This study was undertaken to investigate the structure-function relationship of VP6 protein of bluetongue virus (BTV) using molecular cloning techniques. VP6 is present in small quantities in BTV and its enzymatic activity and role in the viral replication cycle have not been studied. Since the availability of large amounts of purified VP6 is essential for the analysis of VP6, a BTV -11 S3 gene was cloned into a prokaryotic protein expression system. VP6 protein was expressed in large amounts and purified to near homogeneity. A series of C-terminal and internal deletion mutants of S3 gene was constructed and the truncated VP6 proteins were expressed and purified. The nucleic acid binding activities of the VP6 protein towards dsRNA, dsDNA, and ssRNA were confirmed and a new ssDNA binding activity was also determined. The binding activities of VP6 were concentration-dependent. The sites responsible for the binding activities were mapped using the truncated proteins and synthetic sequence-specific oligopeptides. Two domains of VP6 were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6. The binding affinities of the middle domain of VP6 towards single-stranded and double-stranded nucleic acid were slightly different. Three synthetic oligopeptides corresponding to these domains exhibited concentration-dependent nucleic acid binding activities. Based on these results I suggest that synthetic oligopeptides might be useful to screen nucleic acid binding activities and domains responsible for these activities. Expressed VP6 was used to produce polyclonal and monoclonal antibodies. Oligoclonal antibodies were raised by synthetic oligopeptides. Ten epitopes of VP6 were mapped and characterized. The amino acid sequences and sizes of six linear epitopes identified by oligoclonal antibodies were determined, and their locations were mapped and confirmed by deletion mutant analyses. These linear epitopes were surface-accessible except one. Based on these results I suggest that synthetic sequence-specific oligopeptides could mimic major components of antigenic determinants. Four epitopes recognized by four monoclonal antibodies were mapped and characterized. Three determinants were surface-accessible and three were conformational epitopes. These four determinants were distinct and different from the six linear epitopes determined using oligoclonal antibodies.

Made available in DSpace on 2014-07-29T15:26:22Z (GMT). No. of bitstreams: 1 Tese Talissa de Morais Tavares.pdf: 2094396 bytes, checksum: cca589203914a2c676b55bcf55e62bbc (MD5) Previous issue date: 2008-04-28 / Group A rotaviruses are the major cause of gastroenteritis in children throughout the world. Epidemiological surveys and molecular analysis of rotavirus strains are required for gastroenteritis control and prevention. Studies using VP6, an important immunogenic structural protein, and NSP4, a transmembrane nonstructural glycoprotein which is critical to rotavirus morphogenesis and pathogenesis, have been performed. In this study, 330 rotavirus-positive fecal samples previously obtained from children with or without diarrhea, between 1987 and 2003, in three cities of Central West Region of Brazil (Goiânia, Brasília and Campo Grande), were characterized for VP6- and NSP4-encoding genes. The VP6 and NSP4 genes were amplified by reverse transcription- polymerase chain reaction followed by sequencing and phylogenetic analysis. Detection rates of 84.8% and 78.5% were observed for VP6 and NSP4 genes, respectively. Two distinct genotypes could be recognized for NSP4 (A and B). It was observed that the G9P[6] samples were associated with genotype A, whereas the G1P[6], G1P[8], G2P[8], G3P[8], G4P[8] and G9P[8] samples were associated with genotype B. The analysis of VP6 gene allowed genogrouping of samples in two clusters, genogroups I and II. The G2P[4], G3P[4] and G9P[6] samples were identified as genogroup I, whereas the G1P[6], G1P[8], G2P[8], G4P[6], G4P[8] and G9P[8] samples were identified as genogroup II. In addition, it was showed that samples identified as VP6 genogroup I were associated with NSP4 genotype A and samples identified as VP6 genogroup II were associated with NSP4 genotype B. This investigation described different genetic groups representing diversity of group A rotavirus samples circulating in the Central West Region of Brazil. / Os rotavírus do grupo A são considerados