Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references.

Translational efficiency is dictated in part by the availability of charged transfer RNA. Depletion of aminoacylated tRNAs (e.g. during recombinant protein expression) can increase translational errors and associated stress responses. Here, the role of tRNAs as regulators of gene expression was explored through development of synthetic, tRNA-regulated gene circuits, and through an investigation of the impact of tRNA aminoacylation on endogenous gene expression. Synthetic gene circuits initially explored the use of dominant negative alleles of the release factor eRF1 to modulate stop codon readthrough and translationally regulate gene expression. Mutant eRF1 proteins exhibited only a six-fold stimulatory effect on stop codon readthrough. The dominant negative phenotype was rescued partially by overexpression of eRF1, but not eRF3. Ultimately the severity of growth inhibition by these eRF1 alleles limited their utility in synthetic gene circuit design. A novel synthetic circuit was then implemented that utilised TetR interaction with a TetR-inducing peptide in order to control the expression of a suppressor tRNA, and thus a luciferase reporter gene. Using a parameterised mathematical model, the promoter configuration of the circuit was successfully optimised, allowing suppressor tRNAs to regulate the production of luciferase in both feedforward and positive feedback modes of operation. The effects of charged tRNA levels on the global translation network were dissected by regulating the S.cerevisiae glutamine tRNA synthetase gene GLN4 using a tet-off doxycyclineregulated promoter. tRNA synthetase depletion caused the activation of the Gcn4 amino acid starvation response due to accumulation of uncharged glutamine tRNAs. Doxycycline GLN4 shut-off caused increased amino acid production, and decreased ribosome biosynthesis at the transcriptomic and proteomic level, and further physiological changes proposed to result from compromised translation of glutamine-rich regulatory proteins. tRNA overexpression in the GLN4 depletion strain successfully caused altered competition between different isoacceptor tRNA types for their cognate synthetase resource. Together, these results support a growing understanding of tRNA as a key modulator of translation and gene expression in synthetic and natural systems.

No description available.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Introdução: O carcinoma de células renais (CCR) representa uma causa crescente de mortalidade por câncer. Apesar da sua curabilidade em estádios iniciais, agentes citotóxicos e imunomodulatórios têm homogeneamente alcançado nenhum ou pouco benefício no tratamento do CCR avançado. Um melhor entendimento da biologia tumoral pode levar ao desenvolvimento de terapias mais eficientes e dirigidas aos mecanismos moleculares de manutenção do tumor. Sinalização aumentada através do receptor do fator de crescimento epitelial (EGFR), sistema de quinases proteicas ativadas por mitógenos (MAPK) e via da fosfatidilinositol 3-quinase (PI3K) assim como estimulação do complexo de início de tradução (CIT) foram caracterizados em linhagens celulares e amostras tumorais de CCR. Nós investigamos o efeito de um inibidor de EGFR (gefitinibe), de um inibidor de MAPK (UO126) e de um inibidor do CIT (rapamicina) nos intermediários de sinalização celular e nos elementos do CIT assim como no crescimento in vitro de linhagens celulares de CCR. Métodos: Linhagens celulares de CCR (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN e KRCY) foram mantidas em condições ideais para a cultura de células de mamíferos e expostas a drogas e/ou inibidores nas concentrações e por períodos de tempo variáveis. A fosforilação de intermediários da sinalização celular foi determinada utilizando-se western blots. Nível de mRNA para genes de interesse foram determinados por qRT-PCR. Crescimento celular foi avaliado por método colorimétrico em diferentes concentrações de gefitinibe, UO126 e rapamicina, isoladamente ou em combinação. Resultados: UO126 levou a defosforilação não apenas de intermediários de MAPK como também de substratos do alvo da rapamicina em mamíferos (mTOR) revelando sinalização cruzada entre essas vias. Nós identificamos ainda que ERK5 é a quinase potencialmente responsável por esta sinalização cruzada. No entanto, tratamento com UO126 não afetou o nível de mRNA para o substrato de mTOR 4EBP1. Gefitinibe foi capaz de bloquear a sinalização iniciada por EGF na via de PI3K em todas as linhagens wt-PTEN e de bloquear a sinalização através de ERK1/2 e ERK5 ao menos parcialmente em todas as linhagens celulares. Rapamicina mostrou-se um potente inibidor do crescimento na maioria das linhagens celulares de CCR e tal efeito foi freqüentemente amplificado com a combinação com UO126 ou gefitinibe.Conclusão: EGFR, MAPK e CIT são alvos promissores no tratamento do CCR / Background: Renal cell carcinoma (RCC) is an increasing cause of cancer mortality. Despite its curability in early stages, conventional cytotoxic and immunomodulatory agents have been homogeneous in providing minimal or no benefit in the treatment of advanced RCC. Better understanding of tumor cell biology may lead to development of more efficient targeted therapies. Signaling intensification through epithelial growth factor receptor (EGFR), mitogenactivated protein kinase (MAPK) pathway, Phosphatidylinositol 3-kinase (PI3K) pathway and overactivation of the translation initiation complex (TIC) has been previously characterized in RCC cell lines and tumor samples. We investigated the effect of an EGFR inhibitor (gefitinib) as well as a MAPK inhibitor (UO126) and a TIC inhibitor (rapamycin) in the intermediates of cell signaling, in the elements of TIC and in the in vitro growth of RCC cell lines. Methods: RCC cell lines (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN and KRCY) were maintained on standard mammalian cells culture conditions and exposed to drugs and/or inhibitors in variable concentrations and for variable periods of time as required in each experiment. Phosphorylation status of signaling intermediates were determined using western blots. Levels of mRNA for genes of interest were determined by qRT-PCR. Cell growth was assessed by colorimetric method in control conditions or in different concentrations of gefitinib, UO126 or rapamycin, alone or in combination. Results: UO126 caused dephosphorylation not only of MAPK intermediates but also of mammalian target of rapamycin (mTOR) substrates revealing crosstalk between these pathways. We also identified ERK5 as a kinase potentially responsible for such cross talk. However, treatment with UO126 did not affect mRNA levels of the downstream target of mTOR 4EBP1. Gefitinib was able to block EGF signaling through PI3K in all wt- PTEN cell lines and the signaling through ERK1/2 and ERK5 at least partially in all cell lines. Rapamycin was found to be a potent growth inhibitor in most RCC cell lines and such effect was often increased by its combination with UO126 or gefitinib. Conclusion: EGFR, MAPK and CIT are promising targets for the treatment of RCC

