This thesis describes a comprehensive study of understanding the mechanism of rigidity sensing by quantitative analysis using submicron pillar array substrates. From mechanobiology perspective, we explore and study molecular pathways involved in rigidity and force sensing at cell-matrix adhesions with regard to cancer, regeneration, and development by quantification methods. In Chapter 2 and 3, we developed fabrication and imaging techniques to enhance the performance of a submicron pillar device in terms of spatial and temporal measurement ability, and we discovered a correlation of rigidity sensing forces and corresponding proteins involved in the early rigidity sensing events. In Chapter 2, we introduced optical effect arising from submicron structure imaging, and we described a technique to identify the correct focal plane of pillar tip by fabricating a substrate with designed-offset pillars. From calibration result, we identified the correct focal plane that was previously overlooked, and verified our findings by other imaging techniques. In Chapter 3, we described several techniques to selectively functionalize elastomeric pillars top and compared these techniques in terms of purposes and fabrication complexity. Techniques introduced in this chapter included direct labeling, such as stamping of fluorescent substances (organic dye, nano-diamond, q-dot) to pillars top, as well as indirect labeling that selectively modify the surface of molds with either metal or fluorescent substances. In Chapter 4, we examined the characteristics of local contractility forces and identified the components formed a sarcomere like contractile unit (CU) that cells use to sense rigidity. CUs were found to be assembled at cell edge, contain myosin II, α-actinin, tropomodulin and tropomyosin (Tm), and resemble sarcomeres in size (~2 μm) and function. Then we performed quantitative analysis of CUs to evaluate rigidity sensing activity over ~8 hours time course and found that density of CUs decrease with time after spreading on stiff substrate. However addition of EGF dramatically increased local contraction activity such that about 30% of the total contractility was in the contraction units. This stimulatory effect was only observed on stiff substrate not on soft. Moreover, we find that in the early interactions of cells with rigid substrates that EGFR activity is needed for normal spreading and the assembly of local contraction units in media lacking serum and any soluble EGF. In Chapter 5, we performed high temporal- and spatial-resolution tracking of contractile forces exerted by cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like CUs simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.

Kung, Wing Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 92-115). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vii / Table of contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.4 / Chapter 2.1 --- Food allergy and its prevalence --- p.4 / Chapter 2.2 --- Mechanism and clinical symptoms of food allergy --- p.6 / Chapter 2.3 --- Tropomyosin as the major allergen in shellfish --- p.15 / Chapter 2.4 --- Cross reactivity and epitope mapping of tropomyosin --- p.21 / Chapter 2.5 --- Novel approaches for the treatment of food allergy --- p.29 / Chapter Chapter 3. --- Expression of shrimp recombinant tropomyosin and sensitization of mice --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and Methods --- p.40 / Chapter 3.2.1 --- "Recovery of E, coli with tropomyosin-carrying plasmid" --- p.40 / Chapter 3.2.2 --- Preparation of tropomyosin-carrying plasmid --- p.41 / Chapter 3.2.3 --- Confirmation of DNA sequence of the tropomyosin --- p.41 / Chapter 3.2.4 --- Identification of the recombinant protein --- p.43 / Chapter 3.2.5 --- Purification of the recombinant protein --- p.43 / Chapter 3.2.6 --- Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 3.2.7 --- Concentration measurement of the recombinant tropomyosin --- p.45 / Chapter 3.2.8 --- Mice --- p.46 / Chapter 3.2.9 --- Mice sensitization and challenging --- p.46 / Chapter 3.2.10 --- Tropomyosin-specific IgE level in blood --- p.47 / Chapter 3.2.11 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- DNA sequence of the cloned tropomyosin --- p.52 / Chapter 3.3.2 --- Expression and purification of tropomyosin --- p.52 / Chapter 3.3.3 --- Hypersensitivity symptoms after challenge --- p.53 / Chapter 3.3.4 --- Blood tropomyosin-specific IgE level --- p.53 / Chapter 3.4 --- Discussion --- p.62 / Chapter Chapter 4. --- Identification of T cell epitopes --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.67 / Chapter 4.2.1 --- Soluble epitope peptide synthesis --- p.68 / Chapter 4.2.2 --- Isolation of spleen cells from mice --- p.69 / Chapter 4.2.3 --- T cell proliferation assay --- p.70 / Chapter 4.3 --- Results --- p.71 / Chapter 4.3.1 --- Splenocyte proliferation to synthetic peptide --- p.72 / Chapter 4.3.2 --- Splenocyte proliferation to synthetic peptides pool --- p.72 / Chapter 4.4 --- Discussion --- p.77 / Chapter Chapter5 --- General conclusion --- p.89 / References --- p.92

Background: Cardiac Rehabilitation (CR) has been shown to be effective in reducing mortality and morbidity in Coronary Heart Disease (CHD). There is a limited amount of research that evaluates the impact of menu-based CR, in patients with Acute Coronary Syndrome with Low Troponin levels (ACSLT). Aim: This thesis contains a feasibility study and an exploratory study. The feasibility study aimed to examine the feasibility of a Randomised Controlled Trial (RCT) which would test the impact of a menu-based CR programme, on individuals diagnosed with ACSLT, against standard care. This feasibility study included staff views. The exploratory study aimed to explore the impact that ACSLT and CR can have on this client group. Method: The feasibility study was a repeated measures case-control trial of menu-based CR based on the theoretical framework of the Common Sense Model of Self-Regulation (CSM), using a range of health assessments. The areas assessed included misconceptions, symptoms, anxiety, depression and Health Related Quality of Life (HRQoL). In addition, focus groups were held with both ward and specialist CR staff to seek their views on the feasibility of a RCT of menu-based CR for ACSLT. The exploratory study consisted of description and analysis of the data that had been collected from the participants over the two year period as above. In addition it included qualitative data that had been collected during interviews with the participants. Findings: Participants (n=33) were recruited from cardiology wards following an admission with ACSLT. They were assessed at baseline (T1), nine months (T3) and 24 months (T4). Twenty-five participants completed the studies. The feasibility study was successful in its aim of testing the CR intervention and protocols for a further RCT. The intervention was acceptable to the participants and to the specialist staff, although the ward staff did not see the need for a RCT. The measures used, with the exception of the self-reporting measures, were suitable and provided a wide range of data that could be utilised in a RCT. However the changes to diagnostic categories meant that a RCT would no longer be feasible. The exploratory study found that both groups were similar on a range of baseline demographic and clinical factors. There was a tendency to benefit within the exploratory study which favoured the intervention. An additional finding from the exploratory study was the degree of uncertainty experienced by the participants, within the context of a changing political and clinical landscape. Discussion and conclusions: The studies presented in this thesis add to our knowledge by highlighting some of the difficulties in designing a RCT of menu-based CR in a specific subgroup of CHD and by presenting outcome data for a small group of participants that have not previously been studied within the literature. This data suggests that there was a tendency to benefit for the intervention that requires further study. Implications for practice: Patients with ACSLT are now being included in CR programmes due to the changes within the diagnostic criteria. Clinicians have little understanding of the impact of CR on this group of patients, or what type of interventions would work best. Large RCT’s will however be problematic and this thesis has highlighted that further work is required to explore how CR can best improve the well-being of individuals with ACSLT.