Nasopharyngeal carcinoma (NPC) has distinctive ethnic and geographic distributions, with the highest incidence in Southern China. Epstein-Barr virus (EBV) infection, non-viral environmental risk factors, and host genetics contribute to the development of NPC. In our previous studies, Fibulin-2 (FBLN2), located at chromosome 3p25.1, has been identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by using a chromosome 3 NotI genomic microarray screen, followed by functional assays. FBLN2 belongs to the fibulin family of extracellular matrix glycoproteins. It encodes a large protein consisting of cysteinerich and cysteine-free segments, three anaphylatoxin (AT) modules, a series of cbEGFlike repeated, and a fibulin module. Although FBLN2 was also identified as a candidate TSG in other cancers, its molecular characterization is still largely unknown. In the present study, lentiviral constitutive and inducible transgene expression systems, fluorescent protein labelling and reporter systems, and shRNA-mediated knockdown system were optimized and established for studies in NPC. With the use of lentiviral systems, the FBLN2-mediated signaling pathways and the functions of FBLN2-related p65 signaling pathway were revealed. Lentiviral pWPI-FBLN2 infected HONE1, HK1, and C666 cell lines consistently reduced p65 phosphorylation at serine S536. Also, FBLN2 was shown to inactivate RhoA and Cdc42, resulting in decreased stress fiber and filopodia formation. Full-length and truncated FBLN2 fragments, with the exception of anaphylatoxin module, reduced phosphorylation of p65 as well as suppressed HUVEC tube formation. The p65 pathway was then chosen for in-depth studies. Inactivation of p65 by p65 stable knockdown and IκBα super repressor overexpression showed reduced cell migration, invasion, angiogenesis, in vitro cell growth, and in vivo tumor growth. In contrast, overexpression of wild type p65 and phospho-mimic S536E p65 promotes cell migration, invasion, angiogenesis, in vitro cell growth, and cell cycle progression. Molecular studies suggested that tumorassociated angiogenesis is regulated by p65 through expression of pro-angiogenic factors and the p65 activity controls epithelial-to-mesenchymal transition (EMT)-like properties in NPC. Western blotting and qPCR analyses showed that inactivation of p65 reduced expression of pro-angiogenic factors and mesenchymal markers. Overexpression of p65 induced expression of pro-angiogenic factors and mesenchymal markers as well as enhanced EMT-like properties. The elimination of the p65 feedback mechanism by IκBα knockdown largely induced expression of pro-angiogenic factors and mesenchymal markers, as well as changes in cell morphology. In conclusion, these results suggest that FBLN2 suppresses tumor growth, tumor-associated angiogenesis, migration, and invasion through the regulation of Erk1/2, p65, and Rho GTPase pathways. The important roles of the p65 pathway in angiogenesis and EMT were also revealed. These findings provide a strategic new insight into the understanding of mechanistic role of FBLN2 in NPC and provide a better understanding for the molecular genetic basis of NPC tumorigenesis. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