Introdução: O carcinoma de células renais (CCR) representa uma causa crescente de mortalidade por câncer. Apesar da sua curabilidade em estádios iniciais, agentes citotóxicos e imunomodulatórios têm homogeneamente alcançado nenhum ou pouco benefício no tratamento do CCR avançado. Um melhor entendimento da biologia tumoral pode levar ao desenvolvimento de terapias mais eficientes e dirigidas aos mecanismos moleculares de manutenção do tumor. Sinalização aumentada através do receptor do fator de crescimento epitelial (EGFR), sistema de quinases proteicas ativadas por mitógenos (MAPK) e via da fosfatidilinositol 3-quinase (PI3K) assim como estimulação do complexo de início de tradução (CIT) foram caracterizados em linhagens celulares e amostras tumorais de CCR. Nós investigamos o efeito de um inibidor de EGFR (gefitinibe), de um inibidor de MAPK (UO126) e de um inibidor do CIT (rapamicina) nos intermediários de sinalização celular e nos elementos do CIT assim como no crescimento in vitro de linhagens celulares de CCR. Métodos: Linhagens celulares de CCR (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN e KRCY) foram mantidas em condições ideais para a cultura de células de mamíferos e expostas a drogas e/ou inibidores nas concentrações e por períodos de tempo variáveis. A fosforilação de intermediários da sinalização celular foi determinada utilizando-se western blots. Nível de mRNA para genes de interesse foram determinados por qRT-PCR. Crescimento celular foi avaliado por método colorimétrico em diferentes concentrações de gefitinibe, UO126 e rapamicina, isoladamente ou em combinação. Resultados: UO126 levou a defosforilação não apenas de intermediários de MAPK como também de substratos do alvo da rapamicina em mamíferos (mTOR) revelando sinalização cruzada entre essas vias. Nós identificamos ainda que ERK5 é a quinase potencialmente responsável por esta sinalização cruzada. No entanto, tratamento com UO126 não afetou o nível de mRNA para o substrato de mTOR 4EBP1. Gefitinibe foi capaz de bloquear a sinalização iniciada por EGF na via de PI3K em todas as linhagens wt-PTEN e de bloquear a sinalização através de ERK1/2 e ERK5 ao menos parcialmente em todas as linhagens celulares. Rapamicina mostrou-se um potente inibidor do crescimento na maioria das linhagens celulares de CCR e tal efeito foi freqüentemente amplificado com a combinação com UO126 ou gefitinibe.Conclusão: EGFR, MAPK e CIT são alvos promissores no tratamento do CCR / Background: Renal cell carcinoma (RCC) is an increasing cause of cancer mortality. Despite its curability in early stages, conventional cytotoxic and immunomodulatory agents have been homogeneous in providing minimal or no benefit in the treatment of advanced RCC. Better understanding of tumor cell biology may lead to development of more efficient targeted therapies. Signaling intensification through epithelial growth factor receptor (EGFR), mitogenactivated protein kinase (MAPK) pathway, Phosphatidylinositol 3-kinase (PI3K) pathway and overactivation of the translation initiation complex (TIC) has been previously characterized in RCC cell lines and tumor samples. We investigated the effect of an EGFR inhibitor (gefitinib) as well as a MAPK inhibitor (UO126) and a TIC inhibitor (rapamycin) in the intermediates of cell signaling, in the elements of TIC and in the in vitro growth of RCC cell lines. Methods: RCC cell lines (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN and KRCY) were maintained on standard mammalian cells culture conditions and exposed to drugs and/or inhibitors in variable concentrations and for variable periods of time as required in each experiment. Phosphorylation status of signaling intermediates were determined using western blots. Levels of mRNA for genes of interest were determined by qRT-PCR. Cell growth was assessed by colorimetric method in control conditions or in different concentrations of gefitinib, UO126 or rapamycin, alone or in combination. Results: UO126 caused dephosphorylation not only of MAPK intermediates but also of mammalian target of rapamycin (mTOR) substrates revealing crosstalk between these pathways. We also identified ERK5 as a kinase potentially responsible for such cross talk. However, treatment with UO126 did not affect mRNA levels of the downstream target of mTOR 4EBP1. Gefitinib was able to block EGF signaling through PI3K in all wt- PTEN cell lines and the signaling through ERK1/2 and ERK5 at least partially in all cell lines. Rapamycin was found to be a potent growth inhibitor in most RCC cell lines and such effect was often increased by its combination with UO126 or gefitinib. Conclusion: EGFR, MAPK and CIT are promising targets for the treatment of RCC