Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, December 2003. / Includes bibliographical references. Also available on the Internet.

Thesis--University of Helsinki. / Includes bibliographical references.

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

The Actin Related Protein (ARP) 2/3 complex is a major regulator of the actin cytoskeleton that is implicated in cell morphogenesis in plants. However, a similar role is attributed to the endoplasmic reticulum (ER). My research explored the relationship between the two systems by using transgenic plants simultaneously expressing fluorescent proteins highlighting F-actin and ER organization in living cells. A comparison of F-actin organization in cells of wild type Arabidopsis thaliana and mutants with aberrant actin cytoskeleton suggests bundling in the distorted2 mutant but a relatively fine F-actin arrangement in klunker. These differences correlate with ER organization into cisternae, fenestrated sheets and tubules. A model relating ER-organization to the degree of actin bundling in a cell emerges and is supported by drug-induced interference in actin polymerization, altered ionic conditions and temperature. The study adds to the mechanistic understanding of cell morphogenesis in plants.

As nascent secretory and membrane proteins are inserted into the endoplasmic reticulum (ER), they are maintained in folding and/or assembly competent states by molecular chaperones including the Hsp70 and Hsp90 homologues, BiP and GRP94, and the lectin-like chaperones calnexin (CNX) and calreticulin (CRT). Folding is catalyzed by protein disulfide isomerase (PDI), its CNX (and CRT) associated homologue, ERp57, and protein prolyl isomerase (PPI). Moreover, N-linked glycoproteins benefit from a lectin-based "quality control apparatus" that ensures their correct folding or oligomeric assembly. Binding to these lectins occurs through oligosaccharide trimming from Glc 3Man9GlcNAc2 to the monoglucosylated form (Glc 1Man9GlcNAc2). Release and subsequent rebinding occurs though the hydrolysis and reglucosylation of the innermost glucose by glucosidase II and UDP-glucose glycoprotein:glucosyltransferase, respectively. This cyclical process, termed the "Calnexin Cycle", continues until their correct conformation is achieved. / The cloning and characterization of human glucosidase II is reported here. cDNAs for two splice variants of the catalytic alpha subunit and the beta subunit were isolated. Expression of the beta subunit was shown to be required for enzymatic activity, solubility and/or stability, and ER retention of the enzyme. Detailed kinetic analysis on recombinant alpha1/beta and alpha2/beta isoforms, using p-nitrophenyl alpha-D-glucopyranoside as a substrate, reveals that both exhibit kinetic profiles of a two binding site model, and share properties of catalysis and inhibition on this substrate. Moreover, similar rates of hydrolysis of the oligosaccharide substrates rules out the possibility that the two binding site kinetic model, first proposed by Alonso et al. (1999, Biochem J. 278:721--7), is the result of co-purified isoforms of glucosidase II that have different substrate specificities. / Also, an ER protein two-hybrid system, based on Ire1p and the unfolded protein response (UPR) pathway in Saccharomyces cerevisiae, was developed to examine and map the interactions between CNX/CRT and ERp57. Ire1p fusions with CNX and CRT were shown to interact specifically with ERp57, and as expected, PDI did not. Through deletion analysis, new roles were assigned to the proline-rich loop domains of CNX and CRT, and the non-catalytic B thioredoxin domain of ERp57 in mediating their heterodimerization.

No description available.

We investigated whether human lysosomal hydrolases, in common with secretory and plasma membrane glycoproteins, associate with the ER chaperone calnexin. Neither $ alpha$- or $ beta$-chains of $ beta$-hexosaminidase A, cathepsin D, nor the endogenous proteases cathepsins B or L associated with calnexin in COS-I cells. Hex $ alpha$-chains misfolded due to either the incorporation of azetidine-2-carboxylic acid, treatment with dithiothreitol, or the presence of a Tay-Sachs Disease mutation (leading to retention of Hex A $ alpha$-chains in the ER) also did not associate with calnexin. Chemical-crosslinking reagents or long-term labeling also failed to show a Hex A $ alpha$-chain association with calnexin. Lysosomal hydrolases also did not associate with the ER chaperone calreticulin. Surprisingly, $ alpha$-L-iduronidase and Hex A $ alpha$-chains associated with calnexin when overexpressed using a CMV promoter. The segregation of lysosomal hydrolases from secretory proteins thus occurs at an earlier stage than predicted. Hydrolase folding appears to be controlled by a pathway different from that used by secretory and plasma membrane glycoproteins.

Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2007. / "A dissertation in cell biology and biophysics and molecular biology and biochemistry." Advisor: Antony A. Cooper. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Feb. 7, 2008 Includes bibliographical references (leaves 100-134). Online version of the print edition.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 65-74). Also available in electronic version.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.