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dc.contributor.advisorCheng, Kun (Professor)eng
dc.contributor.authorMahato, Rubieng
dc.date.issued2014-09-30eng
dc.date.submitted2014 Falleng
dc.descriptionTitle from PDF of title page, viewed on July 13, 2015eng
dc.descriptionDissertation advisor: Kun Chengeng
dc.descriptionVitaeng
dc.descriptionIncludes bibliographic references (pages 109-118)eng
dc.descriptionThesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2015eng
dc.description.abstractThe objective of this dissertation is to present the various therapeutic approaches for two distinctly different diseases, prostate cancer and type-1 diabetes. Our objective (1) for prostate cancer is to evaluate the siRNA mediated silencing of IKKα expression and examine its effects on tumor cell invasiveness and growth, and (2) for type-1 diabetes is to develop a stable transgene delivery system that can infect dividing as well as quiescent cells, such as pancreatic islets. In chapter 1 and 2 the biological characteristics and molecular mechanism of cancer are described, and the therapeutic strategies and their limitations are summarized; the current therapeutic approach, characteristics and molecular mechanism of type 1 diabetes are also presented. In chapter 3, the molecular mechanism of IKKα, its expression profile in prostate cancer cells and the therapeutic effect of IKKα siRNA on prostate cancer cell lines are described. Three synthetic siRNAs targeting different regions of the IKKα mRNA were designed, and the silencing effect was determined by quantitative real time RT-PCR. Inhibition of IKKα in prostate cancer cells reduced wound healing, inhibition of in vitro cell migration, cell invasion and attachment capabilities. Similar anti-invasive effects were also observed in the presence of RANKL, which is a Receptor activator of NFκB Ligand. However, silencing of IKKα only showed a negligible effect on cell proliferation and cell cycle distribution, estimated by cell viability assay and cell cycle distribution assay, respectively. Although Lipofectamine possesses good transfection efficiency, nevertheless it produces cytotoxic effects. In addition, if siRNA is given alone parenterally it may not reach the target efficiently. To overcome these problems, anti-PSMA aptamer as a targeting moiety to specifically deliver the siRNA to the PSMA overexpressing prostate cancer cells was introduced in chapter 4. Originally developed PSMA aptamer, A9, is a 70mer RNA sequence; however in this study the truncated version of this aptamer which is 41mer RNA, called as A9L aptamer, was used. This truncated aptamer maintain the PSMA binding activity, specificity and functionality, just as the full length anti-PSMA aptamer. In addition, it also retains the ability to inhibit PSMA’s NAALA-Dase activity. In this study, A9L was attached to one end of the sense strand of the IKKα siRNA duplex through a 14mer RNA sticky bridge. The resulting triplex (sense + antisense + aptamer) showed similar silencing effect as duplex (sense + antisense) when transfected the cells using Lipofectamine-2000. Furthermore, the triplex showed comparable cellular uptake and gene silencing effect, but less cytotoxicity compared to transfection with Lipofectamine-2000. In chapter 5, the effect of RNAi mediated CD40 gene silencing on Insulinoma endothelial cells (islet β cell) and rat pancreatic islets was evaluated. Three siRNAs targeting different regions of CD40 mRNA were designed and the silencing efficiency was estimated at mRNA level by real-time RT-PCR. Results showed moderate, but efficient gene silencing with siRNA. The silencing efficiency in the presence of various cytokines was also evaluated, and it was found that siRNA could inhibit CD40 expression in the presence of cytokines compared to the control group. Transfection efficiency in islets was evaluated by confocal microscopy. Although islet is a cluster of non-dividing cells, siRNA could efficiently transfect the islets and it had little effect on islet viability. Further we designed AAV vector encoding CD40 shRNA, and estimated the transfection and silencing efficiency of the vector in INS-1E cells. The AAV vector could stably transfect the cells, for up to 6 days, and down-regulated CD40 efficientlyeng
dc.description.tableofcontentsIntroduction -- Literature review -- Blocking IKKa expression inhibits prostate cancer invasiveness -- PSMA aptamer mediated SIRNA delivery to PSMA positive prostate cancer cells -- Small interfering RNA mediates CD40 gene silencing for the treatment -- Summary and conclusionseng
dc.format.extentxiii, 120 pageseng
dc.identifier.urihttps://hdl.handle.net/10355/43896eng
dc.subject.lcshProstate| -- Cancer -- Treatmenteng
dc.subject.lcshDiabetes -- Treatmenteng
dc.subject.meshDiabetes Mellitus, Type 1 -- therapyeng
dc.subject.meshProstatic Neoplasms -- therapyeng
dc.subject.otherDissertation -- University of Missouri--Kansas City -- Pharmacyeng
dc.subject.otherDissertation -- University of Missouri--Kansas City -- Chemistryeng
dc.titleEvaluation of siRNA mediated Silencing of IKKα in Prostate cancer and CD40 in Type-1 Diabeteseng
dc.typeThesiseng
thesis.degree.disciplinePharmaceutical Sciences (UMKC)eng
thesis.degree.disciplineChemistry (UMKC)
thesis.degree.grantorUniversity of Missouri--Kansas Cityeng
thesis.degree.levelDoctoraleng
thesis.degree.namePh.D.eng


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