Thesis (M.P.H.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 72-75).

published_or_final_version / Community Medicine / Master / Master of Public Health

Resumo Introdução: A Vigilância Sanitária se constitui como campo de intervenção da Saúde Pública tendo como uma de suas responsabilidades, garantir o controle sanitário de aeroportos e a proteção da saúde dos viajantes. Objetivo: Neste sentido, o presente estudo teve como objetivo conhecer, descrever e analisar a prática sanitária adotada frente à Pandemia de Influenza A (H1N1) 2009, pela Vigilância Sanitária no Terminal de Passageiros do Aeroporto de Guarulhos. Metodologia: A pesquisa qualitativa foi adotada, tendo como referencial teórico as representações sociais. Utilizou-se o referencial metodológico da hermenêutica dialética, fazendo uso de uma abordagem interpretativa reconstrutiva das falas dos entrevistados. A construção das três categorias empíricas Trabalho, Comunicação, Intersetorialidade - permitiu resgatar junto às falas dos profissionais pesquisados a prática vivenciada pela Vigilância Sanitária durante a pandemia. Resultados: Pôde-se apreender que as dificuldades evidenciadas durante a Pandemia de H1N1 estiveram relacionadas aos recursos humanos, à estrutura física e de material, ao fluxo de procedimentos e de informações. Conclusões: Os resultados evidenciaram a prática da VISA associada diretamente a sua estrutura organizacional; a uma atuação coadunada com o desenvolvimento atual do país; e uma experiência que serviu como o mais importante e único teste de enfrentamento para uma pandemia de influenza / Abstract Introduction: The Health Surveillance is a field of Public Health with the one of its responsibilities to ensure the sanitary control of airports and heath protection of travelers. Objective: In this sense, the present study aimed to understand, describe and analyze the sanitary practice adopted on the face of Influenza A (H1N1) Pandemic in 2009, by the Health Surveillance Agency in the passengers arrival gates of Guarulhos Airport. Methodology: The qualitative research was adopted in this study, using as a theoretical reference the social representations. In this document it was used the referral method of Hermeneutic Dialectic, using the interpretation of the interviews. It was built three empirics categories, which allowed retrieving in the interviews the practical experience of the employees of the Health Surveillance during the Pandemic period. Outcomes: It could be learnt that the difficulties during the A H1N1 Pandemic was related to the human resources, physical and material infrastructure and the process and information flows. Conclusion: The outcomes emphasized the way the Health Surveillance works directly linked to its organizational structure; its behavior, aligned with the current Brazil situation; and the experience which was a unique test of how they face the Influenza pandemic

Resumo Introdução: A Vigilância Sanitária se constitui como campo de intervenção da Saúde Pública tendo como uma de suas responsabilidades, garantir o controle sanitário de aeroportos e a proteção da saúde dos viajantes. Objetivo: Neste sentido, o presente estudo teve como objetivo conhecer, descrever e analisar a prática sanitária adotada frente à Pandemia de Influenza A (H1N1) 2009, pela Vigilância Sanitária no Terminal de Passageiros do Aeroporto de Guarulhos. Metodologia: A pesquisa qualitativa foi adotada, tendo como referencial teórico as representações sociais. Utilizou-se o referencial metodológico da hermenêutica dialética, fazendo uso de uma abordagem interpretativa reconstrutiva das falas dos entrevistados. A construção das três categorias empíricas Trabalho, Comunicação, Intersetorialidade - permitiu resgatar junto às falas dos profissionais pesquisados a prática vivenciada pela Vigilância Sanitária durante a pandemia. Resultados: Pôde-se apreender que as dificuldades evidenciadas durante a Pandemia de H1N1 estiveram relacionadas aos recursos humanos, à estrutura física e de material, ao fluxo de procedimentos e de informações. Conclusões: Os resultados evidenciaram a prática da VISA associada diretamente a sua estrutura organizacional; a uma atuação coadunada com o desenvolvimento atual do país; e uma experiência que serviu como o mais importante e único teste de enfrentamento para uma pandemia de influenza / Abstract Introduction: The Health Surveillance is a field of Public Health with the one of its responsibilities to ensure the sanitary control of airports and heath protection of travelers. Objective: In this sense, the present study aimed to understand, describe and analyze the sanitary practice adopted on the face of Influenza A (H1N1) Pandemic in 2009, by the Health Surveillance Agency in the passengers arrival gates of Guarulhos Airport. Methodology: The qualitative research was adopted in this study, using as a theoretical reference the social representations. In this document it was used the referral method of Hermeneutic Dialectic, using the interpretation of the interviews. It was built three empirics categories, which allowed retrieving in the interviews the practical experience of the employees of the Health Surveillance during the Pandemic period. Outcomes: It could be learnt that the difficulties during the A H1N1 Pandemic was related to the human resources, physical and material infrastructure and the process and information flows. Conclusion: The outcomes emphasized the way the Health Surveillance works directly linked to its organizational structure; its behavior, aligned with the current Brazil situation; and the experience which was a unique test of how they face the Influenza pandemic

Thesis (M.Sc.). / Written for the Dept. of Epidemiology, Biostatistics and Occupational Health. Title from title page of PDF (viewed 2009/06/19). Includes bibliographical references.

