"Research conducted at the Department of Haematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science."--T.p. Includes bibliographical references (leaves 170-214). Elevated levels of receptor tyrosine kinases have been implicated in carcinogenesis. It is possible that high expression of c-Kit by the leukaemic cell provides them with a growth advantage over their normal counterparts in the bone marrow microenvironment. Thus, a means of inhibiting the interaction of c-Kit on these cells with ligand Steel Factor may remove proliferation and survival signals. Main aim of the study was to produce a biological inhibitor of this interaction and evaluate its ability to prevent ligand Steel Factor from binding to c-Kit on live cells.

Copy of author's previously published work inserted. Includes bibliographical references (leaves 160-182). Concentrates mainly on the characterisation of the molecular mechanism of action of the tec protein tyrosine kinase using biochemical and genetic approaches.

Bibliography: leaves 139-165.

The implication of the transmembrane signalling Receptor Tyrosine Kinases (RTKs) in cancer has accelerated the pursuit for drugs to target these molecules. In the process our understanding of how these membrane bound molecules are entangled in cell signalling has significantly expanded. There is now evidence that RTKs can facilitate the formation of a lattice-type network of signalling molecules to elicit whole cell responses to external ligand stimuli. Although beginning to be unravelled, knowledge pertaining to the mechanisms of molecular control that initiate these signalling pathways is still in its infancy. In this thesis, a random mutagenesis approach allowed the identification of the crucial interaction surfaces between membrane-bound EphA3 and its preferential binding partner ephrinA5, that are required to induce the formation of higher-order Eph signalling complexes. Modelling and experimental dissection of this co-ordinated receptor aggregation has provided detailed insights into the molecular mechanisms of Eph receptor activation, which in some aspects may also apply to other members of the RTK family. In particular, the importance of certain molecular interfaces in determining preferential and non-preferential Eph/ephrin interactions, suggests their role in the selection of biologically important binding partners. In addition to the assignment of the ephrin-interaction surfaces, the random mutagenesis strategy also identified a continuous conformational epitope as binding site for an anti-EphA3 monoclonal antibody. Fortuitously, antibody binding to this site functionally mimics ephrin stimulation of EphA3 positive cells, and in particular together with divalent ephrinA5, yields synergistically enhanced EphA3 activation. Elucidation of the underlying mechanism has provided opportunities to develop an efficient EphA3 targeting mechanism that is based on increased affinity and accelerated ephrinA5 uptake as consequence of this unique activation mechanism. On a genetic level, novel oligonucleotide analogues known as Peptide Nucleic Acids (PNAs) were analysed for their ability to sterically inhibit EphA3 DNA transcription and suggest a dosedependent downregulation of EphA3 expression, in malignant melanoma cells. Combined, ephrinA5, the anti-EphA3 MAb (IIIA4) and PNA, offer the possibility to investigate the specific machinery involved in Eph receptor expression and signalling for the specific targeting of EphA3 expressing tumour cells.

Bibliography: leaves 139-165. / ix, 183 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 2001

Copy of author's previously published work inserted. / Includes bibliographical references (leaves 160-182). / xi, 182 leaves, [57] 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. / Concentrates mainly on the characterisation of the molecular mechanism of action of the tec protein tyrosine kinase using biochemical and genetic approaches. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001?

"Research conducted at the Department of Haematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science."--T.p. / Includes bibliographical references (leaves 170-214). / xiv, 221 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Elevated levels of receptor tyrosine kinases have been implicated in carcinogenesis. It is possible that high expression of c-Kit by the leukaemic cell provides them with a growth advantage over their normal counterparts in the bone marrow microenvironment. Thus, a means of inhibiting the interaction of c-Kit on these cells with ligand Steel Factor may remove proliferation and survival signals. Main aim of the study was to produce a biological inhibitor of this interaction and evaluate its ability to prevent ligand Steel Factor from binding to c-Kit on live cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001

Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 104-121).

Nitric oxide (NO) is a short-lived free radical that plays a critical role in the regulation of cellular signalling. Mast cell (MC) derived NO and exogenous NO regulate MC activities including the inhibition of MC degranulation. At a molecular level the intermediate metabolites of NO modify protein structure and function through several mechanisms including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione (SNOG). Using two dimensional gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MC. S-nitrosoglutathione treatment also reduced the Vmax of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance (NMR) analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that SNOG treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose-1,6-bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation and intracellular Ca2+ levels in LAD2 cells. In addition to aldolase, 15-hydroxy prostaglandin dehydrogenase (PGDH), a critical enzyme in the metabolism of PGE2, was identified as a prominent target for tyrosine nitration in LAD2 cells. Thus for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target in HMC-1 and LAD2; and PGDH in LAD2 cells. The post translational nitration of aldolase A and PGDH may be important pathways that regulate MC phenotype and function. / Experimental Medicine

Recently, the zebrafish has been considered an important animal model that can be used to investigate human diseases and drug development. Parkinson¡¦s disease (PD), an important neurodegenerative disorder, is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. However, current treatments for PD are limited and mainly improve only the clinical symptoms of the disease. Thus, a neurodegenerative rat model has been widely used for a long while to search for a new treatment for PD. However, the use of rats as an animal model has certain limitations such as breeding, efficiency, and high dosage. Recently, researchers indicated that neurotoxins such as rotenone, 6-hydroxydopamine (6-OHDA), and paraquat can induce Parkinson¡¦s-like symptoms in zebrafish, and this may be a useful PD model because of the complete development of the zebrafish nervous system, low costs, and low dosage. In this study, we treated zebrafish with 6-OHDA and analyzed their locomotor activity to establish an in vivo animal model of PD. Then, we analyzed the mRNA expression of parkin and PINK1 by reverse transcription¡Vpolymerase chain reaction (RT-PCR).Moreover, we observed tyrosine hydroxylase (TH) expression by immunohistochemical (IHC) staining to confirm if this can be used as a PD model. Finally, we found that treatment with 6-OHDA significantly reduced TH expression. We observed a similar declining trend in the case of mammals. Likewise, parkin and PINK1 mRNA expressions were also decreased after treatment with 6-OHDA. In summary, our study provides a feasible in vivo Parkinson¡¦s model, and a small volume of drugs or compounds can be screened using this model.