Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M. Res.)--University of Hong Kong, 2006.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Background: Wheezing disorders have increased worldwide. The respiratory epithelium plays an important role in the pathogenesis of wheeze. Nasal epithelial cells (NEC) are a valid surrogate for bronchial airway epithelial cells, are accessible and could be a valuable tool in translational epidemiological studies. A better understanding of this layer may decrease the burden of wheezing disorders. Objectives: To determine the feasibility of sampling and culturing NEC from children and adults with and without different wheezing conditions in epidemiological studies. To study NEC mediator release in these individuals following different environmental exposures. Methods: NEC were sampled from unsedated children and adults with and without a wheezing condition by brushing. NEC were cultured in media and also exposed to interleukin-1 (IL-1) & tumour necrosis factor alpha (TNF-), house dust mite (HDM) extract, lipopolysaccharide (LPS) and extracted tobacco smoke (ETS) for 24 hours. Resulting supernatants were analysed via Enzyme – linked immunosorbent assay (ELISA) and cytometric bead array (CBA) for mediator release. Results: 287 individuals including 164 children and 123 adults where phenotyped and brushed. 81 samples reached tertiary passage. Decreased release of vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein -1 (MCP-1) was observed in wheezing individuals when compared to healthy controls. These cytokines were increased in individuals with chronic obstructive pulmonary disease (COPD) relative to both asthmatic and healthy adults. Individuals with/without allergic rhinitis demonstrated different mediator release. Conclusions: It is feasible to obtain NEC in adults and children for both epidemiological and translational research, although the presence of allergic rhinitis may act as a potential confounder. Differences are present in adults and children with asthma compared to healthy controls. Contrasting differences between COPD and asthma suggest that these are different conditions.

Luminal epithelial cells in the mammary gland require two types of signals to proliferate: soluble signals (growth factor signals) and signals from the extracellular matrix (ECM). The composition of the ECM is sensed by adhesion receptors such as integrins. Integrins modulate cell behaviour and play a key role in cell cycle entry. Altered integrin expression and signalling has been associated with breast cancer and studies using mouse mammary epithelial cells (MECs) have shown that the absence of β1-integrin induces growth arrest. However, it is not completely understood how integrins transduce the signals from the plasma membrane to the nucleus to induce cell cycle entry. Thus, the first aim of this project was to determine how β1-integrin controls proliferation in MECs. I established a model to study the effects of depleting β1-integrin using the FSK7 mammary epithelial cell line. The proliferation defect observed in this β1-integrin knockdown model was rescued by expressing a constitutively active Rac1 or Pak. Moreover, inhibiting Rac1 or Pak prevented normal proliferation in MECs in a similar fashion as β1-integrin depletion. Furthermore, in this thesis I have identified the complex comprised of Src, paxillin and p130Cas as a potential link between β1-integrin and Rac1. These results provide an insight into the mechanism that regulates proliferation downstream of β1-integrin. During breast cancer initiation, β1-integrin signals are disrupted. This indicates that additional signals must be driving proliferation during tumorigenesis. Therefore, the second aim of this project was to test whether expression of breast oncogenes can overcome the proliferation defect present in β1-integrin null cells. In order to do so, an oncogenic ErbB2, a constitutively active form of Akt (myrAkt) and the Notch1 intracellular domain (NICD) were transfected in the β1-integrin knockdown MECs. The results showed that ErbB2 overcomes the need for β1-integrin by signalling to Pak. NICD does not require β1-integrin to drive proliferation by an unknown mechanism. Expression of myrAkt did not restore normal levels of proliferation in β1-integrin depleted MECs. This finding suggests that Akt is not sufficient to induce cell cycle entry by itself and instead, both Akt and Erk signalling are needed to exert this function. This work has further delineated the specific signals controlling proliferation downstream of β1-integrin, and has provided a model to test the dependence of oncogenes for β1-integrin to drive proliferation in MECs. These studies are important to understand the role of β1-integrin in breast cancer formation and to define the types of breast cancer where β1-integrin can be used as an effective therapeutic target.