como os principais agentes de gastroenterite em crianças em todo o mundo. Investigações de vigilância epidemiológica e molecular são importantes para o controle e prevenção da doença. Nesse sentido, destacam-se os estudos utilizando VP6, proteína estrutural que se tem mostrado como a mais imunogênica, e NSP4, uma glicoproteína não estrutural transmembrana envolvida na morfogênese e patogênese viral. No presente estudo, 330 amostras de rotavírus coletadas de 1987 a 2003, provenientes de espécimes fecais de crianças com ou sem diarréia, em três cidades da Região Centro-Oeste do Brasil (Goiânia, Brasília e Campo Grande), foram caracterizadas em relação aos genes que codificam as proteínas VP6 e NSP4. Inicialmente, foi feita a amplificação dos genes de VP6 e NSP4 pela reação em cadeia pela polimerase pós-transcrição reversa, seguida do seqüenciamento genômico e de análise filogenética. Os genes de VP6 e NSP4 foram detectados em 84,8% e 78,5% das amostras, respectivamente. Dois genótipos de NSP4 foram identificados (A e B). Foi observado que as amostras G9P[6] associaram-se ao genótipo A e as amostras G1P[6], G1P[8], G2P[8], G3P[8], G4P[8] e G9P[8] associaram-se ao genótipo B. A análise do gene VP6 permitiu a identificação de dois genogrupos distintos (I e II). As amostras G2P[4], G3P[4] e G9P[6] foram caracterizadas como genogrupo I, enquanto as amostras G1P[6], G1P[8], G2P[8], G4P[6], G4P[8] e G9P[8] foram caracterizadas como genogrupo II. Ainda foi evidenciado que as amostras genogrupo I de VP6 estavam associadas com genótipo A de NSP4, e as amostras genogrupo II de VP6 estavam associadas com genótipo B de NSP4. A presente investigação identificou diferentes variantes genéticas, mostrando a diversidade dos rotavírus do grupo A circulantes na Região Centro-Oeste do Brasil.

Rotavirus is responsible for the death of many children annually, and current vaccines have lower efficiency in developing countries. A reverse translated consensus gene sequence of the rotavirus VP6 cloned into a pET-28a(+) plasmid was used to transform BL21 and KRX Escherichia coli cells. Optimal expression of soluble protein was induced in KRX cells by adding 0.05% L-rhamnose and 0.0001 M IPTG, with an incubation temperature of 25ºC for 6 h. VP6 was purified by combining anion exchange chromatography followed by affinity chromatography. Far-UV circular dichroism and intrinsic fluorescence were used as probes to assess the native structure of VP6 and structural in the presence of a denaturant, high sodium chloride concentrations and varying temperatures. The 0.2 M sodium chloride had an impact on the VP6’s tertiary structure and also influenced the proteins conformational changes as detected during thermal unfolding to 90ºC. Although treatment with 3 M urea showed tertiary structural changes no secondary structural loss occurred due to the presence of a denaturant. / Life Sciences / M. Sc. (Life Sciences)

Im Rahmen der vorliegenden Diplomarbeit werden browserbasierte Videoüber-tragungslösungen bezüglich ihrer Verwendbarkeit zur Übertragung des Fernsehprogramms regionaler Fernsehsender auf das Medium Internet untersucht, mit dem Ziel eine Prototypanwendung auf Basis der gemäß den Anforderungen der Regionalsender am besten geeigneten Technik zu implementieren. Grundlage der Untersuchung bildet eine Gegenüberstellung der am Markt be-findlichen Videosysteme mit Webbrowserunterstützung QuickTime, RealVideo, Windows Media, Adobe Flash und Java. Die Systeme werden bezüglich ihres Leistungsumfangs, der Flexibilität ihrer Einsatzmöglichkeiten, der Qualität der zugrundeliegenden Videocodecs und ihrer Verbreitung im Internet verglichen. Anhand der gemeinsam mit den Regionalsendern KabelJournal und Sachsenfernsehen ermittelten Anforderungen an ein Videosystem zur Übertragung ihres Programms im Internet, erweist sich Adobe Flash Video durch seine hohe Verbreitung, seine flexiblen Einsatzmöglichkeiten und seiner Unterstützung von Bewegtbild-, Standbild- und interaktiven Animationsmedien als am besten geeignet. Die im Rahmen dieser Arbeit erstellte Implementierung einer Web-TV Anwen-dung auf Basis von Adobe Flash zeigt bei der Ermittlung von konkreten Einsatzszenarien bei den Sendern KabelJournal und Sachsenfernsehen, dass durch die multimediale Auslegung, die flexiblen Medienkombinationsmöglichkeiten und die offene XML-Schnittstelle mehr als eine bloße Portierung des TV-Programms