Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb 18, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 78-98).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 121-135).

Pericentrin is a molecular scaffold protein. It anchors protein kinases, (PKB, (Purohit, personal communication), PKC, (Chen et al., 2004), PKA Diviani et al., 2000), the γ tubulin ring complex, (γ TuRC) (Zimmerman et al., 2004), and possibly dynein (Purohit et al., 1999) to the spindle pole. The γ TuRC is a ~ 2 MDa complex which binds the minus ends of microtubules and nucleates microtubules in vitro, (Zheng et al., 1995). Prior to this work, nothing was known about the association of the γTuRC with pericentrin. Herein I report the biochemical identification of a large protein complex in Xenopus extracts containing pericentrin, the γ TuRC, and other as yet unidentified proteins. Immunodepletion of γ tubulin results in co-depletion of pericentrin, indicating that virtually all the pericentrin in a Xenopus extract is associated with γ tubulin. However, pericentrin is not a member of the, γ TuRC, since isolated γ TuRCs do not contain pericentrin. The association of pericentrin with the γ TuRC is readily disrupted, resulting in two separable complexes, a small pericentrin containing complex of approximately 740 KDa and the the γ TuRC, 1.9 MDa in Xenopus. Co overexpression/ coimmunoprecipitation and yeast two hybrid studies demonstrate that pericentrin binds the γTuRC through interactions with both GCP2 and GCP3. When added to Xenopus mitotic extracts, the GCP2/3 binding domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly and induced rapid disassembly of pre-assembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding, and were specific for mitotic centro somal asters as I observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Overexpression of the GCP2/3 binding domain of pericentrin in somatic cells perturbed mitotic astral microtubules and spindle bipolarity. Likewise pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. I conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death. Additionally, I provide functional and in vitro evidence to suggest that the larger pericentrin isoform (pericentrin B/ Kendrin) is not functionally homologous to pericentrin/pericentrin A in regard to it's interaction with the γ TuRC.

All-trans retinoic acid (atRA) is an important morphogen in many developmental processes, including apoptosis, growth, organogenesis and differentiation. During the early embryonic development, atRA is synthesized in an irreversible reaction from all-trans retinal (atRAL), catalyzed mainly by retinal dehydrogenase 2 (RALDH2). The upstream metabolic pathway, including the redox reaction between all-trans retinol (atROL) and atRAL, mediated by short-chain dehydrogenase/reductase, however, is less understood during embryonic development. / Previously a Xenopus laevis short-chain dehydrogenase/reductase 3 (dhrs3) was identified as a gene differentially expressed in the Spemann-Mangold Organizer. In this study, dhrs3 was found to be expressed in the circumblastoporal ring, neuroectoderm and pronephros region, and was up-regulated by atRA signalling. By using loss-of-function and gain-of-function approaches, it was found that the phenotype induced by knockdown of dhrs3 mimicked those with an elevated level of atRA signalling, and overexpression of dhrs3 enhanced the phenotype of cyp26a1, which functions in degradation of atRA. In dhrs3 knock-down embryos (morphants), expression domain of the mesoderm markers brachyury was disrupted, and that of organizer marker lim1 were significantly expanded, suggesting altered mesoderm induction. Overexpression of dhrs3, on the other hand, exerted an opposite effect on lim1 by reducing its expression. dhrs3 also rescued the phenotype following raldh2 overexpression induced by exogenous atRAL, suggesting that dhrs3 competed with raldh2 for the same substrate, atRAL. In line with these findings, expression of the mid-brain, hindbrain and neural crest markers was posteriorized in dhrs3-overexpressing embryos, similar to the phenotype of atRA-deficient embryos induced by cyp26a1. These findings indicate that dhrs3 participates in the retinoid metabolism by reducing atRAL to atROL. / Xenopus dhrs3 morphants displayed a shortened anteroposterior axis, similar to that of atRA toxicity. Examination of convergent extension (CE) markers papc indicated a defect in the CE movement, which was also evidenced by the disrupted bra and not expression. Overall, the results of the present study suggest that dhrs3 regulates proper mesoderm patterning through regulating the CE movement. / Kam, Kin Ting. / Advisers: Yu Pang Eric Cho; Wood Yee Chan; Hui Zhao. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves [158]-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 82-96).

