Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.

Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.

The purpose of this study was to determine whether Endothelin-1 (ET-1) induced cellular responses in bovine corneal endothelial cells (BCECs) involves MAPK pathway by phosphorylating ERK1/2 protein kinase and to find out the phosphorylation patterns of ERK1/2 in confluent and sub-confluent cells. BCECs were isolated from bovine corneas and cultured in medium supplemented with 10% serum. Confluent (contact inhibited) and sub-confluent (actively growing cells) serum starved cells grown in T-75 flasks were treated with 10nM Endothelin-1. The control cells were left untreated. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of phosphorylated ERK1/2 (pp44, pp42) proteins relative to overall ERK1/2 (p44, p42) was determined by western blotting technique. Densitometry analysis of immunoblots revealed differential phosphorylation patterns in confluent and sub-confluent cultures. The pERK1/2 levels were significantly increased at 15 min and 24 hrs after post incubation with ET-1, whereas following the initial rise levels declined to 6hrs of incubation with ET-1 in confluent cultures. In sub-confluent cultures pERK1/2 levels increased gradually to 6hrs of incubation with ET-1, returning to pre-incubation levels at 24hrs. In conclusion, ET-1 treatment was shown to induce phosphorylation of ERK1/2 in BCEC. ET-1 treatment in confluent and sub confluent BCEC exhibited time dependent phosphorylation of ERK1/2. ET-1 treatment affected the phosphorylation pattern distinctively in confluent and sub-confluent BCEC. These observations led to the conclusion that ET-1 induced cellular events in BCEC may involve the MAPK cascade and that these ET-1 induced MAPK cascades may exhibit a negative feedback mechanism, suggested by a distinctive oscillations in pERK 1/2 levels. The contrasting effects of ET-1 in confluent and subconfluent cells may suggest a density dependent phosphatase activity.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 107-130).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves [202]-230). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 87-95). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves [202]-230). Also available online.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-117). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references (p. 38-42).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 129-153). Also available in print.