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.

Part 1: Prophage induction has been demonstrated to be a sensitive indicator for a wide variety of toxic and mutagenic compounds and, as a consequence, has been utilized for biologically based carcinogen screenings. Fourteen marine bacterial isolates were screened for development into the Marine Prophage Induction Assay (MPIA), for marine samples. The selected isolate (P99-4S3) was identified by 16S rDNA sequencing as Pseudomonas aeruginosa. This isolate demonstrated a log-linear response to increasing dose of mutagens, and sensitivity to known environmental contaminants. Field-testing of the assay over two years demonstrated the MPIA would be a useful screening tool for environmental contamination. Part 2: The observed resistance of natural populations of Synechococcus to viral infection may be due to lysogeny with associated homoimmunity. A thirteen-month study of lysogeny in natural populations of Synechococcus demonstrated that lysogeny does occur and exhibits a seasonal pattern. Experiments were performed along a transect of the Mississippi River plume, which provided a variety of ambient nutrient regimes for comparison of lysogeny in Synechococcus. Nutrient amendments did not enable induction and often led to a decrease in viral production. Lysogeny in Synechococcus was primarily correlated with ambient host and cyanophage abundance. Cross-infectivity studies demonstrated cyanophage isolates possess variable virulence. The 35 isolates were examined by transmission electron microscopy (TEM), with 33 identified as myoviruses and two as podoviruses. This dominance of myovirus lytic cyanophage is consistent with prior observations. Twenty-five Synechococcus isolates were screened for prophage induction utilizing the inducing agent Mitomycin C. Eleven isolates demonstrated a statistically significant increase in virus-like particles (VLP’s) in treatment samples. No correlation was observed between their resistance to lytic viral infection and prophage induction. Isolate P99-14, with consistently high levels of prophage induction, was investigated further. In contrast to lytic cyanophage, the induced cyanophage is non-tailed. Differential staining and nuclease digestion experiments indicate that the induced particle contains single-stranded DNA. Environmental conditions potentially leading to prophage induction were investigated with Synechococcus cultures and natural populations. The isolate P99-14 demonstrated that high, continuous light caused prophage induction. Natural populations determined that shifts in salinity, temperature and phosphate are not triggers of prophage induction.

Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions exhibit a substantial export of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. In the current report, we have defined nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1 dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.

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<p>Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.</p>

<p>Mushrooms are believed to have a great potential for production of bioactive metabolites e. g. terpenes, a group of interesting compounds with diverse chemical properties such as antitumour and antibacterial activity. Cop6 is a terpene cyclase isolated from the mushroom <em>Coprinus cinereus</em> that catalyzes the cyclization of farnesyl diphosphate (FPP) to mainly α-cuprenene. In this study gas chromatography combined with mass spectroscopy (GC-MS) is used to analyze the product profile of Cop6 mutants created by PCR based site directed mutagenesis. The goal is to produce trichodiene, the parent hydrocarbon in the biosynthesis of trichothecene antibiotics and mycotoxins. Valine instead of tyrosine in amino acid position 195 resulted in cyclisation of (E)-β-Farnesene and (3Z,6E)-α-Farnesene besides the products of the wild type enzyme. Another mutant with aspartic acid instead of asparagine in position 224 resulted in the synthesis of β-Bisabolene except for α-cuprenene and methionine in position 74 instead of isoleucine killed the activity of the cyclase. Furthermore, an attempt to saturation of position 98 was made, resulting in four mutants. Two of them essentially killed the activity of the cyclase whereas two had minor effect of the product profile compared to the wild type. </p>