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Retroviruses are viruses which are able to integrate to genome of host cell. Nonrandom integration of provirus near or inside some cellular genes may result in their deregulation, activation or silencing. This can later lead to cell transformation and tumor formation. This thesis discusses identification of viral integration sites (VIS) and common integration sites (CIS) in tumors originating from different organs (mostly kidneys, lungs and liver) with using mostly avian retroviruses subgroup J, specifically first natural isolate HPRS-103 and laboratory made virus MAV-J, which was made by replacing gene envB by envJ. Infection was made in ovo using chicken breeds Brown Leghorn and White Leghorn and tumors were isolated from 8 to 28 weeks after infection. For molecular analyses was used inverse PCR method and sequencing. From 74 molecularly analyzed tumors there was detected 373 VIS and 6 CIS with statistical significance over 2.10-2 . Gene with the highest number of hits was FRK (14 times), then TERT (5 times), CTDSPL (5 times), EGFR/ERBB1 (3 times), MYB (3 times) and MYC (3 times). Except 6 CIS there were other genes found, which had smaller statistical significance. Keywords: retrovirus, insertional mutagenesis, subgroup J, oncogenesis, oncogenes, MAV-J, HPRS-103, proviral integration sites, tumors

Cancer is the second most common cause of death in the United States. It is a complex disease with environmental, genetic, and lifestyle factors influencing the likelihood of getting cancer and the development of any resulting tumor. Understanding the genetics of cancer is integral to developing novel patient-specific treatments. However, due to complexity, hundreds to thousands of tumors are required for sufficient power to identify the network of relationships among these genes. Animal models of cancer are commonly used to reduce cost and to control experimental variables allowing for more specific hypothesis testing. The Sleeping Beauty transposon mutagenesis system can be used to model cancer in mice. While the Sleeping Beauty mutagenesis system is an important tool in understanding cancer, it has specific computational needs. Experiments need to be analyzed in a fast, unbiased, and efficient manner. A computational method must also accurately model the system allowing for validation and interpretation. Here I present an updated Integration Analysis System and use this system to validate the assumptions present in forward genetic screens of cancer using the Sleeping Beauty. This system allows for rapid identification of cancer genes, but does not directly aid in understanding the relationship between the genes. Given the complexity of cancer, understanding the relationship between cancer genes is very difficult. I have created a connectedness network utilizing the STRING database to better derive an understanding of cancer genes. STRING is a database of known and predicted protein-protein interactions. The connectedness between pairs of genes is calculated using a network reliability metric. This database allows for increased power to detect known pathways when compared to STRING alone. Combining this connectivity network with the set of cancer genes identified by the Integration Analysis System is a strategy for rapid and efficient interpretation of the genetic results.

Gynecological cancers present a tremendous disease burden worldwide. Endometrial cancer, the most common gynecological malignancy, is predominantly a disease of deranged glandular function. The mechanisms by which known environmental risk factors influence the mutational profile of endometrial cancer are poorly understood. Non-HPV vulvar cancer, on the other hand, is a very rare gynecological malignancy of vulvar squamous cells with little known about its pathogenesis. Surgical resection of vulvar cancer is associated with high post-surgical morbidity. Pivotal to improving treatment and outcomes for patients with gynecological cancers is an understanding of the molecular drivers unique to each tumor type. To inform our understanding of endometrial gland regulation, I began my investigations with an assessment of normal endometrial adenogenesis in vivo and present the first evidence implicating the necessity of Sox17 in endometrial gland development. My data suggest Sox17 mediates adenogenesis via a non-cell autonomous mechanism from within the stromal compartment of the endometrium. I then interrogated the contribution of SOX17 to dysregulated glandular function in Type I endometrial adenocarcinoma in vitro. My findings reveal an oncogenic role of SOX17 in the Ishikawa Type 1 endometrial cancer cell line, with homozygous loss of SOX17 impairing cellular proliferation, blunting the cancer phenotype of these cells. The majority of cancers, including gynecological cancers, develop from the accumulation of genetic mutations that occur sporadically in cells over time. The complexity and heterogeneity of solid tumors, however, renders the identification of mutations responsible for driving tumorigenesis difficult. The Sleeping Beauty (SB) insertional mutagenesis system can be used to streamline sporadic tumor formation and driver mutation identification. I present results from an initial attempt to develop an SB model of endometrial cancer and discuss ways in which the SB system can be harnessed to evaluate tumorigenesis in a variety of tissue types and microenvironmental contexts. Finally, I present an SB model of metastatic vulvar cancer. Primary tumors from this model resulted in the identification of 76 novel candidate drivers of vulvar cancer, with the ubiquitin-specific peptidase, Usp9x, the most commonly disrupted gene in our screen. I show data suggesting that differential expression of Usp9x isoforms may underlie Usp9x-mediated tumorigenesis and preliminary data demonstrating the relevance of USP9X to human vulvar cancer. Taken as a whole, these data contribute to our scientific understanding of gynecological tissue homeostasis and cancers, lay the foundation for the development of an SB model of endometrial cancer, and describe the first reported model system for studying HPV-naive vulvar cancer in vivo.

