Sia Sin Fun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 105-121). / Abstracts in English and Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vi / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xv / LIST OF TABLES --- p.xvi / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- The interferon / Chapter 1.1.1 --- Classification of interferons --- p.1 / Chapter 1.1.1.1 --- Type IIFN --- p.2 / Chapter 1.1.1.2 --- Type II IFN --- p.3 / Chapter 1.1.2 --- Biosynthesis of IFN --- p.3 / Chapter 1.1.3 --- IFN-α/β receptor and signal transduction --- p.8 / Chapter 1.1.4 --- Functions induced by IFN / Chapter 1.1.4.1 --- Antiviral activity of IFN-α/β --- p.11 / Chapter 1.1.4.1.1 --- PKR (double-stranded RNA-dependent protein kinase) --- p.11 / Chapter 1.1.4.1.2 --- The 2-5A synthetase/RNase L system (The 2-5A system) --- p.16 / Chapter 1.1.4.1.3 --- Mx proteins --- p.17 / Chapter 1.1.4.2 --- Immunomodulatory function of IFN-α/β --- p.18 / Chapter 1.1.4.3 --- Inhibition of cell growth --- p.18 / Chapter 1.1.4.4 --- Control of apoptosis --- p.19 / Chapter 1.1.5 --- The significance of IFN system --- p.20 / Chapter 1.1.6 --- Subtype of murine IFN-α --- p.21 / Chapter 1.1.7 --- Production of IFN in response to infection --- p.23 / Chapter 1.1.8 --- Existing methods to determine the IFN-α subtypes productionin response to stimulus --- p.24 / Chapter 1.2 --- Influenza virus --- p.27 / Chapter 1.2.1 --- Classification --- p.27 / Chapter 1.2.2 --- The structure of influenza virus --- p.29 / Chapter 1.2.3 --- The viral genome and proteins --- p.29 / Chapter 1.2.4 --- Replicative cycle of influenza virus / Chapter 1.2.4.1 --- "Virus adsorption, entry and uncoating" --- p.31 / Chapter 1.2.4.2 --- Transcription and replication of vRNA --- p.31 / Chapter 1.2.4.3 --- Synthesis of viral proteins --- p.32 / Chapter 1.2.4.4 --- Packaging and budding of progeny virus --- p.33 / Chapter 1.2.5 --- Viral inhibition of the IFN response --- p.33 / Chapter 1.3 --- Aim of study --- p.35 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Overall procedures --- p.37 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- "Cell line, bacterial strain and vector" --- p.40 / Chapter 2.2.2 --- Chemicals --- p.40 / Chapter 2.2.3 --- "Culture media, buffer and other solutions" --- p.41 / Chapter 2.2.4 --- Reagents and nucleic acids --- p.41 / Chapter 2.2.5 --- Reaction kits --- p.42 / Chapter 2.2.6 --- Solutions --- p.42 / Chapter 2.2.7 --- Solutions of reaction kits --- p.43 / Chapter 2.2.8 --- Major equipments --- p.44 / Chapter 2.2.9 --- Primers --- p.44 / Chapter 2.2.10 --- Cell lysate --- p.45 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Design of IFN-α whole coding region and subtype specific primers using OLIGO´ёØ ver 50 --- p.46 / Chapter 2.3.2 --- Construction of plasmid DNA with the whole coding region of IFN-α gene / Chapter 2.3.2.1 --- Isolation of genomic DNA from L929 cells --- p.46 / Chapter 2.3.2.2 --- Amplification of whole coding region of IFN-α subtypes --- p.47 / Chapter 2.3.2.3 --- Preparation of plasmid DNA --- p.48 / Chapter 2.3.2.4 --- Restriction digestion of the vector pBluescript SKII (-) with SmaI --- p.48 / Chapter 2.3.2.5 --- Purification of DNA fragments from agarose gel --- p.49 / Chapter 2.3.2.6 --- Blunt end ligation --- p.49 / Chapter 2.3.2.7 --- Transformation --- p.49 / Chapter 2.3.2.8 --- Screening by polymerase chain reaction --- p.50 / Chapter 2.3.2.9 --- DNA sequencing --- p.50 / Chapter 2.3.3 --- Test on IFN-α subtype specific primers / Chapter 2.3.3.1 --- Determination of the specificity of IFN-α subtype specific primers by PCR --- p.51 / Chapter 2.3.3.2 --- Determination of the amplification of a particular subtype in the presence of excess of other templates --- p.51 / Chapter 2.3.4 --- Induction of expression of IFN in fibroblast L929 cells / Chapter 2.3.4.1 --- By chemical agents Poly(I) Poly(C) and DEAE --- p.52 / Chapter 2.3.4.2 --- By infection with influenza virus (A/NWS/33 and B/Lee/40) --- p.52 / Chapter 2.3.5 --- Detection of IFN-α subtypes expression / Chapter 2.3.5.1 --- Isolation of total RNA by guandidium thiocyanate - cesium chloride ultracentrifugation --- p.53 / Chapter 2.3.5.2 --- Synthesis of first strand cDNA --- p.54 / Chapter 2.3.5.3 --- Normalization of RNA samples --- p.54 / Chapter 2.3.5.4 --- RT-PCR amplification of the IFN-α subtypes --- p.54 / Chapter 2.3.5.5 --- "RT-PCR amplification of IFN-γ, IFN-α receptor 1，IFN-α receptor 2 (transmembrane and soluble form) and IFN-(3" --- p.55 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Designing of primers for IFN-α genes --- p.56 / Chapter 3.1.1 --- Design of IFN-α primers for amplification of whole coding region --- p.56 / Chapter 3.1.2 --- Design of IFN-α subtype specific primers --- p.58 / Chapter 3.2 --- Cloning of the IFN-α subtype genes / Chapter 3.2.1 --- Purification of genomic DNA --- p.59 / Chapter 3.2.2 --- PCR conditions used for amplification of whole coding region of mIFN-α subtypes --- p.61 / Chapter 3.2.3 --- Subcloning the whole coding region of IFN-α genes from amplified fragments --- p.63 / Chapter 3.3 --- Optimization of specific amplification condition for subtype specific primers by cross-PCR --- p.64 / Chapter 3.4 --- Determination of the amplification of a particular subtype in the excess of other templates --- p.67 / Chapter 3.5 --- Determination of the gene expression in poly (I) poly (C) treated L929 cells / Chapter 3.5.1 --- Spectrophotometric analysis of total RNA --- p.70 / Chapter 3.5.2 --- Agarose gel electrophoresis of RNA --- p.72 / Chapter 3.5.3 --- Normalization of RNA sample --- p.73 / Chapter 3.5.4 --- Detection of IFN-α subtypes mRNA in L929 cell induced with poly(I) poly(C)-DEAE dextran --- p.74 / Chapter 3.5.5 --- "Detection of IFN-γ, IFN-α/β receptor and IFN-β expressionin Poly(I) Poly(C)-DEAE dextran or DEAE dextran treated L929 cells" --- p.70 / Chapter 3.6 --- Measurement of gene expression in L929 cells infected with influenza virus / Chapter 3.6.1 --- Spectrophotometric analysis of total RNA --- p.83 / Chapter 3.6.2 --- Agarose gel electrophoresis of RNA samples --- p.84 / Chapter 3.6.3 --- Normalization of RNA samples --- p.86 / Chapter 3.6.4 --- Detection of IFN-α subtypes mRNA in L929 cell infected with influenza A/NWS/33 virus --- p.87 / Chapter 3.6.5 --- Detection of IFN-α subtypes mRNA in L929 cells infected with influenza B/Lee/40 virus --- p.90 / Chapter 3.6.6 --- "Detection of IFN-γ, IFN-α/β receptor and IFN-β expression in L929 cells infected with A/NWS/33 and B/Lee/40" --- p.93 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.97 / REFERENCES --- p.105 / APPENDIX --- p.122

