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(2021)ThesisExercise following a breast cancer diagnosis is known to improve outcomes. In multiple pre-clinical cancer models, exercise alone has shown to inhibit tumour growth. Some of the suggested mechanisms for this benefit include exercise-induced recruitment and activation of anti-cancer immune cells, release of myokines and alterations in intrinsic metabolic cellular pathways. Exercise has also shown to improve tumour blood flow, which may translate to improved chemotherapy delivery to otherwise hypo-perfused tumour regions. Whether exercise acts an adjunct to our current systemic therapies in improving breast cancer outcomes is a question that remains unanswered. The overall hypothesis of this thesis is that exercise suppresses breast cancer tumour growth. We confirmed this in two different in vivo models of breast cancer. Given our positive results, we proceeded to test a series of questions necessary to move our findings into clinical practice; 1. Does exercise act as an adjunct to systemic breast cancer therapy, 2. Does exercise shift the tumour immune microenvironment (TIME) to an immunosuppressive phenotype, 3. Is it feasible to exercise immediately after and during neoadjuvant chemotherapy for early stage breast cancer. We tested the combination of exercise and doxorubicin chemotherapy in two different in vivo models of breast cancer, and found no additional benefit on tumour growth with the combination treatment compared to doxorubicin alone. We did find an exercise-induced shift in the TIME to include immune cells with cytotoxic properties, associated with tumour growth suppression in one in vivo model of breast cancer. Concordantly, where exercise showed to be ineffective in suppressing breast cancer growth in vivo, no clear shift in the TIME was observed. Our clinical study confirmed that exercising immediately after, and during neoadjuvant chemotherapy using a partially supervised exercise program was feasible, safe and well received by patients. We showed that our exercise intervention maintained quality of life, improved muscle strength and reduced sedentary behaviour. Exploratory findings from our study also suggest that exercise acutely may abrogate immunosuppressive effects of chemotherapy on circulating immune cells. Due to the methodological challenges and complexities involved with conducting exercise-studies in in vivo models, we propose that future studies interrogating the effects of exercise on breast cancer focus on clinical studies in the neoadjuvant setting in order to better understand the underlying mechanisms for the benefits already observed with exercise after a breast cancer diagnosis.
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(2021)ThesisCurrent gene delivery systems suffer from poor genetic delivery into resting T-cells and therefore, compensate for this by using multiple viral challenges, by activating and expanding the cells ex vivo. However, cells activated and expanded in this manner have lower engraftment, proliferative potential and potency. In contrast, the use of resting T-cells overcomes many of these short-comings, but comes with restrictions: Firstly, at the plasma membrane, due to the lack of receptor for the commonly used lentiviral envelope pseudotype, VSVg. Secondly, at reverse transcription (RTN), by intracellular restriction factor, SAMHD1. At the membrane restriction, we focused on the abundant expression of CXCR4 on resting T-cells, by testing a panel of lentiviral systems expressing various CXCR4-dependent, CD4-independent HIV envelopes. Results revealed that the above panel fused poorly or not at all within resting T-cells. The lead envelope was initially derived from HIV-2 VCP isolate, which was dependent on HIV-2 Gag to maintain high fusogenicity. Whilst HIV-2 based lentiviral systems are advantageous; they are not as developed as HIV-1 systems. Thus, we re-investigated a HIV-1 system based on a derivate of iR3A isolate that enters resting T-cells primarily through CXCR4, but to do so, it required modifications to its matrix. Fortunately, SIV and HIV-2 have evolved with the accessory gene, vpx, that directly targets SAMHD1 for degradation and can be readily incorporated into lentiviral particles. Yet it is presently unknown if all vpx genes are equal in their ability to antagonise SAMHD1 and/or enhance RTN. Through bioprospecting a genetically diverse panel of vpx, we tested SAMHD1 antagonism across several cell types. Majority of the Vpx proteins could overcome SAMHD1 restriction in dendritic cells and macrophages, but only one lead Vpx could efficiently achieve this in resting T-cells, and this was due to an additional Lysine within its nuclear localisation signal. Results further revealed that, in resting T-cells, lentiviral genetic cargo is prone to heavy deletions during the process of RTN. Inclusion of Vpx within lentiviral particles increased preservation of intact gene cargo and supports a role for SAMHD1 in viral restriction, through promoting deletions in incoming reversed transcribed DNA prior to genomic integration. Taken together, these observations serve as a viable platform to build towards the generation of resting T-cell product for use in vivo.