Cancer is one of the leading causes of death in the world. Approximately one fifth of deaths in the western industrial nations are caused by cancer. Every year several hundreds of thousands of new patients are diagnosed with cancer and several thousands die of cancer. Scientists have been conducting research from different angles for effective prevention, diagnosis and cure of Cancer. Ever since the genetic basis of cancer has been demonstrated, a race has been ignited globally in the scientific community to identify potential oncogenes and tumor suppressor genes. The genetics of the tumors are complex in nature where combinations of loss of function mutations in tumor suppressor genes and gain of function mutations in oncogenes cause cancers. The identification of these genes is extremely important to devise effective therapies to treat cancer. Insertional mutagenesis systems such as sleeping beauty provide an elegant way to identify genes involved in cancers. More and more researchers are adopting the Sleeping Beauty system for their insertional mutagenesis experiments to identify potential cancer causing genes. Given next generation sequence technologies and the vast amount of data they generate requires novel bioinformatics techniques to process, analyze and meaningfully interpret the data. The goal of this project is to develop a publicly available system for researchers worldwide to analyze the sequence data resulting from insertional mutagenesis experiments. This system will identify and annotate all the insertion sites resulting from the sequencing of the experiment. It will also identify the Common Insertion sites (CIS) and genes with Common Insertion Sites (gCIS). The Common Insertion Sites being the regions in the genome that are targeted more often than by chance. The whole system is accessible as a web application for use by researchers worldwide performing insertional mutagenesis experiments.

Made available in DSpace on 2014-06-11T19:32:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-12Bitstream added on 2014-06-13T20:43:09Z : No. of bitstreams: 1 castellane_tcl_dr_jabo.pdf: 1115288 bytes, checksum: c46d10396d02bdc45c92887415f5fd8f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Rhizobium tropici e outras bactérias pertencentes à ordem Rhizobiales são produtores de polissacarídeos extracelulares (EPS), que possuem a função de molécula receptora do micro simbionte, fazendo uma interação célula/célula e desencadeando o processo de nodulação. A diversidade de estruturas e composição química apresentadas pelas moléculas de EPS é refletida pela diversidade de enzimas responsáveis pela sua síntese. Neste trabalho, buscou-se a inativação através da mutagênese insercional por transposição in vitro dos genes exoZ da estirpe selvagem Rhizobium tropici SEMIA 4080 envolvidas na síntese das subunidades repetitivas do EPS e no tanden phbAB, responsáveis pela síntese do PHB, com o intuito de obter microrganismos com aspecto altamente mucoso, produtor superior de EPS em relação a estirpe selvagem. Os mutantes obtidos apresentaram colônias mucosas quando cultivados nos meios de culturas PSYA, demonstrando que estirpes de rizóbio mutantes nos genes exoZ e phbAB são capazes de formar biofilme “in vitro” e, ao avaliar a eficiência relativa na produção de EPS, o mutante 4080 Z03 apresentou o melhor resultado. Para as três amostras de EPS foi possível observar um comportamento de fluxo de líquidos pseudoplástico e, também, a influência da concentração de EPS sobre a viscosidade aparente das soluções aquosas. A estirpe selvagem e todos os mutantes induziram a formação de nódulos em feijoeiro (fenótipo Fix+), sugerindo que os genes exoZ e phbAB não estão envolvidos nos processos de infecção e formação nodular mas, sendo necessários para a fixação biológica de nitrogênio / Rhizobium tropici and other bacteria belonging to the order Rhizobiales are extracellular polysaccharides (EPS) producers, which possess the function of the receptor molecule to function as micro-symbiont, making an interaction cell / cell and triggering the nodulation process. The diversity of structures and chemical composition of EPS presented by molecules is reflected by the diversity of enzymes responsible for its synthesis. In this study, we sought to inactivation by insertional mutagenesis by “in vitro” transposition of genes from wild type exoZ Rhizobium tropici SEMIA 4080 involved in the synthesis of the EPS repeating subunits and phbAB tanden, responsible for synthesis of PHB, in order to obtain microrganisms with high mucosal aspect producer of EPS compared to wild type. The mutants showed mucous colonies when grown in culture media PsyA, demonstrating that mutant strains in genes exoZ and phbAB from rhizobia are able to form biofilm “in vitro”, and to evaluate the relative efficiency in the production of EPS, the mutant 4080 Z03 showed the best result. For the three samples of EPS was possible to observe a flow behavior of pseudoplastic fluids, and also the influence of EPS concentration on the apparent viscosity of aqueous solutions. The wild type and mutants induced the formation of nodules in common bean (Fix+ phenotype), suggesting that genes exoZ and phbAB are not involved in the processes of infection and nodule formation, but are necessary for nitrogen fixation

