Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 128-141). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 128-141). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 161 pages. Includes Vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains ix, 70 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 63-68).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 15, 2010). Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 130-141.

A potential approach to in vivo gene therapy is to target retrovirus to specific receptors through a ligand-receptor interaction. Previous studies have placed a ligand at or close to the N-terminus of the ecotropic Moloney murine leukemia virus envelope and require co-expression of a wild type envelope on the pseudotyped virus for successful transduction of human cells. In this study, over forty chimeric envelopes were generated, which have single or multiple insertions of a 13 or 21 amino acid RGD containing sequence, flanked by cysteine residues, that target the cellular integrin receptors (Chapter III). Virus displaying only the chimeric envelopes was generated from packaging cell lines that express the gag and pol genes. Many of the mutant envelopes demonstrated the formation of syncytia when they were transfected into the XC indicator cell line, which is frequently used to determine envelope binding and fusion capabilities. Pseudotyped virus for several of the chimeric envelopes, transduced both NIH 3T3 mouse fibroblasts and human A375 melanoma cells. Ligands placed in the N-terminal region, within the VRA variable domain, and close to the N-terminus of the proline-rich region (PRR), demonstrated transduction into human melanoma cells. Ligands placed within the PRR and the C-terminus of the envelope did not demonstrate transduction into melanoma cells, although host cell transduction was demonstrated. Pseudotyped virus expressing an RGE containing target sequence, replacing the RGD sequence, had significantly lower transduction efficiency of melanoma cells. These data indicate that the MLV envelope tropism can be altered by insertion of short ligands at various locations throughout the envelope. These initial results were promising and helped to define regions within the envelope that could accommodate the insertion of small targeting ligands, that could redirect the tropism of pseudo typed virus to human cells. In the second part of this study, the focus shifted to targeting receptors that were expressed on specific cells, such as carcinoma cells. We inserted short ligands, flanked with cysteines, into the envelope to generate numerous targeting constructs that bind to receptors over-expressed on a variety of carcinoma cells. These pseudotyped retroviral vectors were generated by packaging cell lines that express only the viral Gag and Pol genes, with no wild-type envelope present. Select chimeric envelopes that express the 21 amino acid bombesin (BN)/gastrin releasing protein (GRP) binding sequence successfully transduced human melanoma cells, breast cancer cells, and cells that express the cloned GRP receptor gene. Nine additional chimeric envelopes were generated, that express a modified 56 amino acid heregulin sequence (HRG), that targets c-rbB-3 (Her-3) and c-erbB-4 (Her-4) receptors on breast carcinoma cells. Pseudotyped virus expressing only the BN/GRP mutant envelopes, transduced NIH 3T3 host cells, and two human carcinoma cell lines; A375 melanoma and MDA-MB-231 breast cells. The HRG chimeric envelopes demonstrated transduction of NIH 3T3 cells and human MDA-MB-453 breast carcinoma cells. Finally, a pseudotyped virus that expressed the chimeric BN/GRP envelopes and packaged the thymidine kinase gene, transduced melenoma and breast carcinoma cells and demonstrated ganciclovir cytotoxicity. Collectively, these data indicate that ligands of various sizes can be used to target pseudotyped virus to a variety of human cancer cells and transfer genes of interest. These findings may expand the feasibility and potential scope of gene therapy.