Thesis (M.N.) -- Memorial University of Newfoundland, 2003. / Typescript. Bibliography: leaves 125-131. Also available online.

Orientador: Maria Rita Donalísio Cordeiro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T16:23:15Z (GMT). No. of bitstreams: 1 Freitas_AndreRicardoRibasde_D.pdf: 4270845 bytes, checksum: 09078cb28a971b59a0103ccfbe6a3bee (MD5) Previous issue date: 2014 / Resumo: Introdução e objetivos: As infecções respiratórias estão entre as mais importantes causas de morbimortalidade no mundo. A sua alta incidência tem relevante impacto nos óbitos, como também na sobrecarga do sistema de saúde e absenteísmo no trabalho e escola Todas as faixas etárias são acometidas, porém, as mais afetadas são as crianças e os idosos. Também são particularmente susceptíveis os imunocomprometidos e os portadores de doenças crônicas em geral. Os vírus são os agentes responsáveis pela maior parte das infecções respiratórias, os principais vírus causadores de infecções respiratórias são o influenza A e B e o Vírus Sincicial Respiratório (VSR). Estes vírus têm comportamento biológicos distintos e o conhecimento de como estes vírus afetam a saúde da população é fundamental para embasar as ações de prevenção, profilaxia e tratamento de pacientes permitindo uma alocação adequada de recursos em quantidade e tempo adequados. No Brasil, no ano 2000, para monitorar a ocorrência destes vírus foi implantada a vigilância de síndromes gripais SIVEP-GRIPE, que através de 128 unidades sentinelas distribuídas em todas as regiões do país coletam semanalmente amostras de secreção de nasofaringe por semana de pacientes com síndromes gripais. Neste trabalho estudamos o impacto do influenza na mortalidade no estado de São Paulo, nas diferentes faixas etárias no período entre 2002 e 2011, incluindo o período da pandemia de 2009. Estudamos também a sazonalidade do VSR nas 5 diferentes regiões brasileiras e o impacto deste vírus nas internações por doenças respiratórias entre menores de 5 anos. Metodologia: Para o estudo da mortalidade associada ao influenza utilizamos o método de regressão de Serfling adaptado para dados semanais extraindo da série histórica os períodos de maior circulação viral a partir dos resultados do sistema de vigilância sentinela SIVEP-GRIPE. Comparar a mortalidade associada à pandemia de influenza de 2009, às epidemias prévias anuais de influenza nas diferentes faixas etárias e com diferentes subtipos de vírus influenza circulantes no estado de São Paulo. Para o estudo da sazonalidade do VSR utilizamos análise de Wavelets, análise de Fourier, análise simplificada de estações anuais comparando os resultados nas 5 regiões administrativas do Brasil. Para identificar possíveis correlações temporais entre a circulação dos vírus respiratórios utilizamos métodos de regressão de ranque de Spearman e de regressão parcial. Resultados e conclusões: A mortalidade por pneumonia e influenza associada à pandemia de 2009 no estado de São Paulo foi ligeiramente mais alta que nos outros anos de influenza sazonal, considerando a mortalidade geral, sem distinção de faixa etária. Houve diferenças no risco de morrer entre as faixas etárias. Entre os indivíduos de 5 a 19 anos, a mortalidade associada à pandemia de 2009 foi 2,6 maior (0,6 óbitos/100.000hab) que a de anos não pandêmicos. Na faixa etária de 20 a 59 anos, a mortalidade associada à pandemia de 2009 foi 5,1 maior (2,8 óbitos/100.000hab) que nos anos não pandêmicos. As taxas de mortalidade entre menores de 5 anos foi 0,9 óbitos/100.000hab e na população de mais 60 anos foi 13,1 óbitos/100.000hab, ou seja, foram menores que nos anos não pandêmicos. O método de análise utilizado permitiu a diferenciação entre a mortalidade associada a subtipos virais (A(H3N2), B ou sazonal A(H1N1) e A(H1N1) pdm 2009). Foi possível a comparação entre a mortalidade associada à pandemia de influenza de 2009 em São Paulo, às epidemias anuais de influenza nas diferentes faixas etárias e com diferentes subtipos de vírus influenza circulando. Isto é, o impacto da circulação do vírus pandêmico influenza A(H1N1) foi maior na