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(1996)Conference PaperThe Mungi single address space operating system provides a protected procedure call mechanism named protection domain extension (PDX). The PDX call executes in a protection domain which is the union of (a subset of) the caller`s, and a fixed domain associated with the procedure. On return, the caller`s original protection domain is reestablished. Extensive caching of validation data allows amortisation of setup costs over a possibly large number of invocations. The PDX mechanism forms the basis for object support in Mungi, particularly encapsulation. It is also used for accessing devices, and to implement user-level page fault handlers.
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(2007)ThesisRelapsed ALL remams one of the most common causes of death from disease in children. Broad-range drug resistance is often associated with relapse, although its underlying molecular mechanisms remained poorly understood. The aim of this thesis was to establish an in vivo model using the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse strain, to facilitate the engraftment, expansion and characterisation of childhood ALL cells, obtained from patients at diagnosis or relapse. Mice were inoculated with leukaemia cells from patients' biopsies and engraftment was monitored by the proportion of human CD45+ cells in the blood. Successful leukaemia engraftment was achieved for 20/20 patient biopsies. Continuous passaging of ten xenografts has also been achieved. Immunophenotypic analysis showed only minor changes in cell surface markers after passage in mice. Leukaemia dissemination in murine bone marrow, liver, spleen and blood was consistent with the human disease. The in vivo responses of ten continuous xenografts to dexamethasone and vincristine, but not methotrexate, significantly correlated with patient outcome (p<0.05). Xenograft sub-lines resistant to vincristine, dexamethasone, methotrexate and cytosine arabinoside were also selected by in vivo drug treatments, although these sublines were not found to be cross resistant to structurally unrelated drugs. Resistance to vincristine, either in in vivo selected sub-lines or inherently resistant xenografts, was not associated with increased activity of drug efflux pumps such as P-gp or MRPl. Class I β tubulin levels remained unchanged when compared between vincristine resistant sublines and their parental xenografts. Decreased expression of stathmin and increased polymerised tubulin were observed in vincristine resistant sub-lines, suggesting a possible mechanism of counteracting the depolymerising effects of vincristine. In summary, this study has shown that primary ALL cells engraft efficiently into NOD/SCID mice, and indicates that their response to vincristine and dexamethasone mimics the clinical situation. This model appears to be highly relevant for the study of childhood ALL and will provide the foundation to delineate clinically relevant mechanisms of drug resistance.
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(2007)ThesisMultidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.
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(2019)ThesisThe development of drug resistance remains a major limitation to the successful treatment of high-risk neuroblastoma, with the survival rate still below 50%. Multidrug resistance is the simultaneous resistance to multiple unrelated drugs and can be conferred by the expression of proteins from the ABC transporter superfamily. Multidrug resistance-associated protein 1 (MRP1) is frequently overexpressed in tumours and effluxes chemotherapeutic agents from tumour cells, protecting them from chemotherapy. In neuroblastoma, elevated MRP1 expression is highly prognostic of poor clinical outcome and contributes to chemoresistance in the Th-MYCN transgenic mouse model of high-risk neuroblastoma. Based on the previously identified MRP1 modulator Reversan, a series of highly promising new MRP1 modulators were developed with a view to identifying candidate molecules suitable for clinical development as chemosensitising agents. These second-generation modulators were highly selective for MRP1 over P-glycoprotein, ABCG2, MRP2 and MRP3, and sensitised the neuroblastoma cell lines Kelly and IMR32 at least two-fold to the substrate drugs etoposide, vincristine and arsenic trioxide in vitro (P<0.001). The second-generation modulators were active in the Th-MYCN transgenic mouse neuroblastoma model, doubling survival of etoposide-treated animals without impacting on etoposide pharmacokinetics, but conferred only minimal survival advantage in Kelly human cell line xenografts. Additional studies with independent MRP1 inhibitors suggest that inhibition of MRP1 alone is not sufficient to sensitise human neuroblastoma xenografts to standard-of-care agents. In addition to blocking efflux of chemotherapy, the second-generation modulators stimulated MRP1-dependent efflux of reduced glutathione (GSH), leading to depletion of intracellular GSH. MRP1 modulators synergised with the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) in MRP1-expressing cells, leading to near-complete intracellular GSH depletion, abolition of colony formation and sensitisation to chemotherapeutics. This dual activity may be beneficial for the treatment of neuroblastoma and other MRP1-expressing tumours. In conclusion, MRP1 inhibition alone may be insufficient to sensitise high-risk neuroblastoma to chemotherapy, however highly selective second-generation MRP1 modulators may increase the therapeutic window for standard-of-care drugs through combined inhibition of drug efflux and depletion of GSH.
