Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 176-207). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 176-207). Also available in print.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a protein with multiple functions that include regulating the turnover of the extracellular matrix (ECM) by inhibiting members of the metzincin family. Extracellular levels of soluble TIMP-3 are low, reflecting its binding to components of the ECM including sulfated glycosaminoglycans (SGAGs) and its endocytosis by low density lipoprotein receptor-related protein 1. Because TIMP-3 inhibits ECM-degrading enzymes, the ability of SGAG mimetics to elevate extracellular concentrations of TIMP3 is of interest for osteoarthritis treatment. However, previous studies of such interactions have utilized immobilized forms of the protein or ligands. Here we have quantified the thermodynamics of the interactions of the inhibitory domain of TIMP-3 with chondroitin sulfate (CS), pentosan polysulfate (PPS) and suramin in solution using isothermal titration calorimetry. All three interactions are driven by a (favorable) negative enthalpy ychange combined with an unfavorable decrease in entropy. The heat capacity change (ΔCp) for the interaction of N-TIMP-3 with CS, PPS, or suramin is essentially zero, indicating an insignificant contribution from the hydrophobic effect. Based on the effects of ionic strength on the interaction of N-TIMP-3 with suramin, their interaction appears to be driven by electrostatic interactions. Modeling supports the view that the negatively charged sulfates of CS, PPS, and suramin interact with a cationic region on N-TIMP-3 that includes Lys -26, -27, -30, and -possibly 76 on the opposite face of TIMP-3 from its reactive site for metalloproteases. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Bellamkonda, Ravi; Committee Member: LaPlaca, Michelle; Committee Member: McKeon, Robert

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Ravi V. Bellamkonda; Committee Member: Andreas Bommarius; Committee Member: Andrés J. García; Committee Member: Niren Murthy; Committee Member: Robert J. McKeon. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Injury to the central nervous system (CNS) results in the formation of a highly inhibitory glial scar consisting mainly of chondroitin sulfate proteoglycans (CSPGs). CSPGs are comprised of a protein core with covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. CSPGs and CS-GAGs have been implicated in the regenerative failure of the CNS, though the mechanism underlying inhibition is unclear. Sulfation affects both the physical and chemical characteristics of CS-GAGs and, therefore, it has been hypothesized that certain sulfation patterns are more inhibitory than others. To investigate this hypothesis, specific chondroitin sulfate sulfotransferases (CSSTs), the enzymes responsible for CS-GAG sulfation, were knocked down in vitro using siRNA. C4ST-1, C4ST-2, and C46ST were chosen as targets for gene knockdown in this study based on their expression in neural tissue and the extent of inhibition caused by their respective CS-GAG. It was hypothesized that transfection of primary rat astrocytes with siRNAs designed to prevent the expression of C4ST-1, C4ST-2, and C46ST would decrease specific sulfation patterns of CSPGs, resulting in improved neurite extension in a neurite guidance assay. Through optimization of siRNA dose, astrocyte viability was maintained while successfully knocking down mRNA levels of C4ST-1, C4ST-2, and C46ST and significantly reducing total levels of secreted CS-GAGs. However, no increase in the incidence of neurite extension was observed using conditioned media collected from siRNA transfected astrocytes compared to non-transfected controls. These data suggest that sulfation does not contribute to CSPG-mediated neurite inhibition, though further investigation is necessary to confirm these findings. Significantly, this work has established a paradigm for investigating the role of CSPG sulfation patterns in CNS regeneration.

M.Tech. (Homoeopathy) / Osteoarthritis (OA) is a chronic and debilitating condition, characterized by irreversible damage to the joint space, most commonly affecting the knees, hips, hands and spine (Colledge et al., 2010). OA is the leading cause of joint pain and disability in middle-aged and elderly persons (Long et al., 2001). The prevalence of OA of the knee in adults living in the United Sates has grown from a reported 21 million in 1990 to a total estimate of 26.9 million in 2005 (CDC, 2011). By the age of 65 years, 80% of the total population has been reported as showing radiographic evidence of OA; while a 20-30% of the total population is symptomatic with radiographic evidence of OA (Doherty et al., 2006). Conventional treatment for OA of the knee is aimed at pain management by use of analgesics and non-steroidal anti-inflammatory drugs (NSAIDs). Some negative effects of these drugs include drug dependency, liver and kidney damage, cardiovascular pathologies, gastric upset and depression. Corticosteroid injections are also used to alleviate chronic inflammation and joint pain, but may lead to further joint destruction (Shamoon and Hochberg, 2000; Mayo Foundation for Medical Education and Research, 2011). OsteoEze Gold™ is a nutraceutical product that contains chondroitin sulphate, glucosamine sulphate, vitamin C and manganese. In combination, the constituents of OsteoEze Gold™ have been shown to be useful in the treatment for OA of the knee (Clegg et al., 2006). In addition, studies have shown that these ingredients prove effective in reducing moderate to severe pain in sufferers of OA of the knee (Vidyasagar et al., 2004). The aim of this study was to determine the effect of OsteoEze Gold™ on the inflammatory marker C-reactive protein (CRP) and quality of life in OA of the knee using blood tests and the Arthritic Impact Measurement Scales (AIMS2SF) respectively. This was a 16-week, double blind, placebo-controlled study using matched pairs according to age, gender and severity of symptoms, and formed part of a group study, with another researcher, who utilized the Intermittent and Constant Osteoarthritis Pain scale (ICOAP) Short Physical Performance Battery (SPPB) and the same sample...

