Bibliography: leaves 269-297. / xvii, 297, [10] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Evaluates the efficacy of enzyme replacement therapy (ERT) with artifically produced recombinant human 4S (rh4S) in feline mucopolysaccharidosis Type VI (MPS VI) and tests the hypothesis that this form of therapy would reverse or alter the disease course, particularly the bone dysplasia and connective tissue pathologics. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1998

Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1998. / Bibliography: leaves 269-297.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Paediatrics, 1998. / Errata pasted onto back end paper. Bibliography: p. 187-204.

Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2001. / Addenda slip inserted in back. Includes bibliographical references (leaves 178-231).

Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003. / Addenda page on inside back cover. Bibliography: leaves 153-176.

Human α-galactosidase (α-GAL; EC 3.2.1.22) is a lysosomal enzyme that hydrolyzes of terminal alpha-linked galactosyl residue of glycosphingolipids. Deficiencies in α-GAL leads to Fabry disease, which is characterized by the build-up of globotriaosylceramide and other neutral substrates in cells, ultimately leading to a multi-systemic organ failure in patients. Hundreds of distinct mutations have been found in the α-GAL gene of Fabry disease patients. One current treatment for Fabry disease is Enzyme Replacement Therapy (ERT), which restores the missing α-GAL function. An alternative treatment, called Pharmacological Chaperone Therapy (PCT), utilizes a small molecule substrate analogue, 1-deoxygalactonojirimycin (DGJ). In order to better understand molecular basis of Fabry disease, this work addresses structural and mechanistic studies of the α-GAL glycoprotein. First, we have determined crystal structures of each stage in the catalytic mechanism of the α-GAL enzymatic reaction. These studies reveal a novel strained conformation of the sugar when it is covalently bound to the enzyme. Second, we examine the molecular mechanism of chaperoning by pharmacological chaperones. A combination of biochemical and biophysical approaches reveals that the high potency of the DGJ chaperone is due to an interaction with α-GAL residue D170. Third, we have investigated mutant α-GAL proteins for their response to pharmacological chaperones, leading to a set of structure-based rules for predicting the effect of pharmacological chaperone on every Fabry disease patient. Fourth, we use rational design approaches to interconvert the specificity of α-GAL into that of a related enzyme, α-N-acetylgalactosaminidase (α-NAGAL). Structural and enzymatic experiments show that the engineered enzyme contains new substrate specificity, as predicted by the design. The structural and mechanistic details we present in this thesis provide better understanding of the catalysis of the human α-galactosidase enzyme as well as define the molecular basis for pharmacological chaperone therapy in Fabry patients. Since α-GAL is one of the best studied lysosomal storage disease, it might be used as a model to better understand other lysosomal storage diseases and as well as other diseases related to misfolded proteins, including Alzheimer's and Parkinson's diseases.

Lysosomal storage diseases (LSDs) are metabolic diseases which occur as a result of a deficiency of one of the essential lysosomal enzymes, called glycohydrolases. A mutation in the gene encoding one of these enzymes leads to an accumulation of unwanted substrates, resulting in a variety of clinical manifestations. A common symptom found in LSDs is skeletal muscle dysfunction, which includes muscle weakness, atrophy and loss of muscle mass. The genes for lysosomal hydrolases are well characterized; however, much less is known about how mutations in these genes affect the cell and lead to the muscle dysfunction observed. One pathway of interest is autophagy; it has been shown to be essential for maintenance of skeletal muscles. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of Sialidosis, Sandhoff Disease, and GM1-Gangliosidosis in order to assess autophagy and its impact on myogenic differentiation in skeletal muscles. Our results demonstrated that autophagy is induced upstream (ULK1 phosphorylation) but is inhibited at the autophagosome to lysosome fusion (p62 upregulation) in LSDs. We also found that myoblast fusion and myogenic differentiation are impaired. We conclude that blocking autophagy impairs myogenic differentiation, which potentiates the muscle dysfunction observed in LSDs. This work highlights autophagy as a new pathway of interest and possible therapeutic target to alleviate muscle dysfunction in LSDs, and other similar neurodegenerative diseases. / Thesis / Master of Science (MSc) / Lysosomal storage diseases (LSDs) occur because of a deficiency of lysosomal glycohydrolases. A common symptom found in LSDs is skeletal muscle dysfunction. Little is known about how a deficiency of these enzymes leads to the clinical manifestations observed. However, one pathway of interest is autophagy. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of LSDs to assess autophagy and its impact on myogenic differentiation in skeletal muscles. We demonstrated that autophagy is induced and blocked, and that myoblast fusion and myogenic differentiation is impaired. We concluded that the induction and block of autophagy impairs myogenic differentiation, which potentiates muscle dysfunction.

