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Chromosome alignment and orientation within the spindle in mitosis and meiosis are determined by chromosome-microtubule interaction. Evidence suggests that within the acentrosomal spindle the mechanism of chromosome positioning is different from in mitotic spindle but its molecular bases are not well understood. I investigated how chromosome-microtubule interactions position the chromosomes within the spindle using Drosophila oocytes. I addressed the role and molecular mechanisms of kinetochore and chromosome interaction with microtubules in this process. I developed new live imaging reagents to observe dynamic chromosome-microtubule interaction. Live imaging combined with inactivation of kinetochores in oocytes revealed that kinetochore-microtubule attachment is required for three-step chromosome positioning in Drosophila oocytes: de-congression, change of orientation and re-congression. Augmin, a γ-tubulin recruiting complex, has been previously shown to be important for chromosome congression specifically in oocytes. Live imaging further showed that Augmin facilitates chromosome congression particularly in early stages of spindle assembly. Study of Augmin dynamics revealed that Augmin stably associates with spindle polar regions, specifically in oocytes. This meiotic regulation of Augmin function may contribute to generation of force pushing chromosomes toward spindle equator. Sentin protein has been shown to be important for microtubule plus end dynamics in mitosis. In meiosis, sentin mutant results in reduced distance between centromeres of homologous chromosomes. However, its meiotic role is unknown. Live imaging of the sentin mutant showed that in oocytes Sentin is required for preventing premature stabilization of kinetochore-microtubule attachments. As conclusion, I have used live imaging to reveal molecular basis of the interaction between chromosomes and microtubules particularly important for oocytes.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: pp. 88-101.

The Xenopus laevis oocyte was examined as a model system for investigating insulin receptor function. The role of extracellular calcium on insulin-stimulated deoxyglucose uptake (ISDU) in the 𝘟𝘦𝘯𝘰𝘱𝘶𝘴 𝘭𝘢𝘦𝘷𝘪𝘴 oocyte was investigated. It was determined that removal of calcium from the medium did not alter the rate of ⁴⁵Ca²⁺ release from oocytes preloaded with ⁴⁵CaCl₂ In contrast to earlier reports using tissue explants and cultured cells, the insulin response in oocytes is not sensitive to a range of extracellular calcium concentrations from 1 μM to 10 mM. However, treatment of oocytes with 1 mM EGTA, in the absence of Ca²⁺, prior to, during or within 5 minutes of insulin addition resulted in a 2-4 fold inhibition of ISDU. To further investigate the event(s) in insulin signalling inhibited by EGTA, the number of receptors for insulin on the oocyte must be increased. To this end we have investigated the effects of the 5' and 3' untranslated regions as well as the coding region of mRNA on translational efficiency in reticulocyte lysate and oocytes. The results obtained in Xenopus oocytes are consistent with earlier cell-free data (Falcone and Andrews, 1991). We have demonstrated that replacing the cognate 5' UTR with the Xenopus beta globin 5' UTR appropriately linked to a consensus sequence for efficient translation initiation (ACCATGG) results in increased in translation in Xenopus oocytes. In vitro synthesized preprolactin transcript injected into oocytes was found to be functionally stable for several days (D. Andrews unpublished data). Stabilization of the preprolactin 3' transcript was localized to the UTR. Furthermore, inserting the preprolactin 3' UTR downstream of another coding region resulted in stabilization of the modified transcript. These results provided a basis for improving expression of cloned human insulin receptor in Xenopus oocytes. By optimizing the 5' and 3' UTR's of the insulin receptor clone we were successful in expressing high levels of insulin receptors in Xenopus oocytes. Effects of the coding region on translation were also investigated and we provide evidence that sequences in the coding region modulate translational efficiency. / Thesis / Master of Science (MSc)

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by Chiu Tak Yu Tony. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 156-182). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.