As doenças cardiovasculares representam a maior causa de mortalidade a nível mundial. Desde o conhecimento da importância da mitocôndria no metabolismo do cardiomiócito, alterações no funcionamento desta organela têm sido associadas a um dos principais causadores do infarto do miocárdio e consequente morte celular. O cofator de transcrição PGC-1alfa tem sido alvo de diversos estudos relacionados com o metabolismo celular devido à sua forte participação na biogênese mitocondrial. Considerando a limitação de material biológico para o estudo de doenças cardíacas, muito se tem investido no estudo de células-tronco pluripotentes induzidas (iPSCs). Esta tese teve como principal objetivo a avaliação dos efeitos da variação da expressão de PGC-1alfa em iPSCs e na sua diferenciação em cardiomiócitos. Após estabelecimento de um protocolo de reprogramação celular, em que ocorre geração de iPSCs a partir de fibroblastos humanos, induzimos a inibição da expressão de PGC-1alfa em 50% e 70% pelo uso de vetores lentivirais, e analisamos o estado de pluripotência através da avaliação de expressão genica e proteica dos principais marcadores - SSEA4, TRA-1-60, OCT4, NANOG, SOX2, REX1, TRA-1-81. Não observamos diferenças significativas no conteúdo destes marcadores entre os clones de iPSC controle e inibidos. Estabelecemos um protocolo de diferenciação de iPSCs em cardiomiócitos com elevada taxa de reprodutibilidade, através da adaptação de protocolos descritos na literatura, e submetemos estas iPSCs à diferenciação. As células geradas pela diferenciação do clone controle apresentaram características típicas de cardiomiócito: contratilidade e alta expressão molecular de troponina T e troponina I. Em contraste, as células com 70% de inibição de PGC-1alfa se mostraram incapazes de contrair e com baixa expressão de troponina. Através de uma análise dos níveis de expressão genica e proteica de diversos marcadores expressos durante o processo de diferenciação (T, NKX2.5, MIXL1, MYL7, ISL1), observamos que o clone com maior inibição de PGC-1alfa apresentou sempre níveis de expressão diminuídos em relação aos clones controle. Em conclusão, podemos afirmar que o PGC-1alfa não interfere com as características de auto-renovação e pluripotência das iPSCs mas possui um papel essencial na diferenciação de células-tronco pluriotentes induzidas em cardiomiócitos. Os resultados obtidos contribuem para informações preliminares acerca do desenvolvimento de iPSCs com inibição da expressão de PGC-1alfa durante a diferenciação cardíaca, mas estudos relativos ao potencial papel deste cofator durante o desenvolvimento cardíaco in vivo ainda precisam ser aprofundados, utilizando outros modelos de estudo / Cardiovascular diseases are the leading cause of mortality worldwide. Since the knowledge of the importance of mitochondria in the cardiomyocyte metabolism, changes in the functioning of this organelle has been associated with one of the main causes of myocardial infarction and subsequent cell death. The transcriptional cofactor PGC-1alpha has been subjected to several studies related to cell metabolism due to its strong involvement in mitochondrial biogenesis. Considering the limitations of biological material in the study of heart disease, there has been a lot of investment in the study of induced pluripotent stem cells (iPSCs). The main objective of this thesis was to evaluate the effects of the variation in expression of PGC-1alpha in iPSCs and it\'s differentiation in cardiomyocytes. After the estabilshment of a cellular reprogramming protocol, where iPSCs is generated from human fibroblasts, the expression of PGC-1alpha was induced by 50% and 70% with the use of lentiviral vectors and the state of pluripotency was determined by analyzing the gene and protein expression of the main markers - SSEA4, TRA- 1-60, OCT4, NANOG, SOX2, REX1, TRA- 1- 81. There were no significant differences observed in the content of these markers between the iPSC clones control and inhibited. A protocol for the differentiation of iPSCs into cardyomyocites was established with a high reproducibility rate, by adapting existing protocols in the general literature, submiting these iPSCs into diferentiation. The cells generated from the differentiation of the control clone showed typical characteristis of cardiomyocytes: contractility and high molecular expression of troponin T and troponin I. In contrast, the cells with 70% inhibition PGC-1alpha were unable to contract and had low troponin expression. Through an analysis of gene expression and protein levels of several markers expressed during the differentiation process (T, Nkx2.5, MIXL1, MYL7, ISL1), the clone with greater inhibition of PGC-1alpha always showed decreased expression levels compared to control clones. In conclusion, we can say that the PGC-1alpha does not interfere with the characteristics of self-renewal and pluripotency of iPSCs but has an essential role in the differentiation of pluripotent stem cells induced into cardiomyocytes. These results were obtained thanks an original approche based on iPSC technology enabling genetic modifications of the cells and controled differentiation into cardiomyocytes, but the potential role of PGC-1alpha on in vivo cardiac development or cardiomyocytes maturation remaisn to be evaluated using other models