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(2008)ThesisBackground: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats. Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined ≤ 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3β, TGF-β, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-β expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-β expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities.
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(2008)ThesisPerlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.
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(2005)ThesisBinary image processing algorithms have been implemented in this study to create a background subtraction mask for the segmentation of cellular time lapse images. The complexity in the development of the background subtraction mask stems from the inherent difficulties in contrast resolution at the cellular boundaries. Coupling the background subtraction mask with the path reconstruction method via superposition of overlapping binary segmented objects in sequential time lapse images produces a semi-automatic method for cellular tracking. In addition to the traditional center of mass or centroid approximation, a novel quasi-center of mass (QCM) derived from the local maxima of the distance transformation (DT) has also been proposed in this study. Furthermore, image isolation and separation between spreading/motile and mitotic cells allows the extraction of both migratory and divisional cellular information. DT application to isolated mitotic cells permits the ability to identify distinct morphologic phases of cellular division. Application of standard bivariate statistics allows the characterization of cellular migration and growth. Determination of Hotelling’s confidence ellipse from cellular trajectory data elucidates the biased or unbiased migration of cellular populations. We investigated whether it was possible to describe the trajectory as a simple binomial process, where trajectory directions are classified into a sequence of (8) discrete states. A significant proportion of trajectories did not follow the binomial model. Additionally, a preliminary relationship between the image background area, approximate number of counted cells in an image frame, and imaging time is proposed from the segmentation of confluent monolayer cellular cultures.
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(2007)ThesisCell attachment is vital for the integration of biomaterials in the body. Surface modification using cell adhesive peptides, such as Arginine-Glycine-Aspartic (RGD), has showed promise for enhancing cell adhesion. Cell adhesion on glass and polyethylene glycol (PEGylated) surfaces modified with active RGD and Proline- Histidine-Serine-Arginine-Asparagine (PHSRN) peptides as well as inactive RDG and HRPSN control peptides was investigated in serum free conditions using three cell lines; NIH3T3 fibroblasts, MC3T3 pre-osteoblasts and C2C12 pre-myoblasts. Peptide attachment to glass surfaces was confirmed by x-ray photoelectron spectroscopy and contact angle measurements. Cell attachment and spreading was equivalent on all peptide and fibronectin coated glass surfaces and was significantly higher than on unmodified glass after 3 hours. Cell attachment to the peptide modified glass was reduced in the presence of soluble RGD and RDG peptides, indicating that cell attachment to these surfaces may be integrin mediated, but not specific for RGD. Inhibition of protein synthesis with cycloheximide revealed that endogenous protein synthesis did not influence the specificity of cell attachment to the peptide modified glass surfaces in all cell types within a 3 hour period. However, cycloheximide treatment inhibited cell spreading on the peptide modified glass surfaces, suggesting that proteins synthesis was required for spreading. Long term adhesion studies, within a 24 hour period, showed that all cell lines were able to remain attached to the peptide modified glass surfaces, while C2C12 and MC3T3 cells were also able to form focal adhesions during this period. Cell attachment to peptide modified PEGylated surfaces over a 3 hour period showed that NIH3T3 and C2C12 cells experienced significantly higher levels of cell attachment on the RGD modified surface compared to the other peptides. MC3T3 cells attached to all the peptide modified PEGylated surfaces to the same extent, suggesting that cell attachment to peptide modified PEGlyated surfaces, can be cell type dependent. In conclusion all the peptides were able to promote cell adhesion on glass surfaces in the absence of a PEG linker. In the presence of a PEG linker cellular response to the peptide surfaces was both peptide and cell type dependent.
