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(2016)ThesisOsteoarthritis is a degenerative joint disease that affects millions of people worldwide. The aims of this study were (1) to quantitatively characterise the boundary and surface features of wear particles present in the synovial fluid of patients, (2) to select key numerical parameters that describe distinctive particle features and enable osteoarthritis assessment and (3) to develop a model to assess osteoarthritis conditions using comprehensive wear debris information. Discriminant analysis was used to statistically group particles based on differences in their numerical parameters. The analysis methods agreed with the clinical osteoarthritis grades in 63%, 50% and 61% of particles for no osteoarthritis, mild osteoarthritis and severe osteoarthritis, respectively. This study has revealed particle features specific to different osteoarthritis grades and provided further understanding of the cartilage degradation process through wear particle analysis – the technique that has the potential to be developed as an objective and minimally invasive method for osteoarthritis diagnosis.
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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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(2017)ThesisChronic wounds fail to undergo an orderly and timely reparative process. Currently, there are no effective treatments for chronic wounds. The heparan sulphate (HS) proteoglycan (HSPG), perlecan, plays an important role in healing chronic wounds through binding and signalling of growth factors. Biomimetic materials that mimic naturally occurring HS may find application in the delivery of growth factors for skin wound healing. Chitosan is a polysaccharide with a similar structure to HS, with the exception of sulphate modifications. This thesis explored the effect of chitosan-arginine (CH-Arg), a water soluble form of chitosan, modified with different levels of sulphate substitution on skin wound healing. Specifically, this thesis investigated the effect of sulphated CH-Arg on the expression of extracellular matrix (ECM) components including perlecan produced by human keratinocytes dermal fibroblasts in 2D and 3D skin models. Perlecan produced by both skin cell types was full-length perlecan decorated with HS and chondroitin sulphate (CS) chains. In addition, smaller fragments of perlecan were present in the fibroblast samples. Exposure to highly sulphated CH-Arg (HS-CH-Arg) increased the perlecan expression in both skin cell types. CH-Arg-based materials did not alter the proliferation of either skin cell type, while sulphated CH-Arg significantly (p <0.05) enhanced keratinocyte migration, demonstrating that the sulphated CH-Arg materials promoted keratinocyte migration. An in vitro skin model was established with similar expression in basement membrane components, proteoglycans and glycosaminoglycans to human adult skin, as well as a well-developed mimicked epidermis. CH-Arg-based materials significantly (p <0.05) enhanced the epidermal thickness, indicating their role in epidermal formation. In addition, sulphated CH-Arg enhanced the expression of perlecan, laminin β1, collagen type IV, syndecans-1 and -4 and HS with the exception of serglycin. Thus, CH-Arg-based materials have the potential to mimic HS and promote skin wound healing with respect to enhancing re-epithelialisation and the formation of ECM molecules.
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(2016)ThesisThe aim of visual prostheses is to return a perception of sight for blind people. From the outset, designing a visual prosthesis system that minimizes platinum dissolution remains a challenge. Recently, laser surface modification was introduced to reduce platinum dissolution. This study will investigate the performance of laser-pattern for platinum electrodes, in particular structured laser interference patterning (SLIP), both in inorganic saline and biologically relevant solution. In this study, a new platinum electrode laser fabrication technique based on enamel was proposed, which can produce more accurate electrode exposure areas and a higher degree of control on surface morphology of microelectrodes. This new method describes the advantages of laser fabrication, which requires no clean room facilities and offers short design-to-prototype time. The feasibility of fabricating implantable electrodes with this method using medical grade enamel will require additional testing. An artificial suprachoroidal interstitial fluid (ASIF) preparation was further developed as a more appropriate test electrolyte for visual prostheses in vitro experiments. This artificial fluid was prepared by analysing residual solution on the surface of electrodes which were explanted from sheep suprachoroidal space. The extent of platinum dissolution occurring on laser-patterned electrodes stimulated at clinically-relevant levels in both Dulbecco’s phosphate buffered saline (DPBS) and ASIF was also quantified. Laser surface modification on electrodes minimised platinum dissolution during electrical stimulation and enhanced charge storage capacity of electrodes both in DPBS and ASIF. Furthermore, SLIP pattern showed the great improvement on the performance of electrodes, and that improvement was not simply related to the increase of electrode surface morphology variation but potentially because of the laser surface plasmon interference effect.