The CLEC16A locus has been identified as a susceptibility gene for multiple autoimmune diseases, including multiple sclerosis, type-I diabetes and systemic lupus erythematosus (SLE), in genome-wide association studies. CLEC16A encodes a novel C-type lectin-like protein, by virtue of a predicted C-type lectinlike domain (CTLD), with unclear function. Studies on the disease-associated SNPs have suggested that CLEC16A polymorphisms affect the expression of neighboring genes, while the effect on its own expression is unclear. Several functional studies have interrogated the physiological role(s) of CLEC16A in disparate directions. The Drosophila ortholog of CLEC16A, Ema, has been reported to regulate endosomal protein trafficking and the autophagic process, while CLEC16A has been found to participate in LPS-induced inflammatory cytokine response in rat astrocytes. Since there is not a consenting role ascribed to CLEC16A, this study was undertaken to investigate the functional involvement(s) of CLEC16A in mammalian cells and the expression of CLEC16A in lupus patients, with the attempt to comprehend the association between CLEC16A and SLE. By overexpressing in non-immune epithelial cells, CLEC16A was revealed to be an intracellular protein of ~130 kDa in size. CLEC16A displayed a punctated expression pattern, which did not co-localize with endosomes, lysosomes, autophagosomes or endoplasmic reticulum in steady state. When treated with rapamycin or serum-starved, CLEC16A-overexpressing cells exhibited a reduced autophagic response, suggesting that CLEC16A may have an inhibitory role in autophagy. Besides the predicted CTLD, motif prediction has also implicated an immunomodulatory role for CLEC16A. Due to the observed inhibition on autophagy, coupled with recent findings linking autophagy and inflammasome activation, the involvement of CLEC16A in NLRP3 inflammasome was investigated. By knocking down CLEC16A in the human macrophage-like THP-1 cells, CLEC16A was found to potentially regulate NLRP3 inflammasome activation via inhibiting the LPS-induced pro-IL-1aasynthesis. Finally, the expressions of the long and short isoforms, CLEC16A_V1 and CLEC16A_V2 of CLEC16A in PBMCs were compared between healthy controls and SLE patients. A higher CLEC16A_V1 expression was observed in SLE patients, whereas the reverse was found for CLEC16A_V2. The expressions of the isoforms, however, were not correlated with the disease severity and clinical manifestations. The finding that CLEC16A may inhibit autophagy is in contrast with the reported function of Ema in supporting autophagy, and such discrepancy could be because of the different cell systems used. The finding that CLEC16A may downregulate NLRP3 inflammasome activation has not been previously reported, and the mechanism(s) of such regulation warrant(s) future studies. The molecular basis of how CLEC16A regulates autophagy and inflammasome waits to be delineated. Such knowledge, together with information of where endogenous CLEC16A is expressed, shall incite better understanding of the contribution of CLEC16A to SLE development. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Asporin (ASPN) is a risk factor for osteoarthritis and intervertebral disc degeneration. Its expression increases with aging and degeneration. D14 (14 aspartate-repeat polymorphism) is the risk allele and D13 is the most common allele. In vitro studies suggest that Asporin functions as a negative regulator of Tgf-β signaling, an important stimulator of matrix formation in bone and cartilage. However, the in vivo role of Asporin in development or its involvement in the pathogenesis of degenerative cartilage diseases is unclear. Here, we use mouse as a model to study the impact of Asporin in the intervertebral discs of the spine. In wide type mice, we showed that Asporin is expressed and localized in the nucleus pulposus and annulus fibrosis of intervertebral discs, and the articular cartilage in knee joints. Furthermore, Asporin expressing cells in these tissues are active in Tgf-β signaling, suggesting a relationship between Asporin and Tgf-β signaling and a role in disc and articular joint maintenance. Using natural degeneration with aging, and models for induced degeneration in the mouse-tail discs, Asporin expression was shown to be up-regulated in nucleus pulposus and annulus fibrosis cells of degenerating intervertebral discs. These cells are also active in Tgf-β signaling supporting a potential relationship with the pathogenesis of disc degeneration. Transgenic mice overexpressing Asporin in cartilage tissues were generated to study this relationship and the impact on the differentiation and function of disc cells. Interestingly, overexpression of Asporin in the nucleus pulposus leads to enhanced production and deposition of extracellular matrix such as glycosaminoglycans, with concomitant changes in cell morphology, suggesting Asporin altered the extracellular matrix niche of resident nucleus pulposus cells. However, such changes are only observed in discs in the tail region but not in lumbar discs. We propose a relationship to mechanical loading as an environmental factor. Molecular analysis of transgene expressing cells showed Tgf-β signaling is active and its downstream target genes up-regulated. Furthermore, overexpression of Asporin enhances differentiation of notochordal-like cells (NCCs) in mouse nucleus pulposus toward the more mature nucleus pulposus cells (NPCs) and chondrocyte-like cells (CLCs) that are more abundant in the human nucleus pulposus and other larger animals that prompt to intervertebral disc degeneration. This study provided new insights into the function of Asporin in the pathogenesis of intervertebral disc degeneration. We proposed a model whereby Asporin, as a genetic risk factor, alters the extracellular environment of the nucleus pulposus, that in conjunction with environmental factors such as mechanical loading, enhances Tgf-β signaling, and consequentially, promotes the maturation of NCCs towards NPCs and CLCs, a hallmark of degenerative process proposed in human and other larger animal models. These transgenic mice provide the opportunity to better understand the relationship between genetic and environmental factors, and the molecular controls leading to the maturation process of NCCs in intervertebral disc degeneration. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

abstract / Psychiatry / Doctoral / Doctor of Philosophy

Alcoholism is a complex disease determined by both genetic and environmental components that exerts a devastating economic and social impact worldwide. The complexity of this disease makes the elucidation of candidate genes for the susceptibility to alcoholism difficult in human populations, however, mouse model systems replicate many aspects of the disease and represent an excellent system for the investigation of the genetic contributions to alcoholism. One component of alcoholism that can be investigated in mouse models is the predisposition to high alcohol consumption. Selectively bred and inbred mice differ markedly in the level of voluntary alcohol intake using a two-bottle choice paradigm. The phenotype of voluntary alcohol consumption in mice is a complex trait and a genetic comparison between mouse models with similar levels of alcohol intake should identify genes that contribute to the predisposition for alcohol consumption. Three different studies were completed at the University of Texas and candidate genes involved in the predisposition to high alcohol consumption in mice were identified through the use of brain transcriptome analysis. In the first study, 3,800 transcripts were identified that were divergent between 3 selected lines and 6 isogenic strains of mice known to differ in voluntary alcohol consumption. This list was filtered to reveal candidate genes associated with alcohol preference on mouse chromosome 9: Arhgef12, Carm1, Cryab, Cox5a, Dlat, Fxyd6, Limd1, Nicn1, Nmnat3, Pknox2, Rbp1, Sc5d, Scn4b, Tcf12, Vps11, Zfp291. In the second study, analysis of voluntary alcohol intake and brain gene expression between two closely related inbred mouse substrains separated for nearly fifty years revealed divergent alcohol consumption as well as genetic variation between the substrains. Finally, the third study revealed dominant and overdominant patterns of expression in an F1 hybrid that voluntarily consumed more alcohol than either inbred parental strain. The microarray datasets analyzed here represent an important first step in the elucidation of the genetic determinants of high alcohol consumption in mice and will be influential in the discovery of genes that play a role in vulnerability to alcoholism in humans.

No description available.

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Botany / Master / Master of Philosophy

The Best M.Phil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,1997-1999 / published_or_final_version / Pathology / Master / Master of Philosophy