mortalidade em adultos e jovens, enquanto em maiores de 65 anos foi discreto. Por outro lado, o excesso de mortalidade foi expressivo em maiores de 65 anos, nos anos de circulação do influenza A(H3N2). O modelo de Serfling adaptado a dados semanais com validação por meio de dados da vigilância sentinela de síndromes gripais (SIVEP-GRIPE) mostrou-se confiável para detectar picos de maior circulação viral do Influenza e supostos reflexos na mortalidade em diferentes faixas etárias em período pandêmico, epidêmico e de circulação sazonal do vírus Influenza. Sobre o VSR foi possível identificar padrões sazonais do VSR em todas as regiões administrativas do Brasil utilizando-se dados da vigilância de síndromes gripais (SIVEP-GRIPE). Houve diferenças entre os momentos de maior circulação do vírus em algumas das cinco regiões administrativas do Brasil. Os padrões sazonais de internação por doenças sabidamente relacionadas com o VSR [Pneumonia devida a vírus respiratório sincicial, Bronquite aguda devida a vírus sincicial respiratório, Bronquiolite aguda devida a vírus sincicial respiratório, Bronquiolite (aguda, não especificada),] foram semelhantes aos encontrados pela análise da circulação do VSR por meio de dados da vigilância de síndromes gripais (SIVEP-GRIPE). Houve correlação temporal entre a circulação do VSR e as taxas de internação por doenças do aparelho respiratório em geral (Capítulo-X da CID-10) entre menores de 5 anos, nas cinco regiões administrativas do Brasil. Houve correlação temporal entre as taxas de internação entre menores de 5 anos por doenças sabidamente relacionadas com o VSR [Pneumonia devida a vírus respiratório sincicial, Bronquite aguda devida a vírus sincicial respiratório, Bronquiolite aguda devida a vírus sincicial respiratório, Bronquiolite (aguda, não especificada),] e as taxas de internação por doenças respiratórias em geral nesta faixa etária nas cinco regiões administrativas do Brasil, indicando que este é o principal vírus associado às internações de crianças até 5 anos por doenças respiratórias. De acordo com as evidências encontradas neste estudo, os esquemas de profilaxia contra o VSR hoje utilizados precisam ser revistos e particularizados para cada região do país. Entre as ações a serem revistas estão a disponibilização do palivizumabe, bem como medidas de prevenção à circulação do VSR na comunidade / Abstract: Introduction and Objectives: Respiratory infections are amongst the most important causes of morbidity and mortality worldwide. Its high incidence has significant impact on deaths, but also burdens the health system and leads to absenteeism from work and school All age groups are affected, but the most affected are children and the elderly. Are also particularly susceptible immunocompromised and patients with chronic diseases in general. Viruses are the agents responsible for most respiratory infections, the main cause of respiratory virus infections are influenza A and B and Respiratory Syncytial Virus (RSV). These viruses have distinct biological behavior and knowledge of how these viruses affect people's health is fundamental to support the prevention, prophylaxis and treatment of patients allowing an adequate allocation of resources in quantity and adequate time. In Brazil, in 2000, to monitor the occurrence of these viruses was established surveillance of influenza-like syndromes SIVEP-FLU, which through 128 sentinel units distributed in all regions of the country collect weekly samples of nasopharyngeal secretions of patients per week with influenza-like illness. In this work we study the impact of influenza on mortality in the state of São Paulo , in different age groups between 2002 and 2011 , including the period of the 2009 pandemic. We also studied the seasonality of RSV in 5 different Brazilian regions and the impact of this virus in hospitalizations for respiratory diseases among children under 5 years. Methods: To study the mortality associated with influenza used the regression method of Serfling adapted for extracting weekly data of the time series periods of increased viral movement from