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(2021)ThesisMultidrug resistance is a leading contributor to treatment failure in cancer patients. As elevated levels of the antioxidant glutathione (GSH) and increased expression of drug efflux pumps may both contribute to chemotherapy resistance, depleting GSH and blocking drug efflux in cancer cells may improve response to treatment. Multidrug resistance protein 1 (MRP1) is a membrane transporter that is frequently overexpressed in cancer cells. By actively effluxing a wide range of chemotherapeutic agents, MRP1 can protect the cancer cell from chemotherapy. GSH is also transported by MRP1 as a low-affinity endogenous substrate. Some small molecules (MRP1 'modulators'), upon binding to MRP1, greatly enhance this innate GSH transport whilst simultaneously blocking the efflux of chemotherapeutics. As this stimulated GSH efflux can deprive the cell of GSH, in parallel with the blocked chemotherapy efflux enhancing intracellular chemotherapy concentrations, MRP1 modulators might be used to exploit the high expression of MRP1 in cancer cells to improve treatment response. MRP1 was found to be frequently expressed in patient tumours of two difficult to treat cancer types: non-small cell lung (NSCLC) and ovarian cancer. As this MRP1 expression might be leveraged with an MRP1 modulator, two modulators were tested on high MRP1-expressing NSCLC and ovarian cancer cell lines. As single agents, the modulators reduced MRP1 drug transport by >85% and improved the efficacy of MRP1-substrate chemotherapeutics (2 5-fold). In combination with the GSH synthesis inhibitor buthionine sulfoximine, complete GSH depletion, diminished clonogenic capacity, enhanced radiosensitivity, and extended chemosensitivity were achievable selectively in high MRP1-expressing cancer cells. As MRP1 expression in NSCLC was also associated with NRF2 activation, a key driver of treatment resistance in NSCLC, the modulator and buthionine sulfoximine combination was tested and found to be effective against cell lines representative of this highly resistant subset of NSCLC. Given the therapeutic potential for MRP1 modulators, their MRP1 binding site and mechanism of action as GSH transport modulators was investigated to provide a basis for future structure-guided design of more potent and selective modulators. Overall, these findings support that the high expression of MRP1 in cancers can be exploited with MRP1 modulators to chemosensitise and radiosensitise NSCLC and ovarian cancers.
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(2020)ThesisThis thesis examines the clinical translation of two novel therapeutic drugs in rare paediatric neurological disease via expanded access programs (EAP). Patients with rare diseases commonly encounter barriers to care and lack of disease-modifying treatments. EAPs help address this need by providing access to therapeutic drugs before commercial availability, however, there is usually limited knowledge regarding the safety and full impact of the drug. Two drugs were recently made available via an EAP for children in New South Wales: cannabidiol for paediatric drug-resistant epilepsy (DRE) and nusinersen for spinal muscular atrophy (SMA). In a time when there was no evidence regarding the use of cannabidiol in paediatric DRE, an EAP was established due to unprecedented patient demand. Our study analysed the safety, adverse effects and preliminary efficacy of cannabidiol in patients with DRE. Cannabidiol was well tolerated, with sedation commonly reported (37.5%). Elevated transaminases were seen in 5% and demonstrated the necessity of medical monitoring. Although many patients reported improved overall health, indirect measures of seizure control did not show improvement, thus signifying the need for larger, randomised-controlled trials. In children with SMA type 1 (SMA1) treated with nusinersen, respiratory, bulbar and nutritional outcomes were analysed, as previous studies had demonstrated increased survival and motor function but had not assessed the broader impact on the burden of disease. Our study demonstrated substantial ongoing comorbidities due to respiratory and bulbar weakness, with the need for ongoing nocturnal noninvasive ventilation, gastrostomy feeding and recurrent acute hospitalisations. Greater burden of disease was seen more in patients with two SMN2 copies. Most children showed improvement in motor outcome assessments, a stark change in the natural history of SMA. These findings show the impact of nusinersen in modifying the phenotype of children with SMA1, yet ongoing significant morbidities and requirement for multidisciplinary supportive medical care. In conclusion, these studies provide additional real-world clinical data on the implementation and extent of efficacy of two novel drugs for rare neurological diseases. EAPs serves to address an unmet clinical need and facilitate the translation of research into practice to advance future health practice and research in rare diseases.
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(2018)ThesisBackground: Public health organisations in Australia lag behind other sectors using client-facing smartphone applications (apps) to enhance service delivery. Patients are increasingly seeking smartphone-enabled health management resources. Mobile health (mHealth) systems incorporating apps may offer effective means of enabling patient-centred care. mHealth interventions can fail to translate into practice due to: design-reality gaps; real-world change outpacing research; or health service sociotechnical complexity. Pragmatic, participatory research methodologies may help meet these challenges. Tools such as the Consolidated Framework for Implementation Research (CFIR) offer structure around which investigators can build fit to context. Aim: Examine the feasibility and acceptability of a co-produced prototype mHealth intervention and model of care to manage gestational diabetes (GDM) focusing on barriers to translation into practice. Through Proof of Concept offer recommendations for future study. Methods: An mHealth system (SugaMama) was developed using lean, agile methodologies guided by Social Constructivist theory. It comprised a patient app (iPhone, Android); Bluetooth glucometer; and clinician web portal. Environmental analysis situated the study in a real-world context. The study was planned and evaluated by a Quality Improvement Collaborative (QIC) comprising patients, clinicians, health service managers and researchers. Mixed methods and an observational case study design were employed. Results: Sixteen GDM patients and seven clinicians trialled SugaMama over 44 days and found it to be technically feasible, acceptable and aligned with patient lifestyle. Technology functioned as intended. A sole critical issue related to planning for system downtime. Patients preferred supported self-management utilising SugaMama over standard care. Clinicians experienced increased control through viewing real-time patient data. For the next iteration more personalisation was requested by patients; clinicians sought improved medication management and integration with other systems. Conclusion: mHealth-enabled care is likely to support patient-clinician partnerships and extend health service reach. Utilisation of CFIR constructs and a QIC may support implementation. Critical barriers to translation into practice can arise despite an intervention being evidence-based; feasible; acceptable; and supported by sponsors, patients and clinicians in a target context.