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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)ThesisDue to the success of antiretroviral therapy (ART), people living with HIV now experience a controlled chronic disease, compared to uncontrolled disease off-ART that progresses to AIDS. Although ART has revolutionized HIV-1 treatment, it is not a cure. This is due to the latent reservoir that can rebound to pre-therapy levels upon ART cessation. Successful HIV-1 stem cell therapy cures have set a precedent for targeting the HIV-1 co-receptor CCR5 (i.e. the Berlin and London patients). A complementary HIV-1 functional cure strategy is the ‘block and lock’ strategy, where, for example, small interfering (si)RNA targeting the promoter block virus transcription and lock the promoter in a latent state via epigenetic silencing. This thesis will assess a combined gene therapy cure incorporating CCR5 targeting and RNAi-induced epigenetic silencing. A major barrier for RNAi-directed HIV-1 gene therapy is efficient delivery of siRNA into cells. This study assessed two different delivery platforms; nanoparticles and lentiviral vectors. Numerous nanoparticle formulations were screened and identified a lead candidate that successfully delivered siRNA in cell lines and human primary cells in vitro and induced functional silencing of virus replication. In parallel, delivery of shRNAs with an established lentiviral delivery system was assessed. Studies confirmed a novel epigenetic silencing shRNA, sh136/T, and demonstrated successful combination delivery of two shRNAs in a single lentiviral construct (i.e. shPromA and shCCR5). Reduced virus reactivation was reported in multiple J-Lat latent cell lines and shRNAs suppressed virus replication in PM1 cells. In vivo studies were performed in a chronic HIV-1 humanized mouse model following engraftment of shPromA or shPromA-shCCR5 transduced CD34+ hematopoietic stem cells (HSCs). Following HIV-1 infection for 10 weeks and ART treatment for 8, virus rebound was followed for 28 days post-ART treatment interruption. Mice carrying the shPromA-shCCR5 gene modification demonstrated increased CD4+ T cell count and delayed viral rebound post-ART treatment interruption. This thesis provides valuable insights for si/shRNA therapeutic delivery using nanoparticle delivery in primary human cells in vitro and lentiviral delivery in T cell lines (in vitro) and primary CD34+ HSCs (in vivo). Both delivery platforms were capable of delivering si/shRNAs to functionally suppress virus replication.
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(2021)ThesisOral appliances are a common alternative to CPAP for obstructive sleep apnoea (OSA) therapy. However, efficacy varies and current methods to predict favourable treatment responses are inadequate. This thesis aims to advance knowledge on the effect of oral appliance therapy on upper airway physiology and identify physiological characteristics to help predict which patients are most likely to respond favourably to oral appliance therapy. Study 1 examines the role of body posture and mandibular advancement on nasal resistance in people with OSA. Efficacy of a novel titanium-based oral appliance with an in-built oral airway was also quantified. Awake nasal resistance increased systematically from seated, to supine, to lateral but was not altered by acute mandibular advancement. Unlike conventional devices, the novel oral appliance had similar therapeutic efficacy in people with high and low nasal resistance. Study 2 used a detailed physiological approach to carefully quantify therapeutic CPAP requirements during combination therapy with CPAP and an oral appliance in the clinically relevant group of non-responders to oral appliance therapy alone. CPAP requirements reduced by ~40% with combination therapy. Study 3 prospectively explored potential differences in the 4 pathophysiological traits that contribute to OSA between responders and non-responders to oral appliance therapy using gold standard respiratory phenotyping methodology and validated algorithm-based estimates. Efficacy of a next generation nylon-based novel oral appliance with a built-in oral airway was also assessed as was awake nasal resistance. Oral appliance therapy reduced OSA severity by ~40%. OSA severity reduced by >50% in half of the participants in both people with and without high nasal resistance. Responders to therapy tended to have a less collapsible upper airway and better pharyngeal muscle compensation at baseline when these traits were estimated via polysomnography but not when measured directly in this prospectively recruited cohort, none of whom had major anatomical compromise at baseline (Pcrit all <2cmH2O). These findings provide new insight into the effects of a novel oral appliance on upper airway physiology and therapeutic efficacy and the potential for combination therapy for those with an incomplete therapeutic response to monotherapy with an oral appliance and the potential for estimates of OSA pathophysiological traits to help predict a favourable treatment response.