ins Internet mit gleichzeitig geringem Aufwand möglich ist. Die Web-TV Anwendung ist eine einfach einsetzbare aber höchst vielseitige Möglichkeit für Regionalsender neue Märkte zu erschließen. / Within the present diploma thesis, established web browser based video solu-tions are examined for their use as internet broadcasting system for local TV stations, with the intention of implementing a broadcasting solution based on the technique matching the requirements of local TV stations best. Basis of this examination is the comparison of the web video solutions Quick-Time, RealVideo, Windows Media, Adobe Flash and Java. These systems are compared by their multimedia support, flexibility of use, quality of the video co-decs they use and their market penetration. On the basis of the requirements of local TV stations, determined together with KabelJournal and Sachsenfernsehen, adobe flash is the most satisfying solution to build a Web TV application upon. Its market penetration, flexibility of use and its support for still images, videos and interactive animations match those requirements best. The implementation of the Web TV application based on adobe flash proofs its great flexibility, ease of use and its diverse variations in combining different me-dia, at the determination of concrete use cases together with KabelJournal and Sachsenfernsehen. It shows that this is an easy but high potential way to enter new markets for local TV stations.

Im Rahmen der vorliegenden Diplomarbeit werden browserbasierte Videoüber-tragungslösungen bezüglich ihrer Verwendbarkeit zur Übertragung des Fernsehprogramms regionaler Fernsehsender auf das Medium Internet untersucht, mit dem Ziel eine Prototypanwendung auf Basis der gemäß den Anforderungen der Regionalsender am besten geeigneten Technik zu implementieren. Grundlage der Untersuchung bildet eine Gegenüberstellung der am Markt be-findlichen Videosysteme mit Webbrowserunterstützung QuickTime, RealVideo, Windows Media, Adobe Flash und Java. Die Systeme werden bezüglich ihres Leistungsumfangs, der Flexibilität ihrer Einsatzmöglichkeiten, der Qualität der zugrundeliegenden Videocodecs und ihrer Verbreitung im Internet verglichen. Anhand der gemeinsam mit den Regionalsendern KabelJournal und Sachsenfernsehen ermittelten Anforderungen an ein Videosystem zur Übertragung ihres Programms im Internet, erweist sich Adobe Flash Video durch seine hohe Verbreitung, seine flexiblen Einsatzmöglichkeiten und seiner Unterstützung von Bewegtbild-, Standbild- und interaktiven Animationsmedien als am besten geeignet. Die im Rahmen dieser Arbeit erstellte Implementierung einer Web-TV Anwen-dung auf Basis von Adobe Flash zeigt bei der Ermittlung von konkreten Einsatzszenarien bei den Sendern KabelJournal und Sachsenfernsehen, dass durch die multimediale Auslegung, die flexiblen Medienkombinationsmöglichkeiten und die offene XML-Schnittstelle mehr als eine bloße Portierung des TV-Programms ins Internet mit gleichzeitig geringem Aufwand möglich ist. Die Web-TV Anwendung ist eine einfach einsetzbare aber höchst vielseitige Möglichkeit für Regionalsender neue Märkte zu erschließen. / Within the present diploma thesis, established web browser based video solu-tions are examined for their use as internet broadcasting system for local TV stations, with the intention of implementing a broadcasting solution based on the technique matching the requirements of local TV stations best. Basis of this examination is the comparison of the web video solutions Quick-Time, RealVideo, Windows Media, Adobe Flash and Java. These systems are compared by their multimedia support, flexibility of use, quality of the video co-decs they use and their market penetration. On the basis of the requirements of local TV stations, determined together with KabelJournal and Sachsenfernsehen, adobe flash is the most satisfying solution to build a Web TV application upon. Its market penetration, flexibility of use and its support for still images, videos and interactive animations match those requirements best. The implementation of the Web TV application based on adobe flash proofs its great flexibility, ease of use and its diverse variations in combining different me-dia, at the determination of concrete use cases together with KabelJournal and Sachsenfernsehen. It shows that this is an easy but high potential way to enter new markets for local TV stations.