the results of sentinel surveillance system SIVEP - FLU . Compare the mortality associated with pandemic 2009 influenza , annual epidemics of influenza prior at different ages and with different subtypes of influenza viruses circulating in the state of São Paulo . To study the seasonality of RSV , we use wavelet analysis , Fourier analysis , simplified analysis of annual seasons comparing the results in five administrative regions of Brazil . To identify possible temporal correlations between the circulation of respiratory viruses use regression methods of Spearman rank and partial regression. Results and Conclusions: The mortality from pneumonia and influenza associated with the 2009 pandemic in the state of São Paulo was slightly higher than in the other years of seasonal influenza, considering the overall mortality, irrespective of age. There were differences in the risk of dying between age groups. Among individuals 5-19 years, the mortality rate associated with the 2009 pandemic was 2.6 higher than that of non-pandemic years. (0.6 deaths /100,000 inhabitants) In the age group 20-59 years, the rate associated with the 2009 pandemic mortality was 5.1 higher than in non-pandemic years. (2.8 deaths /100,000 inhabitants). Mortality rates among children under five years was 0.9 deaths /100,000 inhabitants and in persons over 60 years was 13.1 deaths /100,000 inhabitants, ie were lower than in non- pandemic years . The method of analysis used allowed the differentiation between mortality associated with viral subtypes (A(H3N2), A(H1N1) and B or seasonal A(H1N1) pdm 2009) . It was possible to compare the mortality associated with the 2009 influenza pandemic in Sao Paulo, annual influenza epidemics in different ages and with different subtypes of influenza viruses circulating. That is, the impact of the circulation of influenza A(H1N1) pandemic virus was higher mortality in adults and children, while in adults over 65 years was low . On the other hand, the excess mortality was significant in adults over 65 years ago, in circulating influenza A H3N2. The Serfling model adapted to weekly data validation using data from sentinel surveillance of influenza-like illness (SIVEP - GRIPE) was reliable for detecting peaks of higher viral circulation of influenza and alleged impacts on mortality in different age groups in pandemic period , epidemic and seasonal circulation of influenza viruses . About RSV was possible to identify seasonal patterns of RSV in all administrative regions of Brazil using surveillance data of influenza syndromes (SIVEP -GRIPE). There were differences between the moments of greatest circulation of the virus in some of the five administrative regions of Brazil. Seasonal patterns of hospitalization for known diseases with RSV [ Pneumonia due to respiratory syncytial virus , acute bronchitis due to respiratory syncytial virus , acute bronchiolitis due to respiratory syncytial virus bronchiolitis ( acute , unspecified ) ] were similar to those found by analysis of the movement of data through RSV surveillance of influenza-like syndromes ( SIVEP - GRIPE) . There was a temporal correlation between the circulation of RSV and the rates of hospitalization for respiratory diseases in general (Chapter X of ICD- 10) among children under 5 diseases in the five administrative regions of Brazil . There was a temporal correlation between the rates of hospitalization among children under 5 years for known diseases with RSV [ Pneumonia due to respiratory syncytial virus , acute bronchitis due to respiratory syncytial virus , acute bronchiolitis due to respiratory syncytial virus bronchiolitis ( acute , unspecified ) ] and rates of hospitalization for respiratory diseases in general in this age group in the five administrative regions of Brazil , indicating that this is the main virus associated with hospitalizations of children under 5 years due to respiratory diseases . According to the evidence found in this study , the schemes of prophylaxis against RSV used today need to be reviewed and individualized for each region of the country . Among the actions to be reviewed are the availability of palivizumab , as well as measures to prevent the circulation of RSV in the community / Doutorado / Epidemiologia / Doutor em Saude Coletiva