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

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 134-154).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves 113-139).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 126-146).

DISSERTATION (PH.D.)--THE UNIVERSITY OF MICHIGAN

Thesis (Ph. D.)--University OF MICHIGAN.

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2014-08-01T14:25:54Z No. of bitstreams: 1 Tese Waldyr.pdf: 1037034 bytes, checksum: 0951d0a31dbee3d54d18141b2e411b8d (MD5) / Made available in DSpace on 2014-08-01T14:25:54Z (GMT). No. of bitstreams: 1 Tese Waldyr.pdf: 1037034 bytes, checksum: 0951d0a31dbee3d54d18141b2e411b8d (MD5) Previous issue date: 2014 / Rio de Janeiro. Secretaria de Estado de Desenvolvimento Regional, Abastecimento e Pesca / No presente trabalho, foi realizada uma revisão bibliográfica sobre o vírus influenza, enfatizando sua ação e correlacionando-o com as espécies aviárias, com enfoque diferenciado para os pombos domésticos (Columba livia domestica). Alguns aspectos tais como as condições ambientais, o estreito contato com o homem e a disseminação destas aves nos centros urbanos e em diferentes pontos do mundo, foram considerados. Durante o estudo da interação entre homens e pombos, outro agente patogênico mereceu atenção, por ocasião da revisão bibliográfica e por ser considerado o impacto financeiro causado na avicultura, o vírus da doença de Newcastle. Concomitante à revisão, foram analisadas 322 amostras de fezes de pombos, coletadas em praças públicas em várias áreas do município do Rio de Janeiro. Para tal análise foram utilizadas diferentes técnicas, tais como: inoculação em ovos embrionados, hemaglutinação, inibição da hemaglutinação e reação da polimerase em cadeia. As amostras testadas não apresentaram atividade hemaglutinante, bem como, não apresentaram positividade para a presença do vírus influenza e nem para o vírus da Doença de Newcastle, quando submetidas à prova de PCR. Com isto, pôde-se verificar a inexistência dos vírus na população de aves estudada, o que demonstrou não oferecer risco potencial para transmissão destas patologias, no presente momento. / In the present work, influenza was carried through a bibliographical revision on the virus, emphasizing its action and correlating it with the avian species, with approach differentiated for the domestic pigeons (Columba livia domestica). Some aspects such as the ambient conditions, the narrow contact with the man and the dissemination of these birds in the urban centers at different points of the world had been considered. During the study of the interaction between men and pigeons, another pathogenic agent had deserved attention, for occasion of the bibliographical revision, to caused financial impact in the poultry keeping, the virus of Newcastle disease. Concomitant to the bibliographical revision, 322 excrement samples had been analyzed of pigeons, collected in public squares in some areas of the city of Rio de Janeiro. For such analysis, different techniques had been used, such as: inoculation in embrionatted eggs, hemaglutination, inhibition of the hemaglutination and polymerase chain reaction. The tested samples had not presented hemaglutination activity, as well as, had not presented positivism for the presence of the virus influenza and nor for the Newcastle virus when submitted to the test of PCR. With this, the inexistence of the viruses in the studied population of birds could not be verified, what it demonstrated not to offer potential risk for transmission of these pathologies, in the present moment.

Objetivos. Determinar las características clínicas y demográficas de la neumonía por el virus de influenza AH1N1/2009 pandémico en un hospital de referencia de Perú. Materiales y métodos. Se realizó un estudio serie de casos en niños hospitalizados por neumonía por influenza AH1N1/2009 pandémico en un hospital de referencia. Revisamos las historias clínicas entre los meses de junio a septiembre 2009. Todos los casos tuvieron confirmación virológica. Resultados. Se encontró 74 casos de neumonía por el virus de Influenza AH1N1/2009 pandémico (NVIp), de los cuales 50 tuvieron el diagnóstico de neumonía adquirida en la comunidad viral (NACv) y 24 con neumonía nosocomial viral (NNv) de los cuales 16 requirieron ventilación mecánica. Fallecieron 12, todos ellos con antecedentes de comorbilidad. Los casos NNv presentaron asociación estadística con mortalidad. En los casos NACv, los menores de 6 años representaron 72 % (36/50). La mediana de tiempo de enfermedad fue de 5 días. Los síntomas más frecuentes fueron fiebre, tos, rinorrea. Recibieron oseltamivir el 82 %. En la radiografía de tórax el 48 % de los casos presentó infiltrado en parches y el 44 % infiltrado intersticial en la radiografía de tórax. La proteína C reactiva (PCR) mayor a 10mg/L tuvo una asociación significativa con insuficiencia respiratoria (p <0,05). Conclusiones. Encontramos casos NNv quienes tuvieron mayor mortalidad, también los que presentaron el PCR elevado y los que presentaron condición preexistente. / ObjectiveTo determine the clinical and demographic characteristics of pneumonia with influenza virus AH1N1/2009 pandemic at the National Institute of Child. Methods. Retrospective case series in children hospitalized for influenza pneumonia pandemic AH1N1/2009 in a pediatric hospital. Reviewed the medical records between the months of June to September 2009. All cases had virological confirmation, we describe the clinical characteristics and conditions of severity. Results. A total of 74 children of pneumonia with influenza virus AH1N1/2009 pandemic (NVIp), of those 50 were community acquire pneumonia viral (NACv) and 24 pneumonia nosocomial viral (NNv), 16 required mechanical ventilation. 12 died, all had preexisting factors. NN cases showed statistical association with mortality. The most frequent factors were malnutrition, respiratory infections, congenital heart disease and neurological deficits In NACv cases the children under 6 years accounted for 72% (36/50). The median disease duration was 5 days. The most frequent symptoms were fever, cough, runny nose. Received oseltamivir 82%. The chest radiograph 48% of cases showed patchy infiltrates and 44% interstitial infiltrate on chest radiograph. Protein c reactive (CRP) more than 10mg / L was significantly associated with respiratory failure (p <0.05). Conclusions. Cases of NN found who had more mortality, even those who had the highest PCR and those with preexisting condition.