção de ácidos nucléicos podem ser utilizados para a detecção qualitativa e quantitativa do RNA-VHC no sangue dos pacientes, de modo a avaliar a dinâmica da infecção e definir a conduta terapêutica. Desta forma, o Ministério da Saúde adotou no Brasil uma metodologia baseada na Reação em Cadeia da Polimerase em Tempo Real (RT-qPCR) precedida de etapa de transcrição reversa para a detecção do material genético viral em amostras de plasma dos infectados. A presença do VHC está documentada em outros compartimentos biológicos como células mononucleares do sangue periférico (PBMC) e plaquetas, o que sugere que o protocolo atualmente adotado no Brasil pode superestimar o número de partículas virais ativas, visto que o plasma preparado para a realização desta análise contém uma grande quantidade de plaquetas em relação à contagem total dos indivíduos. Desta forma, a finalidade da execução deste projeto foi comparar a carga viral do VHC e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hepatitis C is a liver disease of viral etiology whose agent is Hepatitis C Virus (HCV). This virus infection, in the vast majority of cases, progresses to chronicity and is therefore a public health problem due to its great potential for progression to cirrhosis and hepatocarcinoma. Therefore, the use of molecular tests as a complementary diagnosis and disease control, as well as for early detection of HCV infection, is currently essential. In this sense, tests for the detection of nucleic acids can be used for the qualitative and quantitative detection of HCV RNA in the blood of the patients, in order to evaluate the dynamics of the infection and define the therapeutics. Thus the Brazilian Ministry of Health adopted a methodology based on the Real-Time Polymerase Chain Reaction (RT-qPCR) preceded by a reverse transcription step for the detection of the viral genetic material in plasma samples of infected individuals. The presence of HCV is well documented in other biological compartments such as peripheral blood mononuclear cells (PBMC) and platelets, suggesting that the protocol currently adopted in Brazil may overestimate the number of active viral particles, since the plasma samples prepared for this analysis contains a large amount of platelets. Thus, the purpose of this project was to compare the HCV viral load in chronically infected patients, taking into account the protocol currently used by the SUS and another one modified, where the plasma samples are practically ... (Complete abstract click electronic access below) / Mestre