Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. Includes bibliographical references.

Thesis (M.A.)--Trinity International University, 2004. / Abstract. Includes bibliographical references (leaves 140-152).

Thesis (M.S.) in Marine Biology--University of Maine, 2006. / Includes vita. Includes bibliographical references (leaves 74-81).

Thesis (M.A.)--Trinity International University, 2004. / Abstract. Includes bibliographical references (leaves 140-152).

No description available.

No description available.

The ability of Escherichia coli to use the expression signals of mycobacterial genes was tested by inserting fragments of M. bovis BCG DNA into the E. coli promoter-probe plasmid pKK232-8. Comparison with the promoter activity achieved following insertion of restriction fragments of the E. coli host into. pKK232-8 revealed that a significant proportion of M. bovis BCG promoters were functional in E. coli. These results confirmed the suitability of E. coli as a host for the cloning and expression of mycobacterial genes. Using a variety of E. coli cloning vectors (pBR322, pUC13, EMBL4 and gtll), M. bovis BCG and M. leprae DM gene libraries were prepared. Recombinant M. bovis BCG clones were screened with rabbit antiserum and clones expressing M. bovis BCG antigens were identified. A pBR322/M. bovis BCG clone, expressing a 65KD molecule, was isolated and this antigen was shown to be cross-reactive with a 65KD M. leprae antigen. Recombinant gtll clones, expressing antigenic M. bovis BCG molecules, were also detected and a partial DM sequence was determined for one of these molecules. Moreover, recombinant gtll clones expressing (i) an 85KD biotinylated M. leprae molecule and (ii) an 85KD biotinylated M. bovis BCG molecule were also detected. In an attempt to test the feasibility of diagnosing leprosy by the presence of antibodies to specific antigens, antisera samples from leprosy patients and their contacts were screened for antibodies to mycobacterial antigens. Although only a small number of antisera were tested, a number of candidate antigens were identified.

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-22Bitstream added on 2014-06-13T20:29:53Z : No. of bitstreams: 1 jesuino_db_me_botfm.pdf: 3747670 bytes, checksum: f0d0340cc56341df27b2574498d32310 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / 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. / 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.

The obligate methylotroph, Methylophilus methylotrophus, used by ICI for its Single Cell Protein production, may represent a valuable alternative host organism to E. coli for the commercial production of heterologous gene products. The organism has the advantages of being safe, coupled with an ability to grow well, in large quantities, on a cheap carbon source. This thesis describes the construction, characterisation and analysis of a series of plasmid cloning vectors designed for use in M.methylotrophus. The vectors are based on the IncQ plasmid R300B and maintain a broad host range with an increased capacity for easy-to-use cloning sites mainly derived from the E.coli plasmids pBR322 and pBR328. All the vectors thus carry antibiotic- resistance genes containing restriction sites which could lead to insertional inactivation as a means of detecting recombinants. One plasmid in particular, pGSS33, has four antibiotic-resistance genes all of which contain at least one such restriction site. The first demonstration of expression of a eukaryotic coding sequence, murine dihydrofolate reductase (DHFR), in M.methylotrophus has been described. This has been followed by expression of E.coli B-galactosidase and synthetic human ?-1 interferon, all making use of pGSS vectors. The pGSS15-DHFR plasmid, pDHFR2.43, may turn out to be a valuable test plasmid for studying the stability of cloned eukaryotic coding sequences in both E.coli and M.methylotrophus; it is readily detected (trimethoprim-resistant) and can be grown with or without selection. A start has been made towards providing increased expression from vectors carrying strong E.coli promoters (lacUV5 and synthetic trp) which have been demonstrated to work well in M.methylotrophus. Broad host range cosmid vectors have been constructed which have the potential to be used for the production of gene-banks in Gram-negative organisms other than E.coli. Copy numbers of the vector plasmids have been determined in E.coli strains and several different methods of measurement reviewed in an attempt to find one suitable for use with M.methylotrophus. An encouraging lead has been identified in the search for a high copy number plasmid, which coupled with a strong promoter, could provide the basis for a very efficient batch production process. Thus with the availability of easy-to-use cloning vectors, convenient delivery systems and the accumulated evidence of strong E.coli promoters working efficiently in M.methylotrophus, this organism can seriously be considered as a safe alternative host to E.coli.

No description available.