published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Kinesins are motor proteins responsible for the anterograde transport on microtubules. Kinesin-1 is the first characterized kinesin, and it consists of two heavy chains and two light chains. KIF5B is a form of Kinesin-1 heavy chains that is ubiquitously expressed in mammals. The head domain of KIF5B is responsible for ATP-dependent mechanical movement along microtubules, while the tail region is well-known for its interaction with cell specific cargos. Recent studies reveal a second microtubule binding site in the tail, suggesting special functions of KIF5B in microtubule sliding and bundling. To understand the role of KIF5B in long bone development, a conditional knockout mouse model was generated, in which Kif5b is deleted in early limb mesenchyme using Prx1-cre/LoxP mediated recombination. Unlike Col2a1-cre directed Kif5b knockout in chondrocytes, the expression of Prx1-cre in limb mesenchyme results in Kif5b knockout in both chondrocyte and osteoblast lineages. The Prx1-cre mediated Kif5b conditional knockout mice develop malformed long bones characterized by their bowed shape, shortened length and multiple fractures, which reflects a combination of defects in bone matrix and growth plate. The mutant mice demonstrate impaired bone matrix formation, as indicated by both collagen density reduction and collagen matrix disorganization. Also, the growth plate does not retain its normal organization, and the hypertrophic zone is absent. The KIF5B deficient chondrocytes not only lose planar cell polarity, but also undergo early apoptosis and fail in terminal differentiation. Interestingly, the binucleation rate is significantly increased in these chondrocytes, suggesting a severe cytokinesis defect. Besides, the intracellular retention of extracellular matrix (ECM) molecules and the uneven distribution of ECM in the cartilage imply both blockage and inappropriate direction of secretion. Cytokinetic defect in chondrocytes is closely associated with growth plate abnormality and growth retardation. In Kif5b knockout chondrocytes, cytokinetic defect is also one of the earliest and principal phenotypes. Therefore the underlying mechanism of cytokinetic defect was further investigated at cellular level. Since Kif5b knockout chondrocytes cannot survive in primary culture, RNA interference approach was adopted to generate a Kif5b-knockdown chondrogenic cell line. As expected, the Kif5b knockdown cells demonstrate cytokinetic defects characterized by increased binucleation rate and prolonged cytokinesis phase. In control cells, KIF5B becomes concentrated in the midbody during cytokinesis, and the midbody organization is disrupted in Kif5b knockdown cells. Furthermore, transient expression of full-length KIF5B significantly reduces the binucleation rate of these KIF5B deficient cells, whereas over-expression of a truncated KIF5B (without microtubule binding sites in tail region) cannot rescue the defect. Additionally, KIF5B is found to interact with midbody components PRC1 and Aurora B kinase by GST pull-down assay. This study demonstrates the multiple functions of KIF5B in long bone development and emphasizes its significant role as a key modulator in chondrocyte cytokinesis. More importantly, the study also brings new insights into the mechanisms of cytokinesis: we propose that KIF5B may participate in cytokinesis by regulating the midbody organization and stability via microtubule bundling and transporting or anchoring important components to the midbody. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2009. / Title from title screen (site viewed January 5, 2010). PDF text: xiv, 328 p. : ill. (some col.) ; 7.48 Mb. UMI publication number: AAT 3373081. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on February 17, 2010). Includes bibliographical references.

