Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Iδ and Iε (CKIδ/ε) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKIδ/ε while avoiding the embryonic lethality of CKIδ disruption in mice, we used CKIδ-deficient Per2Luc mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKIε (DN-CKIε) in the mutant MEFs. CKIδ-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKIδ loss. When CKIε activity was disrupted by the DN-CKIε in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKIδ/ε are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKIδ/ε was disrupted by overexpressing the CKIδ/ε binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKIε did not, suggesting that CKIδ/ε may have a noncatalytic role in stabilizing PER. Our results show that an essential role of CKIδ/ε is conserved between Drosophila and mammals, but CKIδ/ε and DBT may have divergent non-catalytic functions in the clockwork as well. Since reversible phosphorylation events in the circadian clock are thought to be involved in temporal regulation of clock proteins, a dynamic process of clock proteins mediated by protein kinases and phosphatases may be an essential feature in the time-keeping mechanism in mammals. To address these issues more definitively and extend findings that CKIδ/ε are essential for the clockwork, we proposed to explore the dynamics of reversible PER phosphorylation by studying CKIδ/ε conditional mutant mice / cells and by identifying protein phosphatases in targeting PER and characterizing them using genetic and biochemical approaches. We finally validated that CKIδ/ε are essential protein kinases to facilitate driving clockwork based on our findings that CKIδ/ε double KO cells have no circadian rhythms and x they are rescued by transducing CKIε. Moreover, PP1 is highly associated with PER dephosphorylation based on our results in genetic (dominant negative PP1) and chemical approaches (phosphatase inhibitors: OA vs. CA). Therefore, we propose that dynamic and reversible processes mediated by kinases and phosphatases are essential features in the timedriving/ keeping mechanism in mammals. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2010.. / October 15, 2010. / Mouse embryonic fibroblast, Dominant negative, Casein kinase, Circadian clock / Includes bibliographical references. / Choogon Lee, Professor Directing Dissertation; James Fadool, University Representative; Mohamed Kabbaj, Committee Member; James Olcese, Committee Member; Yoichi Kato, Committee Member.

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Various aspects of the precipitation behaviour of calcium phosphate in aqueous media have been investigated using seeded growth in conjuction with constant-volume and constant-composition techniques under different physical and chemical conditions. In each case, precipitation was allowed to proceed for up to seven days. The solid precipitates thus obtained were characterised by means of scanning electron microscopy, powder X-ray diffractometry and wavelength dispersive spectroscopy. During these precipitation experiments, the formation of the thermodynamically most stable and most supersaturated phase was invariably preceded by the appearance of less supersaturated precursor phase(s). These precursors subsequently underwent step-wise phase transformation into more stable phases. The preferred precursor and the rates of precipitation and phase transformation were dependent on the physical conditions and the chemical composition of the calcifying medium. Under physiological conditions, precipitation experiments were also carried out with the addition of certain non-collagenous bone-specific bio-chemicals. Phosphoserine dramatically accelerated the precipitation of a large quantity of small plate-like crystals, while osteonectin and phosphatidylserine induced the formation of quasi-cubic crystals at a slow rate. Bone protein extract displayed the strongest inhibitory effect on calcification. Bovine serum albumin showed signs of being irreversibly adsorbed to the crystal surface, thereupon inducing a high degree of calcium deficiency in the precipitate stoichiometry. Using a number of phosphorylated amino acids of different molecular masses, it was found that the processes of precipitation and phase transformation were facilitated by organic molecules whose phosphoryl functional groups were sterically accessible and highly electronegative. However, the acceleration brought about by the presence of a phosphorylated amino acid was maximised at an optimum concentration. The existence of such an optimum was very likely to be consequent of the competition for free calcium ions by the ongoing complexation and precipitation reactions. A model has also been developed to describe and predict the precipitation behaviour of calcium phosphate. The model is based on the Avrami-Johnson-Mehl expression for threedimensional nucleation and growth processes. Appropriate modifications to the original equation have been made, in order to adapt to this multi-ionic aqueous system. The resulting model has been found to describe the actual precipitation process accurately. It has also been applied to systems in which organic additives were present, and has again furnished predictions closely resembling the behaviour as observed experimentally.

Thesis (M.S. in mechanical engineering)--Washington State University, December 2009. / Title from PDF title page (viewed on Jan. 22, 2010). "School of Mechanical and Materials Engineering." Includes bibliographical references (p. 123-128).

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Thesis (PhD)--University of South Australia, 2003

Thesis (PhD)--University of South Australia, 2003