Introdução- Em abril de 2009, novo subtipo viral foi identificado, Influenza A(H1N1)pdm09. Em 11 de junho de 2009, a Organização Mundial da Saude anunciou o início de uma pandemia de influenza. Objetivo- Investigar os fatores de risco para óbito por Influenza A(H1N1)pdm09 em pacientes e em gestantes hospitalizados com Doença Respiratória Aguda Grave-DRAG. Nas gestantes, foram analisados também os desfechos gestacionais e neonatais. Metodologia- Foram realizados dois estudos caso-controles, em pacientes e em gestantes hospitalizados com Influenza A(H1N1)pdm09 confirmada laboratorialmente e DRAG. Os casos evoluíram para óbito e os controles para cura. Os casos e controles foram selecionados no Sistema de Informação de Agravos de Notificação-SINANInfluenza- web, sendo sorteados dois controles no estudo dos pacientes, e quatro no das gestantes, pareados por semana epidemiológica da data de internação do caso. O primeiro estudo foi realizado nas regiões Metropolitanas de São Paulo e de Campinas, de 28 de junho a 29 de agosto de 2009. Nas gestantes, o estudo incluiu o Estado de São Paulo, de 09 de junho a 01 de dezembro de 2009. Foram realizadas avaliações dos prontuários hospitalares e entrevistas domiciliares, a partir de formulários padronizados. Foram empregados testes de Mann-Whitney-U ou quiquadrado para comparação das variáveis, e cálculos dos odds ratio brutos-ORb e seus intervalos de confiança-IC95 por cento , para avaliação dos fatores de risco. No primeiro estudo foi definido modelo de regressão logística múltipla para análise dos fatores associados ao óbito. Resultados- No primeiro estudo, foram investigados 193 casos e 386 controles, 73,6 por cento dos casos e 38,1 por cento dos controles tinham alguma condição de risco para complicações relacionadas à influenza. No modelo final, as seguintes variáveis foram fatores de risco para óbito: idade entre 18 a 59 anos, Odds Ratio Ajustado-ORa de 2,31, 95 por cento IC 1,31-4,10, (referência pacientes < 18 anos), presença de pelo menos uma condição de risco (ORa=1,99, 95 por cento IC 1,11-3,57), mais de uma condição de risco (ORa=6,05 95 por cento IC 2,76-13,28), obesidade (ORa=2,73, 95 por cento IC 1,28- 5,83), imunossupressão (ORa=3,43 95 por cento IC 1,28-9,19) e ter tido atendimento prévio à internação (ORa=3,35, 95 por cento IC 1,75-6,40). O tratamento antiviral, quando administrado nas primeiras 48 horas do início dos sintomas foi fator de proteção para óbito, (ORa=0,17, 95 por cento IC 0,08-0,37). Houve benefício também da administração do antiviral entre 48 a 72 horas, (ORa=0,30, 95 por cento IC 0,11-0,81). Em gestantes, foram investigados 48 casos e 185 controles. Foram fatores de risco para óbito: ter tido atendimento prévio à internação, (ORb de 8,03, 95 por cento IC 2,38-27,09) e terceiro trimestre de gestação, (ORb=4,45, 95 por cento IC 1,15-29,25). Tratamento antiviral foi fator de proteção, quando administrado até 48 horas do início dos sintomas (ORb=0,14, 95 por cento IC 0,05-0,37), e de 48 a 72 horas, (ORb=0,13, 95 por cento IC 0,02-0,68). Em relação aos desfechos gestacionais, houve maior proporção de perdas fetais e partos prematuros entre os casos, p=0,001. As gestantes que evoluíram para óbitos tiveram recém nascidos vivos com mais baixo peso e índices inferiores no Apgar do primeiro minuto, p=0,016, quando comparado aos controles que tiveram parto durante a internação, p<0,001. Conclusão: A identificação dos pacientes de maior risco e o tratamento precoce são fatores importantes para a redução da morbimortalidade por influenza. / Introduction - In April 2009, a new viral subtype was identified, influenza A (H1N1)pdm09. On June 11, the World Health Organization announced the beginning of the influenza pandemic. Objective - To investigate the risk factors for death from influenza A(H1N1)pdm09 in hospitalized patients and pregnant women with Severe Acute Respiratory Infections-SARI. In