Cystic fibrosis (CF) is an autosomal recessive genetic disorder of which lung disease is the leading cause of morbidity and mortality. One attractive strategy for the treatment of CF lung disease is to directly deliver CF transmembrane conductance regulator gene to airway epithelia. Although promising results have been reported, barriers present in the lung make successful gene transfer to the respiratory tract difficult. In order to improve gene transfer strategies in the intrapulmonary airways, we need to identify the bottlenecks of transduction for the vector system. A previous study reported that feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways in mice than the intrapulmonary airways (Sinn, P.L. et al. 2008, J. Viol). Our first goal was to identify barriers to lentiviral gene transfer in the murine airways. We demonstrate that host immune response is not the major barrier preventing efficient FIV-mediated transduction in the intrapulmonary airways. We show that the FIV vector transduces murine primary nasal epithelial cell cultures with greater efficiency than murine primary tracheal epithelial cell cultures. In addition, GP64 pseudotyped vesicular stomatitis virus (VSV) transduces better in nasal epithelia compared to intrapulmonary airways in mice. On the other hand, we observed that VSVG glycoprotein-pseudotyped VSV transduces the intrapulmonary airway as well as nasal epithelia in mice with similar efficiency. Our results suggest that differentially expressed cellular factor(s) specific for GP64 or FIV vector may be the major barrier(s) for FIV vector-mediated gene transfer in the murine intrapulmonary airways. The recent development of CF porcine models prompted us to investigate possible barriers for lentiviral vector-mediated gene transfer in porcine cells. Our preliminary results showed that HIV transduction was restricted in porcine but not human lung-derived cell lines. Porcine TRIM5 has sequences similar to restrictive bovine TRIM5 orthologs. Therefore, our second goal was to investigate the possible restriction of lentiviral vectors by porcine TRIM5. We demonstrate that transient overexpression or knockdown of endogenously expressed porcine TRIM5 does not affect HIV or FIV transduction. Lastly, we characterized a mucin domain-deleted EBOV (EBOVΔO) glycoprotein mutant with increased transduction. This EBOVΔO 5-mer mutant was generated based on mutants with an increased transduction as identified by alanine scanning mutagenesis (Brindlay, M.A. et al. 2007. J. Viol). We show that VSV pseudotyped with the 5-mer mutant increased transduction both in vitro and in mice when compared to the wild-type EBOVΔO. Structural analysis demonstrated that 5 mutations were located proximal to the GP1-GP2 interface. Enhanced transduction likely results from a lower energy metastable state of the glycoprotein. FIV pseudotyped with 5-mer also shows increased transduction in multiple cell lines. Identification of barriers in intrapulmonary airways and improvements of vector systems will help the advancement of gene therapy for CF.

Malignant mesothelioma (MM) is an aggressive malignancy that originates from mesothelial cells and is highly resistant to conventional forms of anti-cancer therapy. Defects in apoptotic pathways are believed to play a major role in determining resistance to chemotherapy. The characterization of these pathways in mesothelioma is essential in order to develop more effective therapies. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that regulate apoptosis and have been implicated in the resistance of malignant cells. There is evidence that upregulation of specific IAP molecules can influence tumour progression and response to chemotherapy. In this study we examined the apoptotic signalling in MM cells and the potential role of IAPs in both cell proliferation and chemosensitivity. We examined expression of six IAP genes or isoforms in both malignant and normal mesothelial cells. Results demonstrated that XIAP, IAP-1, IAP-2, survivin and Bruce were expressed in all four MM cell lines and four primary mesothelial cultures. There was no evidence for differential expression of these genes between MM and mesothelial cultures. Livin expression was detected in only one MM cell line. Various aspects of apoptotic signalling pathways in response to the chemotherapeutic drug cisplatin were also analysed including: a) the mitochondrial integrity, b) caspase activation, c) cell viability and d) phosphatidylserine translocation. In order to further characterize the role of IAPs, the transcriptional regulation of these genes in response to cisplatin was investigated using real-time RT-PCR. The results of these experiments indicated that there was no significant regulation of IAPs at the transcriptional level in the cells examined during cisplatin-induced apoptosis. Overall the data was consistent with cisplatin inducing apoptosis in MM cells via intrinsic signalling pathways in a dose dependent manner. Regulation of IAP expression was not seen at the RNA transcription level as has been described in other tumour types but may occur through protein posttranslational events. In order to further investigate IAP function we performed analyses of two IAPs which had previously been proposed as having a role in mesothelioma: XIAP and survivin. Protocols for RNAi knockdown at the protein expression level were established. Although the data indicated significant reduction in protein expression, the effects on cell survival after treatment with cisplatin were moderate. These studies were then extended to other molecules that are known to interact with and modulate the function of IAPs. We characterized the expression of the proteins: XAF1, HTRA2, ARTS in MM cells. RT-PCR data showed that HTRA2 and XAF1 genes were expressed in MM cell lines, however we did not see expression of ARTS. On the basis of recently published data we examined the XAF1 splice variants expressed in MM cell lines by sequence determination and PCR screening.

Addenda page on inside back cover. Bibliography: leaves 153-176. Mucopolysaccharideosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is an autosomal recessive lysosomal storage disease, with a prevalence in Australia of 1 in 114,000. MPS IIIA is caused by a deficiency of the lysosomal enzyme sulphamidase which is needed together with other exohydrolases and a N-acetyltransferase to break down the glycosaminoglycan heparan sulphate to sulphate and monosaccharides. Patients are characterised by severe central nervous systems degeneration together with mild somatic involvement; this disproportionate correlation is unique amongst the mucopolysaccharidoses. Features include severe behavioural disturbances, such as hyperactivity and aggressiveness, coarse hair and mild hepatosplenomegaly. Death is usually in the mid- to late-teenage years. Enzyme replacement therapy by intravenous administration of recombinant human NS (rhNS) has been proposed as a potential therapy for MPS IIIA. This thesis suggests that rhNS, entering the brain in the first few weeks of life, is able to retard the behaviour and learning difficulties in MPS IIIA mice.

