Thesis (Ph.D.)--University of Delaware, 2005. / Principal faculty advisor: Simmons, Daniel T., Dept. of Biological Sciences. Includes bibliographical references.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Daniel T. Simmons, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2002. / Includes bibliography.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / "July 2004" Typescript. Includes bibliographical references (l. 64-66). Also issued on the Internet.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Daniel T. Simmons, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 64-66). Also issued on the Internet.

Expression of the Simian Virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. The results of this study demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids either lacking the SV40 origin of DNA replication, or encoding a large T mutant defective in its ability to bind the retinoblastoma gene product (Rb) were as proficient as wild type plasmids, indicating that both viral DNA replication and binding of T antigen to Rb are not required for cytogenic damage. On the other hand, preliminary results with a plasmid encoding a T antigen mutant unable to bind the cellular p53 protein suggest that formation fo this complex may be important for cytogenetic damage. This study has also shown that chromosomal aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. These results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization. / Thesis / Master of Science (MS)

Human cells transformed by SV40 large T antigen achieve an extended lifespan and continue to grow and divide past the normal growth limit. This extended lifespan often ends at crisis when the cells die through fatal cell division. A few cells will survive this crisis and continue to proliferate indefinitely and are therefore considered immortal. Transformation of cells by SV40 large T antigen is associated with the induction of genomic instability at early times. This instability may contribute to a cells surviving crisis and becoming immortal through the chance disruption of genes involved in cell proliferation and regulation of cell death. Genetic instability is also observed in human tumours and the mechanisms by which it occurs both in tumour cells and SV40 transformed cells may be similar. In order to investigate these mechanisms, human and rodent cells were transfected with wild type and mutant forms of SV40 large T antigen and analyzed cytogenetically. The results of this study demonstrate that the amino terminal 147 amino acids of SV40 large T antigen are sufficient for the induction of genomic instability and at least three regions within this amino terminal fragment are necessary. One between amino acids 17 and 27. A second being the retinoblastoma protein binding site, and the third between amino acids 130 and 147. Finally, binding of T antigen to p53 appears to not be required for the induction of genomic instability, but may be necessary for the survival of aberrant cells. There is an apparent correspondence between the ability of T antigen mutants to induce genomic instability, and their abilities to induce cellular DNA synthesis and to transform and immortalize cells. / Thesis / Master of Science (MS)

Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...

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