the pregnant women, the gestational and neonatal outcomes were analyzed. Methodology - Two case control studies were performed in hospitalized patients and pregnant women with laboratory confirmed influenza A (H1N1)pdm09 and SARI. The cases died and the controls recovered. The cases and controls were selected from the Information System for Notifiable Diseases-SINAN-Influenza-web. Two controls were randomly selected in the study of patients and four in the pregnant women, matched by epidemiological week of the date of admission of the case. The first study was conducted in the metropolitan regions of São Paulo and Campinas, from June 28th to August 29th, 2009. The study on pregnant women included the State of São Paulo, from June 09th to December 1th, 2009. Evaluations of the medical records and home interviews were conducted using standardized forms. The Mann-Whitney U test or the chi-square tests were performed to compare the variables, in addition to calculations of crude odds ratio- ORc and their 95 per cent confidence intervals for the assessment of the risk factors. In the first study a multiple logistic regression model was used to analyze factors associated with death. Results - In the first study, 193 cases and 386 controls were investigated, 73.6 per cent of cases and 38.1 per cent of controls presented some risk condition for developing influenza-related complications. In the final model, the following variables were risk factors for death: aged between 18 and 59 , Adjusted Odds Ratio-ORa of 2.31, CI95 per cent 1.31-4.10, (reference patients <18 years), the presence of at least one risk condition (ORa=1.99, CI95 per cent 1.11-3.57), more than one risk condition (ORa=6.05 CI95 per cent 2.76-13.28), obesity (ORa=2.73, CI95 per cent 1.28-5.83), immunosuppression (ORa=3.43 CI95 per cent 1.28-9.19) and being attended prior to hospitalization (ORa=3.35, CI95 per cent 1.75-6.40). Antiviral treatment, when administered during the first 48 hours of onset of symptoms, was a protective factor for death, (ORa=0.17, CI95 per cent 0.08-0.37). There were also benefits from antiviral administration between 48 and 72 hours, (ORa=0.30, CI95 per cent 0.11-0.81). In the pregnant women, 48 cases and 185 controls were investigated. The risk factors for death were: being attended prior to hospitalization, (ORc 8.03, CI95 per cent 2.38-27.09) and third trimester of pregnancy, (ORc=4.45, CI95 per cent 1.15-29.25). Antiviral treatment was a protective factor when administered within 48 hours of onset of symptoms (ORc=0.14, CI95 per cent 0.05-0.37) and between 48 and 72 hours, (ORc= 0.13, CI95 per cent 0.02-0.68). In relation to pregnancy outcomes, there was a higher proportion of fetal losses and premature births among cases, p=0.001. The pregnant women who died had live newborns with lower weight and lower Apgar scores in the first minute, p=0.016 compared to controls who gave birth during hospitalization, p<0.001. Conclusion: The identification of high-risk patients and early treatment are important factors in the reduction of morbidity and mortality from influenza.

Tse, Yuk Tin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-149). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The influenza virus --- p.1 / Chapter 1.1.1 --- Influenza NA and its inhibitors --- p.3 / Chapter 1.1.2 --- Follow-up on the use of NAIs --- p.9 / Chapter 1.2 --- Sources of NA for experimental studies --- p.11 / Chapter 1.2.1 --- Viral sources --- p.11 / Chapter 1.2.2 --- NA isolation --- p.12 / Chapter 1.2.3 --- Recombinant NA expressed in cell lines --- p.12 / Chapter 1.2.4 --- Glycosylation on NA functionality --- p.13 / Chapter 1.2.5 --- Recombinant NA expressed in yeast --- p.15 / Chapter 1.3 --- Research objectives --- p.16 / Chapter 2 --- Cloning of Influenza Neuraminidase and Expression in P. pastoris --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.1.1 --- Full-length cloning of the A/HongKong/483/97(H5N 1) NA --- p.17 / Chapter 2.1.2 --- Identification of the H274 equivalent --- p.19 / Chapter 2.1.3 --- The experiment --- p.22 