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Made available in DSpace on 2014-10-09T12:42:17Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:00Z (GMT). No. of bitstreams: 0 / Com a intenção de estabelecer métodos físico-químicos como uma alternativa ao bioensaio in vivo para determinação de atividade biológica, o conteúdo de hFSH, hTSH e hLH de diferentes preparações, nativas e recombinantes, foi determinado por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) e comparado ao dado obtido pelo clássico bioensaio in vivo em camundongos ou ratos (BA). Para estes hormônios foi encontrada uma relação linear entre os dois métodos: hFSH BAUI = 0,9925 RP-HPLCUI - 1,3165, r = 0,9371, p < 0,001, n = 24; hTSH BA&mu;g = 0,9790 RP-HPLC&mu;g - 0,052, r = 0,8725 , p < 0,001, n = 14; hLH BAUI = 0,8771 RP-HPLCUI + 12,41; r = 0,9786, p < 0,01, n = 5. Para outras nove preparações de hFSH e onze preparações de hTSH foi determinada a diferença média (d) entre a bioatividade predita pela RP-HPLC através destas equações e da média das bioatividades obtidas com os dois métodos. Para o hLH não foi possível determinar esta diferença em virtude das poucas amostras disponíveis. No caso do hFSH, d ± DP = -2,11 ± 3,49 % sendo a precisão de 1,16% e no caso do hTSH, d ± DP = -2,01 ± 5,56 % com precisão de 1,68%. Amostras parcialmente alteradas apresentaram diferentes graus de atividade de hFSH, hTSH e hLH que puderam ser preditas por RP-HPLC com uma aceitável concordância com os bioensaios in vivo. Estes resultados demonstraram que o emprego de um ensaio físico-químico sem o uso de animais, tal como a RP-HPLC, é uma alternativa viável ao uso do bioensaio in vivo para a determinação da potência de hFSH e hTSH, reduzindo assim o número de animais em geral utilizados para assegurar a qualidade e eficácia de um produto farmacêutico. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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A função e a arquitetura dos genes está começando a ser elucidada a partir do estudo de genomas completos tanto de procariotos como de eucariotos. Diversos estudos foram ultimamente realizados a respeito de exon shuffling do ponto de vista evolutivo, fenômeno relacionado à origem de novos genes através de recombinações de DNA mediadas por introns. Apesar de eventos de exon shuffling serem responsáveis pelo aumento da modularidade gênica, outros processos foram desenvolvidos ao longo da evolução para que houvesse o aumento da diversidade do proteoma sem a conseqüente expansão dos genomas, sendo splicing alternativo um dos mais freqüentes. Apresentamos nesta dissertação duas extensivas análises: 1) a análise de uma base de dados de genes eucarióticos contendo pelo menos um intron que apresentou excesso de introns de fase 0 e exons simétricos, dados que suportam exon shuffling como um importante mecanismo de evolução gênica. Avaliamos também a confiabilidade de introns preditos por programas de computador através de alinhamento de ESTs; e 2) a análise do uso alternativo de exons (UAE), um tipo de splicing alternativo, em transcritos humanos detectando que cerca de 51% dos genes humanos possuem mais de uma variante de splicing e que este tipo de processamento pós-transcricional parece ser mais freqüentemente encontrado em tecidos tumorais. / Abstract not available.

Genome editing technologies have advanced from methods that rely on nucleases to catalyze programmed double-strand breaks (DSBs), which are known to cause deleterious side effects, to next-generation reagents that perform more controlled chemistry using DSB-independent approaches. Base editing and prime editing are ideally suited for small-scale modifications, but methods to achieve large-payload gene insertion have been lacking. Recent technology development efforts have advanced strategies that employ eukaryotic transposases, bacterial recombinases/transposases, and retroelements, yet these enzymes broadly suffer from either inflexibility of target site requirements or non-specific, genome-wide insertion profiles. The ability to precisely and safely insert kilobase-scale DNA cargos at user-defined loci remained challenging due to a dearth of programmable transposase tools. CRISPR-associated transposon (CAST) systems represent a unique opportunity to solve this longstanding challenge. CASTs are diverse (types I-B, I-D, I-F, and V-K) heteromeric, macromolecular machineries that require multivalent protein-protein interactions, each of which represents a potential kinetic bottleneck, inefficiency, or failure point when porting from bacteria to mammalian cells. Through meticulous, step-by-step engineering involving functional assays, bioinformatic mining, homolog screening, structure-guided engineering, and directed evolution, we have iteratively improved overall editing efficiencies of type I-F CASTs, identified an essential bacterial co-factor, and reached editing efficiencies that approach therapeutic relevance. Together, this work represents a critical advancement towards a broad platform for targeted genomic integration of large DNA payloads.

The ability of a genetically-engineered <i>Erwirzia carotovora</i> subsp. <i>carotovora</I> (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or <i>Pseudomonas fluorescens</i> was tested on filters, within soil microcosms, and <i>in planta</i>. Ecc was engineered by chromosomal insertion of a disarmed <i>endo</i>-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10⁻² transconjugants per donor (TPD) and to P. <i>fluorescens</i> at a frequency of 2.4 X 10⁻⁵ TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10⁻³ and 2.0 X 10⁻³, while matings on potato slices yielded frequencies of 4.7 X 10⁻² and 2.3 X 10⁻². In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10³ and 8.4 X 10⁻⁵ TPD. / Master of Science

The unfolding of recombinant DNA, from research technique to political issue, is described. As a research technique, recombinant DNA (abbreviated rDNA) has opened up new vistas in biological and other fields of research. But its potential yet unproven hazard has created uneasy feelings toward the technique. The controversial nature of the issue finally launched rDNA into the political sphere, involving scientists, the public at large, and Congress in efforts to control the development of the field. The first group to regulate rDNA was the scientists. The scientific community called for a voluntary moratorium on experiments perceived as potentially dangerous at the time. It was an unprecedented act. The National Institutes of Health subsequently issued guidelines for a safe execution of rDNA experiments to minimize potential dangers to public health and well-being. Efforts of the scientific community to control rDNA was seen, however, as a politics of expertise. Challenges to this "technocratic" approach soon emerged. Vocal members of the public suspected expert decision makers as being biased toward scientific interests, reducing rDNA to a technical issue. They rejected the experts’ tunnel vision and demanded a say in decisions. Public participation in the decision-making process precipitated community debates at locations where rDNA research was ongoing. A democratic approach to decision-making proved to be a viable policy-making mode. The ensuing local and state laws, however, seemed inadequate to cover global consequences of rDNA. In an effort to unify regulations of the field, Congress attempted to legislate on the subject. Resistance from the scientific community, which regard legislative control as rigid and unnecessary, was one of the causes of diminishing congressional interest in the matter. None of the introduced bills was enacted. For complex policy areas with uncertain yet far-reaching scientific and societal consequences -- like rDNA -- this dissertation recommends a policy-making process where scientists, interested lay persons, politicians, public administrators, and other relevant parties participate in structured communications prior to an emerging controversy. To facilitate the process, establishment of National Science Fora is recommended. / Ph. D.