A cell integrates mitogenic signals received at the plasma membrane with intracellular biochemical changes to direct the events of cell division. Oocytes from Xenopus laevis offer a system that allows molecular dissection of pathways controlling cell growth and division in response to extracellular cues. Xenopus oocytes, physiologically arrested in a G2 like state, respond to the hormone progesterone to reinitiate meiosis and mature into a fertilizable egg. Signals received at the oocyte membrane induce translation of dormant maternal mRNAs that not only drive meiotic entry but also maintain the cell cycle arrest in an egg. A major pathway controlling the translation of these mRNAs is cytoplasmic polyadenylation, facilitated by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) through cis-acting elements in their 3'untranslated regions (3'UTRs). Cytoplasmic polyadenylation requires the phosphorylation of serine174 on CPEB by Aurora-A as well as the translation of a hitherto unknown mRNA. The transcript of the RINGO/Spy gene is a putative candidate for this unknown upstream regulator of CPEB function. RINGO/Spy mRNA is translationally repressed in immature oocytes by a ribonucleoprotein (RNP) complex consisting of the repressor Pumilio-2, the putative activator Deleted in Azoospermia-like (DAZl) and embryonic poly A binding protein (ePAB). Progesterone signaling leads to the dissociation of Pumilio-2 from the mRNP and the ensuing RINGO/Spy protein synthesis, in turn, promotes cytoplasmic polyadenylation and oocyte maturation. Pumilio and its associated proteins, such as Drosophila Brain tumor (Brat) and DAZl, in addition to their cytoplasmic roles have ill-defined functions within the nucleus. We detected DAZl within the nucleoli of telomerase-immortalized human retinal pigment epithelial (RPE) cells in interphase and on acrocentric chromosomes during mitosis. DAZl colocalizes with the RNA polymerase I associated Upstream Binding transcription Factor (UBF), most likely through pre-ribosomal RNA and is a likely component of the Nucleolar Organization Region (NOR). Stably knocking down DAZl in RPEs using short hairpin RNAs results in loss of nucleolar segregation, the physiological outcome of which is under investigation. These preliminary findings indicate an additional role for DAZl within the nucleolus, one likely to be independent from cytoplasmic translational control.

Fully grown oocytes' of the African clawed frog, Xenopus laevis, are arrested at the diplotene stage of meiotic prophase I, which resembles the G2 phase of the mitotic cell cycle. Re-entry into the meiotic divisions is initiated by hormonal signaling normally provided by progesterone. Progesterone signaling leads to the activation of maturation promoting factor (MPF), a heterodimer consisting of the protein kinase cdk1 and cyclin B1; this complex promotes the oocyte's entry into M phase of meiosis I. A crucial event required for MPF activation is cytoplasmic polyadenylation element (CPE)-mediated translation of specific dormant mRNAs such as c-mos and cyclin B1. The CPE, which resides in mRNA 3' untranslated region (UTR), is bound by the CPE binding protein (CPEB), which in turn is bound by Maskin. Maskin is bound to the 5' cap binding protein eIF4E. This type of closed-loop mRNA structure inhibits the recruitment and assembly of the translation initiation complex at the 5'UTR of CPE containing mRNAs. To alleviate this inhibition, CPEB undergoes phosphorylation on S174 by the serine/threonine kinase Aurora A. Phosphorylated CPEB promotes the recruitment of specific polyadenylation factors leading to the polyadenylation of the dormant mRNA, resulting in the disassociation of Maskin from eIF4E. eIF4E is subsequently bound by translation initiation factors leading to mRNA assembly into polysomes and synthesis of the encoded protein. Insulin signaling has also been shown to induce oocyte maturation. However, this signaling cascade uniquely requires the activation of two upstream components, PI3 kinase and PKC zeta. In this thesis, I show that insulin induced oocyte maturation requires the same CPE-mediated mRNA translation mechanism as had been described for progesterone signaling. I also show that Aurora A kinase activation and S174 phosphorylation play an essential role in insulin-induced CPE-mediated mRNA translation. Interestingly, inhibition of PI3 kinase and PKC zeta inhibits CPE-mediated polyadenylation only in the insulin-signaling pathway; the progesterone pathway is unaffected. These results clearly indicate that different upstream signaling components control CPE-mediated translation between progesterone and insulin signaling cascades. However, both pathways are antagonized by over expressed GSK-3, leading to inhibition of oocyte maturation. Furthermore, I found that GSK-3 inhibits Aurora A kinase activity by directly phosphorylating Aurora A on serine 290/291, promoting an inhibitory autophosphorylation event on serine 349. The importance of a GSK-3/Aurora A interaction is underscored by the finding that GSK-3, Axin, and Aurora A reside in a complex in immature oocytes. During progesterone or insulin signaling, GSK-3 dissociates from Aurora A allowing Aurora A to become active, leading to CPEB phosphorylation, CPE-mediated mRNA translation and oocyte maturation.