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Preparation of chemically competent Escherichia coli --- p.23 / Chapter 2.2.1.1 --- Reagents --- p.23 / Chapter 2.2.1.2 --- Reagent setup --- p.23 / Chapter 2.2.1.3 --- Equipment --- p.23 / Chapter 2.2.1.4 --- Procedure --- p.23 / Chapter 2.2.2 --- Amplification of N1 NA and EGFP genes --- p.24 / Chapter 2.2.2.1 --- Reagents --- p.24 / Chapter 2.2.2.2 --- Reagent setup --- p.25 / Chapter 2.2.2.3 --- Equipment --- p.25 / Chapter 2.2.2.4 --- Procedure --- p.26 / Chapter 2.2.2.4.1 --- Amplification of the full-length N1 NA gene from cDNA --- p.26 / Chapter 2.2.2.4.2 --- Amplification of the EGFP gene --- p.26 / Chapter 2.2.3 --- TA cloning of PCR products --- p.27 / Chapter 2.2.3.1 --- Reagents --- p.27 / Chapter 2.2.3.2 --- Reagent setup --- p.27 / Chapter 2.2.3.3 --- Equipment --- p.28 / Chapter 2.2.3.4 --- Procedure --- p.28 / Chapter 2.2.3.4.1 --- TA cloning of PCR products --- p.28 / Chapter 2.2.3.4.2 --- Site-directed mutagenesis by overlapping PCR --- p.30 / Chapter 2.2.4 --- Construction of P. pastoris expression vectors --- p.31 / Chapter 2.2.4.1 --- Reagents --- p.31 / Chapter 2.2.4.2 --- Reagent setup --- p.31 / Chapter 2.2.4.3 --- Procedure --- p.32 / Chapter 2.2.4.3.1 --- Generation of N1 NA expression vectors --- p.32 / Chapter 2.2.4.3.2 --- Generation of EGFP expression vectors --- p.34 / Chapter 2.2.5 --- Transformation of P. pastoris --- p.37 / Chapter 2.2.5.1 --- Reagents --- p.37 / Chapter 2.2.5.2 --- Reagent setup --- p.37 / Chapter 2.2.5.3 --- Equipment --- p.38 / Chapter 2.2.5.4 --- Procedure --- p.38 / Chapter 2.2.5.4.1 --- Preparation and transformation of electrocompetent P. pastoris --- p.38 / Chapter 2.2.5.4.2 --- PCR analysis of P. pastoris transformants (colony PCR) --- p.39 / Chapter 2.2.6 --- Expression of N1 NA and EGFP in P. pastoris --- p.40 / Chapter 2.2.6.1 --- Reagents --- p.40 / Chapter 2.2.6.2 --- Reagent setup --- p.40 / Chapter 2.2.6.3 --- Procedure --- p.41 / Chapter 2.2.6.3.1 --- Small-scale protein expression in P. pastoris --- p.41 / Chapter 2.2.6.3.2 --- Sequence alignment --- p.42 / Chapter 2.2.6.3.3 --- Data processing --- p.42 / Chapter 2.3 --- Results and Discussion --- p.43 / Chapter 2.3.1 --- Cloning of NA and EGFP into the pPICZB expression vector --- p.43 / Chapter 2.3.2 --- Growth of P. pastoris transformants --- p.51 / Chapter 3 --- Physical Characterization of Influenza Neuraminidase Expressed in P. pastoris --- p.53 / Chapter 3.1 --- Background --- p.53 / Chapter 3.1.1 --- Structural significance of disulphide bonds in NA --- p.53 / Chapter 3.1.2 --- Localization of recombinant N1 NA in P. pastoris --- p.55 / Chapter 3.1.3 --- The experiment --- p.56 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Differential centrifugation --- p.57 / Chapter 3.2.1.1 --- Reagents --- p.57 / Chapter 3.2.1.2 --- Reagent setup --- p.57 / Chapter 3.2.1.3 --- Equipment --- p.57 / Chapter 3.2.1.4 --- Procedures --- p.58 / Chapter 3.2.1.4.1 --- Cell harvesting and lysis --- p.58 / Chapter 3.2.1.4.2 --- Preparation of crude membrane --- p.58 / Chapter 3.2.1.4.3 --- Preparation of plasma membrane --- p.58 / Chapter 3.2.2 --- Sodium dodecyl sulphate polyaciylamide gel electrophoresis (SDS-PAGE)… --- p.59 / Chapter 3.2.2.1 --- Reagents --- p.59 / Chapter 3.2.2.2 --- Reagent setup --- p.60 / Chapter 3.2.2.3 --- Equipment --- p.61 / Chapter 3.2.2.4 --- Procedure --- p.61 / Chapter 3.2.3 --- Immunoblotting --- p.61 / Chapter 3.2.3.1 --- Reagents --- p.61 / Chapter 3.2.3.2 --- Reagent setup --- p.62 / Chapter 3.2.3.3 --- Equipment --- p.62 / Chapter 3.2.3.4 --- Procedure --- p.62 / Chapter 3.2.3.4.1 --- Electroblotting --- p.62 / Chapter 3.2.3.4.2 --- Blocking and probing --- p.63 / Chapter 3.2.3.4.3 --- Immunodetection --- p.63 / Chapter 3.2.3.4.4 --- Molecular weight determination --- p.63 / Chapter 3.2.4 --- Confocal microscopy --- p.64 / Chapter 3.2.4.1 --- Equipment --- p.64 / Chapter 3.2.4.2 --- Procedure --- p.64 / Chapter 3.2.4.2.1 --- Image acquisition --- p.64 / Chapter 3.2.4.2.2 --- Image processing --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Localization of