Thesis (PhD(HealthSciences))--University of South Australia, 2001.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 117-130). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 117-130). Also available on the Internet.

Thesis (Ph. D.)--University of Hong Kong, 1998. / Cover title: Genomic isolation and molecular analysis of a rat alpha-globin gene cluster. Includes bibliographical references (leaf 163-204).

Thesis (M. Phil.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 100-128).

Thesis (M. Phil.)--University of Hong Kong, 1990.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-21Bitstream added on 2014-06-13T19:43:43Z : No. of bitstreams: 1 000737473.pdf: 4424353 bytes, checksum: 83d4de348d8c1081e547b22ff3eab4d2 (MD5) / Lipoxigenases são enzimas que catalizam a oxido-redução de ácidos graxos poliinsaturados que contém em sua estrutura um ou mais grupos cis 1,4- pentadieno, produzindo hidroperóxidos através da incorporação de um oxigênio molecular. Durante anos a presença de lipoxigenase foi considerada exclusivamente eucariótica, presente em mamíferos, plantas, pequenos invertebrados marinhos e fungos. A função biológica dessas enzimas tem sido amplamente estudada. Porém, pouco tem sido descrito em organismos procariotos. O presente estudo teve por objetivos clonar e caracterizar bioquimicamente duas lipoxigenases de Shewanella woodyi ATCC 51908, caracterizar os produtos de reação destas enzimas e realizar um estudo filogenético e estrutural, comparando-as com lipoxigenases de eucariotos e procariotos. Parâmetros enzimáticos dessas enzimas foram descritos. A influência do pH e temperatura na atividade catalítica foram estudadas. Também foi determinado a preferência por substrato e o efeito da adição de vários íons divalentes que podem interferir na atividade catalítica. A enzima SWPrecLox de S. woodyi apresentou temperatura e pH ótimos de 31ºC e 8,0, respectivamente. Demonstrou afinidade por ácido linoleico, com uma Km de 0,47 mM e Vmax de 2,78 mmols min-1 mg-1. A enzima SWAracLOX teve ácido araquidônico como substrato preferente, com valores de Km 0,20 mM e Vmax 1,6 mmols min-1 mg-1. Atividade ótima foi alcançada com 27ºC e pH 7,0. A partir das estruturas moleculares das lipoxigenases de Plexaura homomalla e Pseudomonas aeruginosa, foram criados hipotéticos modelos estruturais de SWPrecLOX e SWAracLOX. / Lipoxygenases are enzymes that catalyze the oxido-reduction of polyunsaturated fatty acids in their structure that contains one or more groups cis 1,4- pentadiene, producing hydroperoxides from the incorporation of molecular oxygen. For years, the presence of lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates and fungi. The present study aimed to clone and characterize biochemically two lipoxygenases from Shewanella woodyi ATCC 51908, characterize the reaction products of these enzymes and conduct a phylogenetic and structural analysis, comparing them with eukaryotes and prokaryotes lipoxygenases. The biological function of these lipoxygenases has been widely studied. However, only few have been described in prokaryotes. Enzymatic parameters of these enzymes have been described. The influence of the pH and the temperature on the catalytic activity has been studied. Also has been studied the substrate preference and the effect of the addition of several divalent cations that can enhance or eliminate the catalytic activity. The enzyme SWPrecLox of S. woodyi showed temperature and pH optimum were 31 ºC and 8,0, respectively. Demonstrated substrate affinity for linoleic acid, with Km 0,47 mM and Vmax 2,78 mmols min-1 mg-1. The enzyme SWAracLox has arachidonic acid as preferred substrate, with Km 0,20 mM and Vmax 1,6 mmols min-1 mg-1. Optimum activity was reached at 27 ºC and pH 7,0. From the molecular structures of lipoxygenase Plexaura homomalla and Pseudomonas aeruginosa, were created a hypothetical structural model of the SWPrecLox and SWAracLox.

