<p>The small GTPases <italic>HRAS, <italic>NRAS and <italic>KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in <italic>RAS, with 98% of those occurring in the <italic>KRAS isoform. While there have been many advances in the understanding of <italic>KRAS&ndash;driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of <italic>Kras&ndash;driven tumorigenesis in the lung:</p><p>(<italic>I) Despite nearly identical protein sequences, the three <italic>RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, <italic>KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to <italic>HRAS and <italic>NRAS. To determine the consequences of rare codon bias during <italic>de <italic>novo tumorigenesis, we created a knock-in <italic>Kras<super>ex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the <italic>Kras<super>ex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non&ndash;oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in <italic>Kras tumorigenesis.</p><p>(<italic>II) Lung cancer patients exhale higher levels of NO and <italic>iNOS<super>-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes <italic>Kras&ndash;driven lung adenocarcinoma, and NOS inhibition may decrease <italic>Kras&ndash;driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L&ndash;NAME in a genetically engineered mouse model of <italic>Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic <italic>Kras<super>G12D and dominant-negative <italic>Trp53<super>R172H in lung epithelial cells. L&ndash;NAME treatment was provided in the water and continued until survival endpoints. In this model, L&ndash;NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.</p> / Dissertation

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Orientador: Marcelo Menossi Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T20:50:45Z (GMT). No. of bitstreams: 1 Mattioli_MarceloAugustoPortugal_M.pdf: 1336841 bytes, checksum: 296a3b101b6750274ad9c012a8f42161 (MD5) Previous issue date: 2008 / Resumo: O código genético é degenerado, o que permite que um mesmo aminoácido seja codificado por trincas de nucleotídeos distintas. Trincas de um mesmo aminoácido não ocorrem com a mesma freqüência, sugerindo que existe utilização preferencial de codons (codon usage bias). Diferentes organismos geralmente apresentam codon usage bias distintos e estas diferenças podem ser fatores limitantes na expressão heteróloga. Além do codon usage bias existem outros fatores presentes em um gene que podem influenciar a intensidade com que este é traduzido, dentre eles destacam-se: a constituição das regiões gênicas próximas ao códon de iniciação (contexto de iniciação) e a região terminadora da tradução (contexto de terminação). Nosso objetivo foi identificar a presença destes padrões em seqüências ESTs de eucalipto e gerar informações que permitam aumentar os níveis de produção de proteínas de interesse em eucalipto geneticamente modificado. Em concreto, foram alcançados os seguintes objetivos: Identificados os códons mais utilizados em eucalipto; Avaliada as variações na utilização do códon entre genes mais expressos e menos expressos e também entre genes expressos em diferentes tecidos; Identificados os padrões presentes nas regiões iniciadora e terminadora da tradução; Construído um software que indica as alterações necessárias na seqüência de DNA de qualquer gene que se tenha interesse em expressar em eucalipto; Desenhados e construídos genes sintéticos para futura validação in vivo da metodologia de otimização da tradução de proteínas de eucalipto desenvolvida e implementada no software. / Abstract: Most of the amino acids are represented by more than one codon because of that the genetic code is said to be degenerated. Codons from the same amino acid does not occur with the same frequency, suggesting that there is preferential use of codons (codon usage bias). Different organisms often have different codon usage bias and these differences can be limiting factors in heterologous expression. Besides the codon usage bias there are other factors present in a gene that may influence the intensity with which this is translated, among them are: the DNA bases surrounding the initiation codon (initiation context) and the stop codon (termination context). Our goal is to identify the presence of these patterns - codon usage bias, initiation and termination contexts - in eucalyptus EST sets and generate information to increase protein expression in genetically modified eucalyptus. Specifically, the following objectives have been achieved: Identified the set of codons most used in the eucalyptus; Verified changes in the use of codons between genes expressed at different levels and also between genes expressed in different tissues; Identified patterns surrounding the translation initiation and stop sites; Development of a software that indicates the necessary changes in the DNA sequence of any gene to be expressed in eucalyptus Designed and constructed synthetic genes for further validation in vivo of the optimization methodology developed and implemented in the software. / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22356号 / 医博第4597号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 岩田 想, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Translation initiation in the hepatitis C virus (HCV) occurs through a cap- independent mechanism that involves an internal ribosome entry site (IRES) capable of interaction with and utilization of the eukaryotic translational machinery. We focused on the structural configuration of the different HCV-IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. For this purpose we introduced into our laboratory, methods such as denaturing gradient and temperature gradient gel electrophoresis for screening the degree of heterogeneity and total amount of HCV-IRES variability accumulated in HCV infected patients over a period of time. The selected samples showed variable migration pattern of the HCV-IRES (from all the patients) visualized in DGGE and TGGE, were sequenced and evaluated for translation efficiency using flow cytometry. In some cases, we discovered that multiple mutations, even those scattered across different domains of HCV-IRES, led to restoration of the HCV-IRES translational activity, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long- range inter- and/or intra-domain functional interactions. We established a large-scale HCV-IRES...

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