Retinal degenerative diseases are diseases that may severely affect vision of people at different ages. These include retinitis pigmentosa (RP) and age-related macular degeneration (AMD). The current treatments for these diseases are limited. Since dysfunction and atrophy of the RPE are the key factors in the development of retinal degenerative diseases, transplantation of healthy retinal pigment epithelial (RPE) cells might be a promising therapeutic strategy. However, homologous RPE cells may lead to host immune rejection and harvesting autologous RPE cells may cause severe complications. Autologous iris pigment epithelial (IPE) cells, which are relatively easy to obtain, possess the same embryonic origin and share similar characteristics as RPE cells. Therefore, they may be used as a substitute of RPE cells for transplantation. Increasing interests have been demonstrated with the use of substrate to support cell attachment, proliferation and differentiation, so that transplanted cells could maintain the differentiated phenotype and perform their normal functions. However, degradation of biodegradable substrates may cause the breakdown of functional cell monolayer and produce toxic byproducts. Therefore, the aim of current study is to investigate the in vitro characteristics of rat IPE cells cultured on surface modified non-degradable expanded-polytetrafluorethylene (ePTFE) substrates and host response to the substrates without cells. Primary pure IPE cells were successfully isolated from rat eyes, which provided abundant cells for subsequent experiments. IPE cells harvested from both Long Evans rats and Dark Agouti rats proliferated and reached confluence on fibronectin coated n-heptylamine modified (F-HA) ePTFE substrates. These cells exhibited cuboidal or polygonal morphology with heavy pigmentation. In addition to the typical epithelial cell morphology, rat IPE cells grown on F-HA ePTFE substrates were able to form a cell monolayer with functional formation of tight junctional complex between neighboring cells. The IPE cell monolayers also demonstrated increased phagocytosis of photoreceptor outer segments (POS) with time and expression of cellular retinylaldehyde-binding protein (CRALBP) that served an important role in the conversion of all-trans-retinal to 11-cis-retinal in visual cycle. In the in vivo study, F-HA ePTFE substrate was successfully transplanted into the subretinal space of Royal College of Surgeons (RCS) rat, which is a well-recognized animal model of retinal degeneration. The F-HA ePTFE substrate remained flat up to 4 weeks after transplantation and did not induce significant up-regulation of pro-inflammatory cytokines TNFα and IL1β as well as activation of Müller cells and astrocytes which occurred in response to retinal inflammation. In conclusion, rat IPE cells that were grown on F-HA ePTFE substrate were able to establish a monolayer with functional tight junctions and RPE-specific functions. The F-HA ePTFE substrate demonstrated good biocompatibility in the subretinal space of RCS rats. These findings provide a potential therapeutic strategy for retinal degeneration. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease in the elderly. It is currently the fourth leading cause of death and will become the third by 2030. Cigarette smoke is the major cause of COPD pathogenesis, resulting from the burden of oxidants, which stimulates the production of inflammatory chemokines, leading to the influx of inflammatory cells into the airways and causing chronic inflammation. Due to lung infection by bacteria, such as Pseudomonas Aeruginosa during acute exacerbation in COPD, cigarette smoking might induce an immunosuppressive effect, which leads to bacteria colonization in the airways and further contributes to the chronic inflammation in the airway of COPD. Furthermore, cigarette smoke-induced production of reactive oxygen species (ROS) also plays an important role in the pathogenesis of COPD, however, N-acetyl-L-cysteine (NAC), which has been administered for the treatment of COPD as a mucolytic agent, also showed antioxidant and anti-inflammatory effect. The exact mechanism or cellular pathway through which cigarette smoke suppresses bacteria-induced inflammatory response and how NAC acts as an anti-inflammatory agent still remains uncertain. This study aims to investigate the effect of cigarette smoke and lipopolysaccharide (LPS) alone or in combination on the release of pro-inflammatory chemokines and to elucidate cigarette smoke-induced chemokines release in the presence and absence of NAC. Both cigarette smoke and LPS alone induced the release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1).Cigarette smoke suppressed the LPS-induced IL-8 and MCP-1release. NAC reduced both basal and cigarette smoke-induced secretion of these inflammatory chemokines. Moreover, Western blot demonstrated that cigarette smoke activated AMPKα phosphorylation, which was suppressed with NAC pretreatment, suggesting that NAC might have inhibitory effect on the release of chemokine release via the AMPK pathway. Our current data suggests that there may be a link between ROS generation to AMPK activation and chemokine release in BEAS-2B cells. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue. In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix. In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure. To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis. The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion. Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M.S.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 29-33.

Thesis (M.S.)--University of Michigan, 1975.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.