"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.

Human Papillomavirus (HPV) is one of the most common causes of sexually transmitted disease worldwide. Infections by high-risk HPVs, such as HPV-18, have been associated etiologically with cervical cancer. The successful development of HPV vaccines may be beneficial to the HPV-na??ve population, but women that have already been exposed to the virus are still at risk of developing HPV-associated malignancies. A need for a systemic cure for HPV-infection therefore still exists. Gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers; however, few studies have explored this possibility for cervical cancer. The aim of this project is to construct a gene expression vector that can specifically target HPV-infected cervical cancer cells, by making use of the activity and selectivity of the P105 promoter which is determined by transcription control elements within the HPV-18 long control region (LCR). The first part of this study involved the construction of LCR deletion plasmids, and examining the subsequent level of gene expression induced within different mammalian cell lines. The results suggest the LCR to be capable in achieving cervical cancer-specific gene expression. The 3′-end of the viral L1 gene upstream of the LCR appeared to have a repressive effect on the promoter and therefore should be excluded for maximum LCR promoter activity. The second part of the project involved site-directed mutagenesis studies performed on selected transcription factor binding sites with an attempt to further increase the level of LCR promoter activity and specificity towards HPV-infected cervical cancer cells. The results suggest that a GRE/YY1 mutation may significantly enhance promoter activity. In terms of promoter regulation, the E2BSs appeared to be responsible for promoter activation in the absence of viral E2 proteins. The findings of this study suggest a possible gene therapy approach towards the treatment of cervical cancer. By making use of the activity and specificity of the HPV-18 P105 promoter to induce cervical carcinoma-specific expression of appropriate therapeutic genes, suicidal phenotypes can be introduced selectively within HPV-positive cervical cancer cells while normal HPV-negative cells are unaffected.

"16th September 2002." / Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). / Bibliography: leaves xxix-li. / xxvii, 213, li leaves : ill., plates (some col.) ; 30 cm. + 1 CD-ROM (4 3/4 in.) / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

Addenda page on inside back cover. / Bibliography: leaves 153-176. / xiii, 176 leaves ; ill. (some col.) ; 30 cm / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Mucopolysaccharideosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is an autosomal recessive lysosomal storage disease, with a prevalence in Australia of 1 in 114,000. MPS IIIA is caused by a deficiency of the lysosomal enzyme sulphamidase which is needed together with other exohydrolases and a N-acetyltransferase to break down the glycosaminoglycan heparan sulphate to sulphate and monosaccharides. Patients are characterised by severe central nervous systems degeneration together with mild somatic involvement; this disproportionate correlation is unique amongst the mucopolysaccharidoses. Features include severe behavioural disturbances, such as hyperactivity and aggressiveness, coarse hair and mild hepatosplenomegaly. Death is usually in the mid- to late-teenage years. Enzyme replacement therapy by intravenous administration of recombinant human NS (rhNS) has been proposed as a potential therapy for MPS IIIA. This thesis suggests that rhNS, entering the brain in the first few weeks of life, is able to retard the behaviour and learning difficulties in MPS IIIA mice. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

Understanding the factors that regulate the growth and differentiation of haemopoietic stem cells (HSC) remains a major challenge. In this study, the proliferation and differentiation of CD34+ cells from normal donors and chronic myeloid leukaemia (CML) patients was compared. The proliferation and entry of CML cells into the cell cycle was decreased relative to cells from normal donors, and greater heterogeneity in the phenotype of CML cells at the initiation of culture was observed. Analysis of phenotype concomitant with cell division also demonstrated that the differentiation of normal CD34+ cells was consistent between donors, while marked variability was observed in the differentiation of CD34+ cells from CML patients. This included expression of CD13, CD33, CD38 and HLA-DR, which were linked to cell division in normal but not CML cells. The tyrosine kinase inhibitor, imatinib, is a novel drug displaying promising results in the treatment of CML by specifically inhibiting the growth of leukaemic cells. To examine whether myelosuppression observed in patients treated with imatinib may arise from inhibition of normal haemopoiesis, imatinib was added to colony assays established using cells from normal bone marrow. Suppression of monocyte/macrophage growth, but not that of eosinophils or neutrophils, was observed at therapeutic concentrations of imatinib. Inhibition of monocytic differentiation to macrophages was also observed and was associated with decreased functional capacity such as altered antigen uptake, production of proinflammatory cytokines and stimulation of responder cells. The specific suppression of monocyte/macrophage differentiation and function was not due to blockade of tyrosine kinases known to be inhibited by imatinib and was consistent with an inhibition of the M-CSF/c-fms signalling pathway. This hypothesis was tested using a cell line that was dependent on M-CSF for growth and survival. Cell proliferation and phosphorylation of c-fms were inhibited at an IC50 of 1.9μM and 1.4μM imatinib respectively and this was not attributable to decreased c-fms expression. These important findings therefore identify c-fms as a further target of imatinib, and suggest that imatinib should be considered for treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. Potential side effects resulting from imatinib treatment must also be considered. / Thesis (Ph.D.)--School of Medicine, 2004.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.