M.Tech. (Homoeopathy) / Osteoarthritis (OA) is a musculoskeletal condition affecting the synovial joints of the body, most commonly the knee and hip (Colledge et al., 2010). OA is the most prevalent joint disorder worldwide (Ickinger & Tikly, 2010). The prevalence of OA of the knee in developing countries, including South Africa, is expected to increase due to the increase in obesity and life-expectancy (Woolf & Pfleger, 2003). OA not only impacts negatively on many areas of the patient’s personal life, but it also has a considerable impact on health care systems and cost to the patient (Lapsley et al., 2001; Majani et al., 2005). The two main complaints in patients suffering from OA of the knee are knee pain and decreased daily functionality, such as walking (Samson et al., 2007). The main aim of conventional treatment is pain reduction. This treatment does not prevent progression of the OA, and may have negative side-effects (Day & Graham, 2005). Treatments for OA, such as OsteoEze GoldTM, may provide an effective and safer alternative. The aim of this study is to determine the effect of OsteoEze GoldTM on pain and functional ability in osteoarthritis of the knee using the Intermittent and Constant Osteoarthritis Pain (ICOAP) scale: knee version (Appendix D) and the Short Physical Performance Battery (SPPB) test (Appendix E). This was a 16-week study, conducted at the Homoeopathic Health Centre, Doornfontein campus (DFC), University of Johannesburg (UJ). The study was randomised, double blind placebo controlled, and matched pairs were utilised. Sixty-seven participants, who satisfied the inclusion and exclusion criteria, were recruited, and 48 of the participants completed the study. Participants were recruited by advertisements, placed in and around the UJ Homoeopathy Health Centre (with relevant permission given) and by word of mouth. The participants were split into two groups using matched pairs according to age, gender and severity of symptoms (Appendix H). The participants in group A received the OsteoEze GoldTM capsules, and the participants in group B received the placebo capsules. Each capsule of OsteoEze GoldTM contained 500mg glucosamine sulphate, 267mg of chondroitin sulphate, 50mg of vitamin C and 1mg of manganese. The OsteoEze GoldTM or the placebo capsules were distributed at the initial (week-0) and second (week-8) consultations.

Chondroitin sulfate proteoglycans (CSPGs) are one major class of axon growth inhibitors that are upregulated and accumulated around the lesion site after spinal cord injury (SCI), and result in regenerative failure. To overcome CSPG-mediated inhibition, digestion of CSPGs with chondroitinase ABC (chABC) has been explored and it has shown promising results. chABC digests glycosaminoglycan chains on CSPGs and can thereby enhance axonal regeneration and promote functional recovery when delivered at the site of injury. However, chABC has a crucial limitation; it is thermally unstable and loses its enzymatic activity rapidly at 37 ºC. Therefore, it necessitates the use of repeated injections or local infusions with a pump for days to weeks to provide fresh chABC to retain its enzymatic activity. Maintaining these infusion systems is invasive and clinically problematic. In this dissertation, three studies are reported that demonstrate our strategy to overcome current limitations of using chABC and develop a delivery system for facilitating chABC treatment after SCI: First, we enhanced the thermostability of chABC by adding trehalose, a protein stabilizer, and developed a system for its sustained local delivery in vivo. Enzymatic activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dimethylmethylene blue (DMMB), and conformational change of the enzyme was measured via circular dichroism (CD) with and without trehalose. When stabilized with trehalose, chABC remained enzymatically active at 37 ºC for up to 4 weeks in vitro. We developed a lipid microtube-agarose hydrogel delivery system for a sustained release and showed that chABC released from the delivery system is still functionally active and slowly released over 2 weeks in vitro. Second, the hydrogel-microtube system was used to locally deliver chABC over two weeks at the lesion site following a dorsal over hemisection injury at T10. The scaffold consisting of hydrogel and chABC loaded lipid microtubes was implanted at the top of the lesion site immediately following injury. To determine effectiveness of topical delivery of thermostabilized chABC, animal groups treated with single injection or gel scaffold implantation of chABC and penicillinase (P'ase) were included as controls. Two weeks after surgery, the functionality of released chABC and the cellular responses were examined by immunohistological analysis with 3B3, CS-56, GFAP and Wisteria floribunda agglutinin (WFA). The results demonstrated that thermostabilized chABC was successfully delivered slowly and locally without the need for an indwelling catheter by using the hydrogel-microtube delivery system in vivo. The results demonstrated that released chABC from the gel scaffold effectively digested CSPGs, and therefore, there were significant differences in CSPG digestion at the lesion site between groups treated with chABC loaded microtube-hydrogel scaffolds and controls. Third, a long term in vivo study (45 days) was conducted to examine axonal sprouting/regeneration and functional recovery with both a single treatment each of microtube loaded chABC or Neurotrophin-3 (NT-3), and a combination of them by using the hydrogel-microtube delivery system. Over the long term study period, the treated animals showed significant improvement in locomotor function and more sprouting of cholera toxin B subunit (CTB)-positive ascending dorsal column fibers and 5-HT serotonergic fibers around the lesion site. We demonstrated that this significant improvement of chABC thermostability facilitates the development of a minimally invasive method for sustained, local delivery of chABC that is potentially a useful and effective approach for treating SCI. In addition to that, we demonstrated that combinatorial therapy with chABC and neurotrophic factors could provide a synergistic effect on axonal regrowth and functional recovery after SCI.