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(2021)ThesisAs the global population ages, the need for optimal presbyopic solutions increases. Despite many multifocal contact lenses (CLs) marketed over the last 20 years, satisfactory vision and comfort at all distances remains elusive. ‘Phase step’ (PS) optics, a technology offering extended depth of focus (EDOF), could augment presbyopic CL correction. This thesis aimed to confirm whether PS designs can improve presbyopic near vision. The study investigated if PS design parameters can be manipulated to affect visual performance and explored influence of pupil size and spherical aberrations (SAs), on visual performance with PS designs. Furthermore, computational prediction of in vitro and in vivo on PS lens performance was assessed. A three-modality method was employed: theoretical (computational ray-tracing analysis), in vitro (optical bench system with anatomically correct model eye), and in vivo (human clinical study). A systematic battery of PS designs were designed, fabricated on hard CL and phase plates; and trialled in vivo and in vitro. The three-paradigm model confirms PS optics can provide presbyopes with EDOF performance. Theoretical and in vitro results demonstrated optical modified performance through lens parameter optimisation with raw visual acuity (VA) providing qualified in vivo support. Uniformity Index (UI), a novel metric quantifying stability of VA, distinguished EDOF designs as demonstrating the most stable VA. In bifocal PS designs, the interplay between optic zone diameter (OZD) and pupil size is important. Increased pupil size in PS EDOF designs decreased near performance in the theoretical and in vivo results, with qualified in vitro support. Decentration effect correlates with the OZD-pupil size relationship. This is the first study to report SA effect on PS performance is design-dependent; generally increasing SA to the 50th SA percentile results in stable performance across object vergences for EDOF designs. Inducing moderate positive SA may aid in EDOF of these PS designs. Good agreement was found between the theoretical and in vitro results, followed by the in vitro and in vivo and, closely by the theoretical and in vivo agreement. Pupil size and SA affect visual performance and must be considered early in design or during prescribing. Systematic evaluation of theoretical-to-bench-to-clinical studies represents an effective and efficient framework for further development of PS solutions and potentially, future presbyopia designs.
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(2021)ThesisSafe water supplies are fundamental to public health protection. The assessment and control of pathogen risk is of primary concern, as a large disease burden is prevented. Due to the insensitivity of laboratory techniques and public health surveillance, quantitative microbial risk assessment (QMRA) is used to quantify public health risk associated with water supply. This thesis aims to establish new evidence to inform QMRA policy and practice. It does this from the perspective of a practitioner-researcher. This thesis comprises three analytical studies. Each answers a research question arising in the professional practice of the researcher. The first study uses the systematic review method to synthesise usage patterns of QMRA for drinking water supplies globally. The second uses interrupted timeseries analysis to examine an intervention into the sensitivity of protozoal enumeration results used in QMRA. The final study uses stochastic QMRA modelling to examine the fitness of a new method, detailed verification , for the routine quantification of public health risk for water recycling. The studies are contextualised by a narrative literature review, a narrative case study, and an overarching discussion and conclusion. The first study found that current QMRA approaches varied most for deriving dose, that dose was influential in determining risk, and that the complexity of selected QMRA approaches is generally incommensurate with the purpose served. The second study found that the intervention overall provided greater precision of public health risk estimation. The intervention was associated with an around two-thirds reduction in the estimated public health risk associated with Cryptosporidium and Giardia (rate ratio [RR] = 0.35, p < 0.05, and RR = 0.32, p < 0.001, respectively) in one system, and an increased frequency of non-zero protozoal counts in three scenarios (p < 0.05). The third study found that quantitative public health requirements were met. Sensitivity analysis found that assumptions relating pathogen to surrogate levels were influential on the probability of infection (Spearman s rank correlation co-efficient, ρ, = 0.38 to 0.90). This thesis establishes new evidence for QMRA implementation for public water supplies. Its findings will support public health protection through the improved assessment and control of pathogen risk.
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(2021)ThesisIn Australia, non-alcoholic fatty liver disease (NAFLD) has reached epidemic proportions, and in parallel, hepatocellular carcinoma (HCC) has become one of the fastest-growing cancers. NAFLD related HCC (NAFLD-HCC) is associated with poor prognosis and is a leading cause of cancer-related death. The work in this thesis provides insight to host determinants of this disease in New South Wales, with a particular focus on metabolic comorbidities and their impact on survival. Moreover, we provide evidence for interactions between the gut microbiome and its metabolites in orchestrating aberrant peripheral and intrahepatic immune responses associated with poor outcomes. We make several key observations: 1) NAFLD-HCC is a distinct clinical entity, whereby the accumulation of metabolic risk factors and presence of type II diabetes are independent negative predictors of survival 2) Divergence in gut microbiome composition and function occurs in NAFLD-HCC, distinct from NAFLD-cirrhosis, and appear to direct the peripheral immune response toward an immunosuppressive phenotype ex vivo that is associated with poor outcomes and 3) In longitudinal animal studies, gut dysbiosis occurs early in the trajectory of liver injury, and occurs in parallel to immunosuppressed peripheral and intrahepatic immune responses that develop as HCC ensues. This data provides evidence for the identification of patients with NAFLD-HCC who are at risk of poor outcome and serves as a platform for the development of gut-based interventions, which, when timed early, may offset the burden of this deadly disease.