recombinant N1 NA in P. pastoris sub-cellular fractions --- p.66 / Chapter 3.3.2 --- Molecular weight determination for the N1 NA expressed in P. pastoris --- p.69 / Chapter 3.3.3 --- Cellular localization of recombinant N1 NA in P. pastoris --- p.71 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- Molecular weight determination for N1 NA expressed in P. pastoris --- p.77 / Chapter 3.4.2 --- Disulphide bond formation in N1 NA expressed in P. pastoris --- p.78 / Chapter 3.4.3 --- Cell-surface association of recombinant N1 NA in P. pastoris --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter 4 --- Functional Characterization of Influenza Neuraminidase Expressed in P. pastor --- p.is / Chapter 4.1 --- Background --- p.82 / Chapter 4.1.1 --- Fluorometric NA activity assay --- p.82 / Chapter 4.1.2 --- Colorimetric assay of NA activity --- p.84 / Chapter 4.1.3 --- The experiment --- p.85 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Fluorometric assay of N1 NA expressed in P. pastoris --- p.86 / Chapter 4.2.1.1 --- Reagents --- p.86 / Chapter 4.2.1.2 --- Reagent setup --- p.86 / Chapter 4.2.1.3 --- Equipment --- p.86 / Chapter 4.2.1.4 --- Procedure --- p.87 / Chapter 4.2.1.4.1 --- Calibrating cell density with viable cell counts --- p.87 / Chapter 4.2.1.4.2 --- End-point measurement of NA activity --- p.87 / Chapter 4.2.1.4.2.1 --- Determination of expression yield --- p.89 / Chapter 4.2.1.4.2.2 --- End-point assay of NAI sensitivity --- p.89 / Chapter 4.2.1.4.3 --- Kinetic measurement of NA activity --- p.90 / Chapter 4.2.1.4.3.1 --- Derivation ofV0 --- p.92 / Chapter 4.2.1.4.3.2 --- Graphical determination of KM --- p.93 / Chapter 4.2.1.4.3.3 --- Graphical determination of KI --- p.94 / Chapter 4.2.2 --- Colorimetric assay of N1 NA expressed in P. pastoris --- p.96 / Chapter 4.2.2.1 --- Reagents --- p.96 / Chapter 4.2.2.2 --- Reagent setup --- p.96 / Chapter 4.2.2.3 --- Equipment --- p.96 / Chapter 4.2.2.4 --- Procedure --- p.96 / Chapter 4.3 --- Results --- p.98 / Chapter 4.3.1 --- CFU determination --- p.98 / Chapter 4.3.2 --- Fluorescent NA activity assay for N1 NA expressed in P. pastoris --- p.98 / Chapter 4.3.2.1 --- End-point measurement of NA activity --- p.98 / Chapter 4.3.2.1.1 --- Course of N1 NA expression in P. pastoris --- p.102 / Chapter 4.3.2.1.1.1 --- NA activity per unit cell mass --- p.102 / Chapter 4.3.2.1.1.2 --- Yield of NA --- p.102 / Chapter 4.3.2.1.2 --- End-point assay for NAI sensitivity --- p.105 / Chapter 4.3.2.2 --- Kinetic measurement of NA activity and NAI sensitivity --- p.107 / Chapter 4.3.2.2.1 --- Graphical determination of KM --- p.107 / Chapter 4.3.2.2.2 --- Graphical determination of KI --- p.107 / Chapter 4.3.2.3 --- Colorimetric NA activity assay --- p.111 / Chapter 4.4 --- Discussion --- p.114 / Chapter 4.4.1 --- Fluorescent NA activity assay of N1 NA expressed in P. pastoris --- p.115 / Chapter 4.4.1.1 --- End-point measurement of NA activity --- p.115 / Chapter 4.4.1.1.1 --- Time course of expression --- p.115 / Chapter 4.4.1.1.2 --- Effect of H275Y mutation on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.1.3 --- Effect of C-terminal tags on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.2 --- Kinetic measurement of NA activity --- p.118 / Chapter 4.4.1.2.1 --- Graphical determination of KM --- p.119 / Chapter 4.4.1.2.2 --- Graphical determination of KI --- p.120 / Chapter 4.4.1.3 --- Comparison of fluorometric NA activity assays for use with whole P pastoris cells --- p.122 / Chapter 4.4.2 --- Colorimetric NA activity assay --- p.124 / Chapter 4.5 --- Conclusion --- p.126 / Chapter 5 --- Conclusions and Discussions --- p.127 / Chapter 5.1 --- General conclusions --- p.127 / Chapter 5.2 --- Follow-up --- p.127 / Chapter 5.2.1 --- Studies of influenza NA with enhanced activity --- p.128 / Chapter 5.2.2 --- NAI screening using yeast-expressed NA --- p.132 / Appendix --- p.134 / References --- p.141

Thesis (M.P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Masters Abstracts International, Volume: 47-01, page: . Adviser: John Herbold. Includes bibliographical references.