Orientador: Elenice Deffune / Banca: Wirla Maria da Silva Cunha Tamashiro / Banca: Wagner José Fávoro / Resumo: Anticorpos monoclonais (mAbs) são ferramentas-chave na investigação de fenômenos biológicos e uma promissora alternativa para o tratamento de muitas doenças, em especial os tumores. A cadeia produtiva de mAbs apresenta pontos cruciais para o desenvolvimento de um produto de qualidade que justifique os altos custos de produção. O procedimento de clonagem celular é a etapa de produção que garante a monoclonalidade das células produtoras, assegurando a especificidade do mAb. Desde os anos 80 existem tentativas de otimização e automação deste procedimento visando a garantia da monoclonalidade e aumento na eficiência de clonagem. A captura celular sob observação direta ao microscópio pode ser considerada a única metodologia de clonagem celular que garante 100% de crescimento monoclonal. Neste trabalho avaliamos indicadores de eficiência de clonagem, tempo de execução e custos de uma metodologia de clonagem celular por micromanipulação em comparação com a metodologia de diluição limitante. Hibridomas murinos secretores de mAbs de diversas especificidades foram clonados em paralelo por ambas as técnicas. Não houve diferença no tempo de execução das metodologias embora a observação dos clones da micromanipulação seja de 4 a 6 vezes mais rápida. A produção de clones por micromanipulação é 4 vezes superior à diluição limitante com redução de 60% no consumo de meio de cultura, o que justifica os custos da metodologia. Concluímos que a clonagem celular por micromanipulação é uma técnica precisa e que garante 100% de crescimento monoclonal podendo vir a substituir a diluição limitante. Esta garantia elimina testes de comprovação da monoclonalidade e reduz o tempo de cultura necessário para obtenção de mAbs. / Abstract: Monoclonal antibodies (mAbs) are outstanding tools for investigating biological phenomena and a promising alternative to treat several diseases, specially tumors. Production of mAbs has settled critical milestones at the production chain which justifies high development costs. Cell cloning procedure is the milestone that guarantees monoclonality and assures antibody specificity. Since the 80's there have been attempts to optimize and automate this procedure aiming to guarantee monoclonality and enhance cloning efficiency. Celular capture under direct microscope observation is the only method considered to guarantee 100% monoclonal growth. On this work we evaluated cloning efficiency, working time and costs of a micromanipulating cloning technique compared with gold-standard limiting dilution. Murine secretor hybridomas of various specificity were cloned by both techniques in parallel. No differences were observed at both working times although observation of micromanipulated clones was 4 to 6 times faster. Cloning efficiency of micromanipulation cloning was 4 times higher than limiting dilution with 60% reduction on media consume what justifies methodology costs. Cell cloning by micromanipulation is a precise procedure that guarantees 100% monoclonal growth and could substitute limiting dilution. This guarantee eliminates additional tests to assure monoclonality and reduces culture working time on mAbs production. / Mestre

This study was focused on the promoter and non-coding regions of the ribosomal RNA (rrn) operon(s) of mycobacteria; namely, the leader and the intergenic spacer regions. Two clones containing the promoter sequences of M .leprae and M. tuberculosis rrn operon were sequenced, their promoter elements were identified by primer extension experiments and by comparison with E.coli consensus promoter sequences. Their function was tested in E.coli and M. smegma tis . The sequences of the leader and intergenic spacer regions from eight and six species respectively were established after amplification by means of peR. Both leader and spacer regions contain antitermination elements and RNaseIII processing sites. The sequences established for these two regions also showed greater variability than the 168 rRNA gene and are suitable for phylogenetic studies. The sequences of the two rrn operons of M.smegmatis upstream from the 168 rRNA gene were cloned and sequenced. Their sequences showed that rrnI has a Box B element which is typical of slow-growers and that rrnII does not. Primer extension studies revealed that the rrn operon of slow-growers has a single promoter. In contrast multiple promoters were identified in the faster-growing M.smegmatis. Distinctive features, which are absent from slow-growers, were identified in the intergenic spacer regions of M.smegmatis.