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(2021)ThesisWhile MYCN-amplified neuroblastoma has been the focus of neuroblastoma research in the past three decades, most human neuroblastomas do not harbour MYCN oncogene amplification, and their tumorigenic factors are unknown, highlighting the importance of identifying other oncogenic factors. Long noncoding RNAs (lncRNAs) play important roles in cancer oncogenesis. Here, I have shown the lncRNA PRKCQ-AS1 is upregulated by super-enhancers upstream of the PRKCQ-AS1 gene that drive its over-expression only in MYCN non-amplified neuroblastoma cell lines through the well-known super-enhancer components CDK7 and BRD4. Targeting CDK7 with THZ1 or targeting BRD4 with AZD5153 significantly downregulate the expression of the lncRNA PRKCQ-AS1, but not its neighbouring protein-coding gene PRKCQ or the well-documented super-enhancer[1]associated c-Myc oncogene. I have shown that PRKCQ-AS1 was a cytoplasmic lncRNA and bound to the RNA binding protein (RBP) MSI2 in MYCN non-amplified neuroblastoma and that their binding could be blocked by the MSI2 RBP inhibitor Ro 08–2750. I have also shown that MSI2 bound to two fragments of PRKCQ-AS1 RNA spanning about 300bp towards to the 5’ end of PRKCQ[1]AS1. Additionally, RNA immunoprecipitation and subsequent sequencing identified the oncogene BMX as the downstream target most significantly modulated by the PRKCQ-AS1 RNA and MSI2 protein interaction, which in turn activated the MEK/ERK signalling pathway. Functional studies revealed that PRKCQ-AS1 promoted MYCN-non-amplified neuroblastoma cell proliferation in vitro and tumour progression in vivo. The PRKCQ-AS1- binding protein MSI2 and their target BMX also promoted MYCN-non-amplified neuroblastoma cell proliferation. In human neuroblastoma tissues, there was a strong correlation between the expression of PRKCQ-AS1 and MSI2 and the expression of BMX, and high PRKCQ-AS1, MSI2 and BMX expression in human neuroblastoma tissues correlated with poor clinical outcome in patients. Additionally, high levels of both PRKCQ[1]AS1 and MSI2 expression were strong prognostic factors for poor patient outcome independent of the current standard markers including age at diagnosis, disease stage and MYCN gene amplification. My study therefore identified PRKCQ-AS1 and its binding protein MSI2 as factors important for MYCN-non-amplified neuroblastoma cell proliferation and tumorigenesis and the interaction between PRKCQ-AS1 and MSI2 as a valid target for the treatment of MYCN-non-amplified neuroblastoma.
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(2021)ThesisPrimary liver cancer is characterised by poor prognosis, growing disease burden and propensity to develop on a background of chronic liver injury. Delivery of oncogene expression plasmids by hydrodynamic tail vein injection (HTVI) is an emergent method of modelling liver cancer, but does not reproduce chronic injury and fibrosis typical in human liver cancer. Injury and fibrosis contribute to the pathogenesis of liver cancer, although this is not well characterised in plasmid-HTVI liver cancer models. Two previously published plasmid-HTVI models (SB/AKT/c-Met, SB/AKT/NRas) were combined with the hepatotoxin thioacetamide (TAA) to induce liver injury. Consistent with previous characterisation, SB/AKT/c-Met developed steatosis with hepatocellular tumours, while SB/AKT/NRas developed steatosis with both hepatocellular and cholangiocellular tumours. TAA did not increase tumour burden but altered the phenotype of surrounding tissue. TAA reduced plasmid-induced steatosis and increased inflammatory cells and fibrosis with different morphology to TAA alone. In order to find novel gene expression and pathways associated with the cancer-injury combination, liver tissue was subject to whole transcriptome sequencing. Metabolism, inflammation, cell cycling and proliferative signalling pathways were among those that differed by either cancer or injury alone. The cancer-injury combinations had gene expression profiles distinct from cancer or injury alone, with a few differentially expressed genes not explained by either cancer or injury alone. In cancer-injury combinations, some pathways were synergistically upregulated but in many cases cancer and injury were antagonistic. Lipid and xenobiotic metabolism were novel pathways uniquely activated in the cancer-injury combinations. Selected genes were validated by conventional gene expression assays and immunohistochemistry. By characterising a plasmid-HTVI model with concomitant liver injury and fibrosis, it was found that liver injury did not accelerate or increase tumorigenesis but altered the phenotype and transcriptomic profile of the cancer models. In some cases, injury and oncogenes antagonised each other with respect to cancer-associated processes such as fibrosis and steatosis. Novel genes and pathways associated with the cancer-injury combination give insight into how chronic injury interacts with liver cancer in humans.
