Dissertation (Ph.D.)--University of Toledo, 2008. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title in Ohio LINK ETD Center record : Na/K-ATPase signaling : from bench to bedside. Title from title page of PDF document. Bibliography: p. 99-120.

Thesis (Ph. D.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains vii, 350 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 322-346).

Abstract Epithelial cell-cell adhesions have a critical role in morphogenesis, establishment and maintenance of tissue architecture, cell-cell communication, normal cell growth and differentiation. These adhesions are disrupted during malignant transformation and tumour cell invasion. Several kinases, phosphatases and small GTPases regulate cell-cell contacts. In the present work we investigated the dynamics of cell-cell adhesion structures after microinjection of fluorophore tagged vinculin, during transformation caused by an active Src tyrosine kinase and during Helicobacter pylori infection. The regulatory role of Rac GTPase as well as the behaviour of actin and cadherin were analysed in all these conditions. Microinjection of vinculin into bovine kidney epithelial MDBK cells induced release of actin, cadherin and plakoglobin to cytoplasm of the cells, caused disruption of protein complexes at adherens and tight junctions that finally led to formation of polykaryons. Activated Rac GTPase, in turn, enhanced accumulation of cadherin to membranes and thereby diminished the formation of polykaryons, whereas inactive Rac removed cadherin from membranes. Incorporation of vinculin to lateral membranes took place also in acidifying and depolarising conditions where cell fusions were prevented. Thus, the membrane potential seemed to control fusion ability. In src-MDCK cells, activation of Src kinase led to disintegration of adherens junctions. Clusters of junctional components and bundles of actin were seen at the basal surface already within 30 min after Src activation. p120ctn was the only component of adherens junction whose relocation correlated to its phosphorylation. Inhibition of Src by a specific inhibitor PP2 restored the cubic morphology of the cells and accumulated cadherin back to lateral walls. Still p120ctn remained in cytoplasm and thereby was not responsible for the epithelial phenotype. Activation of Rac GTPase by Tiam1 also increased the amount of cadherin at lateral membranes and maintained the morphology of src-MDCK cells practically normal after activation of Src kinase. In the same way, actin cytoskeleton was reorganised in gastric carcinoma cells in response to infection with H. pylori via activation of Rac signalling pathway. Hence, Rac and cadherin seem to be the major players in the maintenance of epithelial cell morphology.

With a five-year survival rate of less than 5%, pancreatic cancer is one of the deadliest cancers. The most common activating mutations in pancreatic cancer are found in the KRAS gene, causing a constitutively-active KRAS protein in approximately 90% of pancreatic ductal adenocarcinomas (PDAC). PDAC-derived cell lines that harbor oncogenic KRAS mutations can be divided into two classes, KRAS dependent (or addicted) cells and KRAS independent cells. Oncogene dependency (or addiction) is a phenomenon where tu-mors require sustained activity of a single aberrantly activated gene despite the accumulation of multiple oncogenic lesions. In the case of PDAC, the single aberrantly activated gene is KRAS. KRAS independent cells have acquired various other oncogenic lesions that confer alternative cell survival signaling pathways to bypass oncogenic KRAS dependency. The Spleen Tyrosine Kinase (Syk) is highly expressed in KRAS dependent cells, while KRAS independent cells have low Syk expression. This pattern suggests that in KRAS dependent cells, constitutively active KRAS and Syk play a role in stimulating pro-survival pathways. One of these pro-survival pathways is known as mTORC1, which causes increased anabolic processes like protein and lipid synthesis. Accordingly, mTORC1 causes suppression of catabolic processes like autophagy. The net effect is in-creased cellular growth and proliferation. However, mTORC1 inhibitors have limited clinical efficacy, and potential therapeutic targets upstream of mTORC1 have drawn interest. Syk is a non-receptor tyrosine kinase that is an upstream activator of the mTORC1 pathway in hematopoietic malignancies. Through Syk inhibition studies using the small molecule PRT062607 (SYKi), we demonstrated that Syk is also involved in activating the mTORC1 pathway in KRAS dependent PDAC cells. However, the mechanism by which Syk-mediated activation of mTORC1 occurs is currently unknown. Moreover, it is unclear whether SYK kinase activity is required for the activation of the mTORC1 pathway. To address this issue, we introduced a single nucleotide mutation in the kinase do-main of Syk to render it kinase-inactive and found that Syk requires its kinase function to activate mTORC1. Studies using Syki also revealed that mTORC1 activity was also inhibited in KRAS independent PDAC cells that lack significant Syk expression. Interestingly, substrate specificity studies indicate that Syki also binds to and inhibits structurally similar protein tyrosine kinases such as the SRC Family Kinases (SFKs). Therefore, we designed an experiment to look for Syk and SFK cooperativity in regards to mTORC1 activation in PDAC cells. Our results indicate that the SFKs, Yes1 and Src display the most significant cooperative effect with Syk in activating the mTORC1 pathway. Src and Yes1 may even be involved in the upstream activation of Syk. To establish the physiological significance of Syk signaling in pancreatic cancer, it is important to establish model organisms that could be used for future studies. Thus, we test-ed Syk expression and function in PDAC cell lines derived from genetically-engineered mouse models (GEMM), which develop pancreatic cancer via oncogenic mutations in KRAS and TP53. We found that Syk is indeed expressed in murine PDAC cell lines and that the use of Syki in the murine PDAC cell lines results in decreased mTORC1 activity. These results recapitulate those obtained in human KRAS dependent PDAC cell lines. In summary, our studies show that Syk is a key regulator of mTORC1 signaling in human and mouse-derived pancreatic cancer cells. Syk kinase activity is required for mTORC1 activation. Finally, SFKs cooperate with Syk to promote robust mTORC1 activation. The mechanisms of SFK and Syk cooperativity in mTORC1 pathway activation will require further investigation. Additionally, our findings provide a strong rationale to study the effects of Syk kinase inhibition in physiologically-relevant murine models of pancreatic cancer. / 2021-06-08T00:00:00Z

No description available.

No description available.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Document formatted into pages: iii, 156 p. Title from title page of PDF document. Bibliography: pages 64-67, 97-100, 116-117, 125-155.