Resumo: Rhizobium tropici e outras bactérias pertencentes à ordem Rhizobiales são produtores de polissacarídeos extracelulares (EPS), que possuem a função de molécula receptora do micro simbionte, fazendo uma interação célula/célula e desencadeando o processo de nodulação. A diversidade de estruturas e composição química apresentadas pelas moléculas de EPS é refletida pela diversidade de enzimas responsáveis pela sua síntese. Neste trabalho, buscou-se a inativação através da mutagênese insercional por transposição in vitro dos genes exoZ da estirpe selvagem Rhizobium tropici SEMIA 4080 envolvidas na síntese das subunidades repetitivas do EPS e no tanden phbAB, responsáveis pela síntese do PHB, com o intuito de obter microrganismos com aspecto altamente mucoso, produtor superior de EPS em relação a estirpe selvagem. Os mutantes obtidos apresentaram colônias mucosas quando cultivados nos meios de culturas PSYA, demonstrando que estirpes de rizóbio mutantes nos genes exoZ e phbAB são capazes de formar biofilme "in vitro" e, ao avaliar a eficiência relativa na produção de EPS, o mutante 4080 Z03 apresentou o melhor resultado. Para as três amostras de EPS foi possível observar um comportamento de fluxo de líquidos pseudoplástico e, também, a influência da concentração de EPS sobre a viscosidade aparente das soluções aquosas. A estirpe selvagem e todos os mutantes induziram a formação de nódulos em feijoeiro (fenótipo Fix+), sugerindo que os genes exoZ e phbAB não estão envolvidos nos processos de infecção e formação nodular mas, sendo necessários para a fixação biológica de nitrogênio / Abstract: Rhizobium tropici and other bacteria belonging to the order Rhizobiales are extracellular polysaccharides (EPS) producers, which possess the function of the receptor molecule to function as micro-symbiont, making an interaction cell / cell and triggering the nodulation process. The diversity of structures and chemical composition of EPS presented by molecules is reflected by the diversity of enzymes responsible for its synthesis. In this study, we sought to inactivation by insertional mutagenesis by "in vitro" transposition of genes from wild type exoZ Rhizobium tropici SEMIA 4080 involved in the synthesis of the EPS repeating subunits and phbAB tanden, responsible for synthesis of PHB, in order to obtain microrganisms with high mucosal aspect producer of EPS compared to wild type. The mutants showed mucous colonies when grown in culture media PsyA, demonstrating that mutant strains in genes exoZ and phbAB from rhizobia are able to form biofilm "in vitro", and to evaluate the relative efficiency in the production of EPS, the mutant 4080 Z03 showed the best result. For the three samples of EPS was possible to observe a flow behavior of pseudoplastic fluids, and also the influence of EPS concentration on the apparent viscosity of aqueous solutions. The wild type and mutants induced the formation of nodules in common bean (Fix+ phenotype), suggesting that genes exoZ and phbAB are not involved in the processes of infection and nodule formation, but are necessary for nitrogen fixation / Orientador: Eliana Gertrudes de Macedo Lemos / Coorientador: Manoel Victor Franco Lemos / Banca: Regiane de Fátima Travensolo Sacomano / Banca: Simone Cristina Picchi / Banca: Jackson Antônio Marcondes de Souza / Banca: Eliamar Aparecida Nascimbem Pedrinho / Doutor

The described experimental model of clonal tumors induced through the insertional mutagenesis with MAV-2 proved to be a valid and rich source of information describing the process of transformation of normal into tumor cell. We have mapped more than 2000 individual clonal VISs from several hundreds of tumor tissue samples. We have analyzed five tumor types of different histology and tissue of origin along with their derivative tissue cultures. Furthermore, we have discovered the industasis phenomenon and described it during the course of the study. The goal of my study was to uncover common reasons for neoplastic transformation of the cell. The results of my study led me to the paradoxical conclusion that the significance of genetic changes as the primary cause of induction of neoplastic transformation is being overestimated. Although studying the functions of individual genes and search for new tumor markers and therapeutical targets are still beneficial, I believe that the traditional perception of tumor formation as a function/result of mutation accumulation and selection is becoming a serious drawback in further investigations. These conclusions are further discussed in the last section of the presented Ph.D. thesis.

<p>Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. </p><p>The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the <i>sis</i>/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. </p><p>Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes.</p><p>An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors.</p><p>The gene <i>Prkg2</i>, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of <i>Prkg2</i> in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the <i>Prkg2</i> protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.</p>

Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the sis/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes. An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors. The gene Prkg2, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of Prkg2 in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the Prkg2 protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.