Addendum inserted at back Includes bibliographical references (leaves 266-287). 1. Introduction -- 2. Materials and general methods -- 3. Characterisation and partial purification of the lysosomal sulphate transporter -- 4. Identification of proteins involved in lysosomal sulphate transport -- 5. The relationship between a sulphate anion transporter family and the lysosomal sulphate transporter -- 6. Investigation of sulphate transport in human skin fibroblasts -- 7. Concluding remarks

Addenda slip inserted in back. Includes bibliographical references (leaves 178-231). To further the understanding of the molecular mechanisms that contribute to the skeletal pathology of mucopolysaccharidosis type VI and to investigate the production of organic matrix by mucopolysaccharidosis VI osteoblasts

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal-recessively inherited disorder caused by the deficiency of lysosomal sulphamidase (NS) enzyme activity, resulting in the accumulation of the glycosaminoglycan (GAG) heparan sulphate (HS). MPS IIIA patients experience progressive and severe neurological deterioration with death usually occurring in the mid-late teenage years. A naturally-occurring mouse model of MPS IIIA has been characterised and the biochemical, histological and behavioural changes closely parallel the human condition. In order to treat the neurological effects of MPS IIIA, it is anticipated that a continual supply of replacement enzyme to affected cells will be required. Consequently, this study aimed to evaluate the efficacy, longevity and safety of gene therapy as a potential treatment for MPS IIIA. Canine adenoviral vectors (CAV-2) were selected on the basis of several important properties. They are non-integrating, are predicted to be less immunogenic in humans than human-derived viral vectors and mediate transgene expression for at least 1 year in vivo. An E1-deleted (∆E1) CAV-2 vector, CAV-NS, co-expressing recombinant human NS (rhNS) and Green Fluorescent Protein (GFP) was constructed and purified. In vitro testing revealed rhNS produced by CAV-NS significantly decreased sulphated GAG storage in human MPS IIIA fibroblasts in a mannose-6-phosphate-dependent manner. Preliminary studies in young adult guinea pigs with CAV-GFP demonstrated widespread GFP expression in the absence of a humoral response. In contrast, minimal GFP expression was found in CAV-injected adult mice due to formation of neutralising antibodies against the CAV-2 capsid. Consequently, intraventricular delivery of CAV-NS was evaluated in newborn mice at various doses. Widespread and dose-dependent GFP expression was observed and the optimal dose for large-scale studies was determined to be 109 CAV-NS particles/hemisphere. Antibodies against CAV-2, rhNS or GFP were not detected. Concurrently, the cognitive function and anxiety-related behaviours of unaffected and MPS IIIA mice were evaluated. MPS IIIA mice had significantly impaired memory and spatial learning in the Morris Water Maze (16-wks) and reduced anxiety in the Elevated Plus Maze (18-wks) when compared to unaffected animals. In a large therapeutic assessment trial, newborn MPS IIIA or unaffected mice received 109 particles of CAV-NS, saline or remained uninjected. GFP expression was visualised for at least 20-wks post-injection. Reductions in the vacuolation of ependymal and choroidal cells of the lateral ventricle and the cerebral cortex of treated MPS IIIA animals were observed in some GFP-positive (and presumably rhNS-expressing) regions. Furthermore, improvements in reactive astrogliosis, but not in the number of activated microglia, were measured in CAVNS- treated MPS IIIA mice. However, insufficient CAV-NS-mediated rhNS expression was generated to improve functional changes as assessed by a behavioural test battery (motor function, open field activity, Elevated Plus Maze, Morris Water Maze), potentially due to chronic inflammatory responses against the CAV-2 vector. Collectively, these data suggest that early intervention with ∆E1 CAV-NS gene therapy was able to improve several components of neuropathology in MPS IIIA animals but was unable to significantly alter the clinical progression of murine MPS IIIA. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1295758 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2007