The aim of this thesis was to study the regulation of proteinase secretion by epithelial cells (E-cells) derived from the epithelial cell rests of Malassez. Since these epithelial cell rests are present only in small numbers in-vivo, E-cells derived from porcine cell rests were cultured according to Brunette et al. (1976) and conditions chosen so that detectable amounts of the proteinases, neutral proteinase and plasminogen activator, could be obtained. The regulation of the secretion of these enzymes was investigated by varying the cell population density, adding E.Coli lipopolysaccharide to the cultures and altering the shape of the E-cells by both chemical and physical means. Cell population density modulated both neutral proteinase and plasminogen activator secretion. Neutral proteinase secretion was highest at low cell population densities and the activity decreased with increasing cell population density. Plasminogen activator secretion followed a similar pattern. Escherichia coli lipopolysaccharide (E.coli LPS) stimulated both neutral proteinase and plasminogen activator secretion. LPS extracted by the phenol method and LPS extracted by the trichloroacetic acid method caused similar increases in neutral proteinase activity but the increase in plasminogen activator activity was greater when the trichloroacetic acid extracted LPS was used. These findings support the proposal that bacterial LPS in contact with periapical tissues could stimulate the epithelial cell rests into increased production of proteinases, thereby contributing to the degradation of connective tissue associated with dental cyst formation. E-cell shape was altered by physical and chemical means. Addition of cholera toxin and dibutyryl cAMP caused E-cells to flatten. Phorbol myristate acetate, however, caused the cells to retract slightly. Mechanical stretching was applied to the cells to cause cell flattening, and cell rounding was effected by mechanical relaxation. Another method made use of E-cells grown on a substrate with V-shaped grooves which caused the cells to adopt a rounder shape more frequently than cells grown on a flat substrate. In addition, dishes coated with increasing concentrations of poly(HEMA) solution, which altered dish adhesivity to the cell, caused the cells to become less well-spread. In all experiments, a more flattened cell shape correlated with a reduced level of neutral proteinase and plasminogen activator secretion while a more rounded shape correlated with increased amounts of neutral proteinase and plasminogen activator secretion. / Dentistry, Faculty of / Graduate

Salmonella is a food-borne pathogen that leads to substantial illness worldwide. The clinical syndromes associated with Salmonella infection are enteric (typhoid) fever and gastroenteritis, in healthy humans. Typhoid fever is caused by host-adapted S. Typhi and S. Paratyphi. Gastroenteritis is caused by serovars usually referred to as non typhoidal Salmonellae (NTS). In recent years, an increasing number of outbreaks due to NTS, despite increased efforts in food safety, were reported because of persistence of Salmonella in the food chain. Thus I hypothesized that Salmonella is able to withstand stresses in the environment and treatments used during food processing for its elimination and thereby able to develop resistance against subsequent stress encounters. The effect of cold, peroxide, and acid was tested on survival of S. Typhimurium and the survival was persistent under cold stress (5°C) for up to 240 h. Pre-adaptation to cold stress (5°C, 5 h) also increased survival of S. Typhimurium during subsequent exposure to acid stress (pH 4.0, 90 min) by repressing hydroxyl radical formation. Cold stress (5°C, 48 h) to S. Typhimurium significantly (p < 0.05) increased its adhesion and invasion in intestinal iv epithelial cells. This phenotype was attributed to a pair of protein-protein interactorsacting as receptors on microbial (STM2699) and host cell surface (SPTAN1). Cold stress significantly (q < 0.05) induced STM2699 in S. Typhimurium and SPTAN1 was significantly (q < 0.05) induced in pithelial cells upon infection with cold-stressed S. Typhimurium. Cold stress to S. Typhimurium also significantly (q < 0.05) induced genes related to virulence such as type 3 secretion system apparatus and effectors genes, prophage genes, and plasmid genes and they remain induced upon infection of epithelial cells with additional induction of spv genes on the plasmid. Infection of epithelial cells with cold-stressed S. Typhimurium significantly (p < 0.05) increased activation of caspase 9 and 3/7. Cold-stressed S. Typhimurium switched metabolism from aerobic respiration to fermentation and it persisted during infection of epithelial cells. As a result, short chain fatty acids formate and acetate, which act as diffusible signal for invasion, were detected in significantly (q < 0.05) high amounts in extracellular media of cells infected with cold-stressed S. Typhimurium supporting the phenotype of high adhesion and invasion of cold-stressed S. Typhimurium in epithelial cells.

No description available.

No description available.