The notochord is a conserved structure in the phylum chordate, which includes all vertebrates and some closely related invertebrates. In mouse embryos, the notochord is a midline structure underneath the neural tube and it consists of a rod of cells constrained by a thick extracellular sheath, which is rich in fibronectin and other extracellular matrix molecule. During notochord formation, two key processes are involved: cell migration and convergent extension. Two molecules are essential for these processes: fibronectin and Protein Kinase C iota (prkci). For cell migration, fibronectin regulates this process by modulating cell protrusion via a signaling pathway in which atypical protein kinase C (aPKC) is an essential factor. For convergent extension, fibronectin has been shown to be important in this process by regulating both cell migration and cell adhesion. The role of aPKCin convergent extension is revealed as it is asymmetrically expressed in Ciona notochord during convergent extension, indicating possible function of aPKC in convergent extension process and in the morphogenesis of notochord. These studies raised the possibility that fibronectin and prkci are important in notochord formation, by regulating cell migration and convergent extension. In this thesis, Cre/loxP system was used to study the function of fibronectin and prkci in the development of notochord. I provided evidence that conditional deletion of fibronectin by Foxa2-Crein the notochord resulted in a notochord of smaller volume and fewer notochordal cells. The nucleus pulposus was also smaller in are and less in cell number. Fibronectin in the notochord was not affected at E9.5, but diminished in the core of the notochord at E12.5 and in nucleus pulposus at E15.5. The phenotypes of smaller notochord and the nucleus pulposus might be the results of reduced notochordal cell proliferation and increased cell death. However, more samples are needed to analyze to confirm this and perform statistical analysis. In addition, convergent extension of notochord seemed less effective. These results are consistent with previous study about fibronectin and α5β1 integrin. The results suggest that fibronectin is required for notochordal cell proliferation, survival, migration and efficient convergent extension, but not for notochordal cell fate determination. The results also demonstrated that prkci seemed not to be important for notochord development. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Endochondral bone development depends on the progression of chondrocyte proliferation, hypertrophy and terminal differentiation, which requires precise transcriptional regulation and signaling coordination. Disturbance of this process would disrupt chondrocyte differentiation and lead to chondrodysplasias. In cells, a highly conserved mechanism, ER stress signaling, has been developed to sense the protein load and maintain the cellular homeostasis. In humans, mutations in COL10A1 induce ER stress and result in metaphyseal chondrodysplasia type Schmid (MCDS). Previous analysis of a MCDS mouse model (13deltg mouse) had revealed a novel mechanism of chondrocyte adaptation to ER stress. The hypertrophic chondrocytes survive ER stress by reverting to a pre-hypertrophic like state (Tsang et al., 2007). To dissect the underlying mechanisms that coordinate chondrocyte survival, reverted differentiation and adaptation to ER stress, different chondrocyte populations in the wild type and 13del growth plates were fractionated for global gene expression analyses. The genome-wide expression profiles of proliferating chondrocytes, prehypertrophic chondrocytes, hypertrophic chondrocytes and terminally differentiated chondrocytes in the wild type growth plate provide molecular bases to understand the processes underlying both physiological and pathological bone growth. Systematic analyses of these transcriptomic data revealed the gene expression patterns and correlation in the dynamics of endochondral ossification. Genes associated with sterol metabolism and cholesterol biosynthesis are enriched in the prehypertrophic chondrocytes. Selected genes (Wwp2, Zbtb20, Ppa1 and Ptgis) that may potentially contribute to endochondral ossification were identified differentially expressed in the growth plate. Bioinformatics approaches were applied to predict regulatory networks in chondrocytes at different differentiation stages, implying the essential and dominant roles of Sox9 in coordination of stage specific gene expression. We further confirmed that Sox9 directly regulates the transcription of Cyr61, Lmo4, Ppa1, Ptch1 and Trps1, suggesting that Sox9 integrates different steps of chondrocyte differentiation via regulation of its target genes and partially crosstalk with IHH signaling pathway. The information on gene expression and regulation from physiological growth plate provides important basis to understand the molecular defects of chondrodysplasia. The hypertrophic zone in 13del growth plate was fractionated into upper, middle and lower parts for microarray profiling, corresponding for the onset of ER stress, onset of reverted differentiation and adaptation phase. Comparative transcriptomics of wild type and 13del growth plates revealed genes related to glucose, amino acid and lipid metabolisms are up regulated in response to ER stress. Fgf21 was identified as a novel ER stress inducible factor regulated by ATF4. Removal of Fgf21 results in increasing cell apoptosis in 13del hypertrophic zone without affecting the reverted differentiation process. Up regulation of genes expression related to hypoxic stress (Slc2a1, Hyou1, Stc2 and Galectin3) in 13del hypertrophic chondrocytes suggested that survival and adaptation of chondrocytes to ER stress involve cross-regulation by other stress pathways. Our findings have provided a new insight into the mechanisms that facilitate chondrocyte survival under ER stress in vivo, and propose the integrative effects of hypoxic stress pathway during the stress adaptation process, which broaden the molecular horizons underlying chondrodysplasias caused by protein folding mutations. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--University of Melbourne, School of Veterinary Science, 2007. / Typescript. Includes bibliographical references (leaves 153-176).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 211-225) Also available in print.