Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.

Thesis (M.S.)--Wake Forest University. Dept. of Biology, 2008. / Vita. Includes bibliographical references (leaves 14-17)

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "July 1999." Typescript. Vita. Includes bibliographical references (leaves 128-129). Also available on the Internet.

No description available.

No description available.

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans. / Medicine, Faculty of / Graduate

Early Xenopus laevis embryonic cell cycles exemplify rapid, non-pathological cell cycles without checkpoint pathways to arrest cell cycles in response to DNA assaults. There is no transcription or apoptosis during these cell cycles, and they continue unperturbed until the 12th cell cycle, marking a period called the midblastula transition (MBT). At the MBT, the embryo undergoes a period of developmental remodeling: gap phases are acquired, zygotic transcription is initiated, and the maternal mRNAs are degraded. After the MBT, checkpoint pathways can be activated in response to unreplicated DNA, and apoptosis initiates when continued embryonic survival is compromised. These studies examine how cell cycle regulation and apoptotic regulation are related. Specifically, the role of two cell cycle components, Chk1 and cyclin A1/Cdk2, during apoptosis was studied during early development of Xenopus embryos. Chk1 is a serine/threonine kinase that inhibits the activity of cyclin-dependent kinases (Cdks) in response to unreplicated DNA. In the pre-MBT embryo, Chk1 is present, but inactive. Injection of mRNA encoding dominant-negative Chk1 (DN-Chk1) into single-celled embryos results in the initiation of apoptosis after the MBT. The loss of Chk1 function also results in the initiation of additional rapid rounds of DNA replication after the MBT. These results suggest that Chk1 has a required function for the embryo after the MBT, possibly through the regulation of a cyclin/Cdk complex responsible for the apoptotic checkpoint. Cyclin A1 is a maternally provided mRNA that is degraded at the MBT. Prior to the MBT, cyclin A1 complexes exclusively with Cdc2 to regulate mitosis. When embryos are treated with ionizing radiation (IR), cyclin A1 activity and protein level persist after the MBT, and cyclin A1 complexes with Cdk2. When treated with aphidicolin, cyclin A1-associated activity and protein level persists. Injection of cyclin A1/Cdk2 into single-cell embryos results in apoptosis after the MBT; however, inhibition of cyclin A1 expression does not abrogate apoptosis. Therefore, cyclin A1/Cdk2 activity is sufficient, but not required, for the initiation of apoptosis in the early Xenopus embryo. These studies show that Chk1 and cyclin A1/Cdk2 have roles in regulating apoptosis in the post-MBT embryo. / Ph. D.

In somatic cells, when DNA is damaged or incompletely replicated, checkpoint pathways arrest the cell cycle prior to M or S phases by inhibiting cyclin-dependent kinases (Cdks). In Xenopus laevis, embryonic cellular divisions (2-12) consist of rapid cleavage cycles in which gap phases, checkpoint engagement, and apoptosis are absent. Upon the completion of the 12th cellular division, the midblastula transition (MBT) begins and the cell cycle lengthens, acquiring gap phases. In addition, cell cycle checkpoint pathways and an apoptotic program become functional. The studies described here were performed to better understand the roles of two protein kinases, Chk2/Cds1 and Wee1, during checkpoint signaling in the developing embryo. The DNA damage checkpoint is mediated by the Chk2/Cds1 kinase. Conflicting evidence implicates Chk2 as an inhibitor or promoter of apoptosis. To better understand the developmental function of Chk2 and its role in apoptosis, we expressed wild-type (wt) and dominant-negative (DN) Chk2 in Xenopus embryos. Wt-Chk2 created a pre-MBT checkpoint by promoting degradation of Cdc25A and phosphorylation of Cdks. Embryos expressing DN-Chk2 developed normally until gastrulation and then underwent apoptosis. Conversely, low doses of wt-Chk2 blocked radiation-induced apoptosis. These data indicate that Chk2 inhibits apoptosis in the early embryo. Therefore, Chk2 operates as a switch between cell cycle arrest and apoptosis in response to genomic assaults. In Xenopus laevis, Wee1 kinase phosphorylates and inhibits Cdks. To determine the role of Wee1 in cell cycle checkpoint signaling and remodeling at the MBT, exogenous Wee1 was expressed in one-cell stage embryos. Modest overexpression of Wee1 created a pre-MBT cell cycle checkpoint, similar to Chk2, characterized by cell cycle delay and phosphorylation of Cdks. Furthermore, overexpression of Wee1 disrupted remodeling events that normally occur at the MBT, including degradation of Cdc25A, cyclin E, and Wee1. Interestingly, overexpression of Wee1 also resulted in post-MBT apoptosis. Taken together, these data suggest the importance of Wee1 as not only a Cdk inhibitory kinase, but also potentially as a promoter of apoptosis during early development of Xenopus laevis. The studies described here provide evidence that Chk2 and Wee1 have both similar and distinct roles in the developing embryo. / Ph. D.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 128-129). Also available on the Internet.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2002. / Includes bibliographical references.