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Elucidating the Mechanisms of RNA-Induced Transcriptional Silencing Machinery in a HIV-1 Therapeutic(2021)ThesisRNA interference (RNAi) is an evolutionarily conserved mechanism that modulates gene expression through homology-dependent short interfering RNA (siRNA). The process consists of two distinct pathways whereby Argonaute proteins i) transiently degrade messenger transcripts or ii) silence genomic DNA via heritable epigenetic modifications. The latter offers artificial control of the human genome. It presents revolutionary therapeutic interventions for currently incurable genetic conditions and viral infections. This process however, is highly controversial. Fundamental mechanisms, including the components of functional epigenetic silencing machinery, are unknown. In order to harness the full potential of RNAi for gene therapy, it is crucial to understand the mechanics of the pathway. This thesis explores RNAi-induced epigenetic silencing in the context of HIV-1 infection. To date, no treatment has been sufficient to surmount the proviral reservoir, making the virus a prime candidate for gene therapy. Using a promoter targeted siRNA developed by our lab, Chapter 3 investigates the intracellular trafficking of the silencing machinery. Through live-cell immunofluorescent microscopy, we show that the actin cytoskeleton is required for dynamic transport of siRNA loaded Argonaute 1 and subsequent nuclear import. In Chapters 4-6, we perform a range of biochemical interaction studies to examine Argonaute 1 complexes and identify potential components of the core machinery. Lead candidates were further explored in Chapter 7, where we generated Argonaute 1 mutants, CRISPR knockout cultures and computational protein models to assess possible RNAi interplay. We demonstrate that HNRNPU and to a lesser extent, CBX3, are involved in siRNA-mediated gene silencing. Further, in a manner that mimics alternate systems, HSP90 may offer a mechanism of regulating the formation of the silencing machinery. This is the first study to identify proteins, additional to the actin cytoskeleton, including HSP90, HNRNPU and CBX3, that are involved in human RNAi-induced epigenetic silencing. This knowledge provides unique avenues for enhanced RNAi therapeutic delivery and warrants further studies to elucidate the precise mechanisms of a pathway that was prior to this study, completely unknown.
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(2021)ThesisThe breakdown of immunological self-tolerance manifests clinically as autoimmune diseases, such as Multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS). Autologous haematopoietic stem cell transplantation (AHSCT) has received increasing attention in recent years as a therapeutic option for treatment refractory MS. AHSCT has been proposed to induce long-term remission by initiating an immune system “reboot” via the chemotherapy-driven ablation of autoreactive immune cells and the regeneration of immunoregulatory lymphocytes, such as CD4+ T regulatory cells (Tregs). Tregs expressing the ectonucleotidase enzyme CD39 exert immunoregulatory function by suppressing T helper (Th)17-related immune responses, which has been significantly associated with MS pathogenesis. However, this immunoregulatory function was reported to be decreased in MS patients. CD39 expression, and its corresponding immunological function, on Tregs is determined by the genetic polymorphism of the CD39 gene (at position rs_10748643), where its expression may be low (AA-genotype), medium (AG-genotype) or high (GG-genotype). Hence, CD39 genetic polymorphism may significantly determine CD39+ Treg function and reconstitution in MS patients and/or influence the immunological shift in patients following AHSCT. Three novel aspects regarding Treg-associated tolerance regeneration in MS patients following AHSCT are presented in this thesis: 1) although there is no direct clinical correlation, this is the first study investigating the role of CD39 genetic polymorphism in immune reconstitution, cytokine evolution, and Treg function following AHSCT in MS patients (Chapters 3, 4, and 6); 2) significant and long-term reductions in Th17- and Th1-related cytokines post-AHSCT further support the ongoing hypothesis that AHSCT induces an anti-inflammatory shift in MS patients (Chapter 4), and 3) to the best of this author’s knowledge, this thesis was the first to report that MS patients without clinical relapse post-AHSCT exhibit significant improvements in CD4+ Treg suppressive function at 24 months post-AHSCT (Chapter 6) and consistent suppression of Th17 cytokines at all timepoints up to 24 months post-AHSCT (Chapter 4). Future studies must address the exact immunoregulatory pathway that is regenerated in Tregs following AHSCT in MS patients.