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(2009)ThesisAtrial fibrillation (AF) is a complex, multifaceted arrhythmia. Pathogenesis of AF is associated with multiple aetiologies and the mechanisms by which it is sustained and perpetuated are similarly diverse. In particular, regional heterogeneity in the electrophysiological properties of normal and pathological tissue plays a critical role in the occurrence of AF. Understanding AF in the context of electrophysiological heterogeneity requires cell-specific ionic models of electrical activity which can then be incorporated into models on larger temporal and spatial scales. Biophysically-based models have typically dominated the study of cellular excitability providing detailed and precise descriptions in the form of complex mathematical formulations. However, such models have limited applicability in multidimensional simulations as the computational expense is too prohibitive. Simplified mathematical models of cardiac cell electrical activity are an alternative approach to these traditional biophysically-detailed models. Utilizing this approach enables the embodiment of cellular excitation characteristics at minimal computational cost such that simulations of arrhythmogensis in atrial tissue are conceivable. In this thesis, a simplified, generic mathematical model is proposed that characterizes and reproduces the action potential waveforms of individual cardiac myocytes. It incorporates three time-dependent ionic currents and an additional time-independent leakage current. The formulation of the three time-dependent ionic currents is based on 4-state Markov schemes with state transition rates expressed as nonlinear sigmoidal functions of the membrane potential. Parameters of the generic model were optimized to fit the action potential waveforms of the Beeler-Reuter model, and, experimental recordings from atrial and sinoatrial cells of rabbits. A nonlinear least-squares optimization routine was employed for the parameter fits. The model was successfully fitted to the Beeler-Reuter waveform (RMS error: 1.4999 mV) and action potentials recorded from atrial tissue (RMS error: 1.3398 mV) and cells of the peripheral (RMS error: 2.4821 mV) and central (RMS error: 2.3126 mV) sinoatrial node. Thus, the model presented here is a mathematical framework by which a wide variety of cell-specific AP morphologies can be reproduced. Such a model offers the potential for insights into possible mechanisms that contribute to heterogeneity and/or arrhythmia.
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(2006)ThesisSuccessful implementation of any technology requires acceptance by the users. Numerous studies in the area of information technology acceptance, based on wellknown theories have been conducted to examine technology acceptance models and predict user adoption/acceptance behaviour. There are several studies dealing with patients’ acceptance of different telemedicine applications, but few about the patients’ acceptance of home telecare. Most existing studies are not based on a strong theoretical framework. In this study, based on an extensive literature review and preliminary qualitative data, a theoretical model of the effect of Home Telecare Management System (HTMS) characteristics and psychological variables associated with technophobia on patients’ acceptance of HTMS is proposed. The proposed model is an augmented Technology Acceptance Model (TAM) of Davis (1986), which is called Home Telecare Management System acceptance model (HTMS-AM), in which TAM has been augmented by two constructs: HTMS anxiety and HTMS self-efficacy. The model is proposed to improve our understanding regarding patients’ acceptance of HTMS, which may lead to successful design and implementation of home telecare systems. In addition, it can be used as a theoretical basis to evaluate new generations of HTMS in terms of users acceptance in the early stage of their design and development even prior to implementation. In order to test the reliability and validity of the measures, video demonstrations of a home telecare system and demonstration of a system prototype to potential users was employed. To propose the HTMS-AM the following five stages were taken: 1. General well-known theoretical models of human behaviour from psychology and technology acceptance models from information technology were reviewed to create a basic template for the proposed model. 2. A preliminary study (focus group interviews, Chapter 5) was conducted to assess patients’ perceptions of HTMS. 3. Based on an extensive literature review and findings from preliminary qualitative studies, HTMS acceptance model was proposed, to improve our understanding about factors, which may affect patients’ intention to use HTMS. Several adaptations were applied in the model to be applicable in the HTMS context, such as augmenting the model with HTMS self-efficacy and HTMS anxiety constructs. 4. To measure the different psychological variables in the proposed model, valid and reliable measures from previous studies were used. However the preliminary study was used to develop measures, which did not exist in the literature. 5. These measures were tested in the final study. The subjects were patients who had been affected with Congestive Heart Failure (CHF) and/or Chronic Obstructive Lung Disease (COPD). Given the chronic nature of these diseases, the necessity for extended monitoring and management and frequent admission to hospitals due to worsening health status, these patients were deemed the most appropriate candidates for the HTMS. Further studies with more cases need to be conducted to test the actual model in which the impact of HTMS characteristics, psychological and demographic factors associated with technophobia upon intention to use the HTMS and the correlation of these factors with each other in appropriate healthcare settings.