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(2021)ThesisResearch on sedentary behaviour, a cardiovascular disease (CVD) risk factor, focuses predominantly on developed countries with little undertaken in low-and-middle-income countries (LMIC). The aim of this thesis is to measure sedentary behaviour in desk-based workers and to develop a concept of a theory-based behaviour change intervention to reduce prolonged occupational sitting time (OST) in Bangladesh. Using a mixed-methods approach, findings from three primary studies were collated. The first study was a cross-sectional survey (n=360 participants) conducted in three workplaces. Regression analyses were used to examine the association of individual level factors with total sedentary time and OST. In the second study, a scoping review (n=43) on workplace health promotion (WHP) interventions for CVD in LMICs was conducted to inform the research, then a descriptive qualitative study was employed to understand the socioecological factors of implementing WHP programs. The third study utilised qualitative methods to identify factors influencing breaking up prolonged OST. Finally, intervention features were developed applying the Behaviour Change Wheel framework. We found total sedentary time to be 9.6 hours/day (SD±2.35) of which half occurred in workplaces. Significant differences in OST were found by sex, income, worksite, and sitting break pattern. All participants showed enthusiasm for the idea of WHP, but the complexities of implementation were poorly understood. Limited awareness of the health consequences of prolonged sitting, work type, productivity concerns, and perceived negative judgement by others were reported as barriers to interrupting prolonged OST. Findings suggest that multiple levels of behaviour (intrapersonal, inter-personal, organisational and societal) must be addressed in designing interventions for OST. Twenty-five behaviour change techniques were identified and translated for delivery through a mobile phone application. The research found that sedentary behaviour is high among desk-based workers in Bangladesh. Given Bangladesh’s rapid urbanisation and growing burden of CVD, more research is required into its risk factors for behaviour change. By identifying opportunities for reducing OST in Bangladesh, this thesis contributes to public health evidence and proposes a potential intervention for LMICs. The recommendations offered will strengthen policy and programs to initiate WHP to tackle the increasing burden of CVD in Bangladesh and similar settings.
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(2021)ThesisBladder cystitis is a chronic lower urinary tract condition characterised by pain perceived to be associated with the bladder filling. The effectiveness of the currently available therapies is low, and new and effective therapeutic options should be developed. There is strong evidence of inflammation in the bladder of cystitis patients and for a role of purinergic P2X7 receptor in the process of inflammation. Therefore, the question arises: could P2X7 antagonism be a possible treatment for bladder cystitis. In this study I will determine whether blockade of purinergic P2X7 receptor prevents urothelial damage induced by either chemical or bacterial stimuli. In this study, an ex-vivo model of bladder cystitis has been established, by perfusing acrolein into the whole porcine bladder. The effect of acrolein and a uropathogenic E. coli strain, UTI89, on the integrity of urothelial cells has also been explored using an in-vitro model of urothelial barrier integrity. A selective P2X7 receptor antagonist, A804598, has been used to determine whether inhibition of P2X7 receptor can protect against cystitis-induced damage to the urothelium in the above mentioned models. As a result, acrolein showed significant damage to urothelial histology, tight junction expression and contractile responses. Acrolein also induced cell apoptosis in the mucosa layer in the ex-vivo model and extreme damage to the urothelial barrier integrity in the in-vitro model. An important finding of this study was the revelation of the role of P2X7 receptor in acrolein-induced urothelial inflammation and that the blockade of the P2X7 receptor by its antagonist remarkably protected the urothelium from this damage. UTI89 has also shown a very strong damaging effect on urothelial integrity, but P2X7 receptor blockade did not show any protective action against this damage. However, UTI89-induced damage was significantly diminished in cells treated with the non-selective P2 receptor antagonist, PPADS. This thesis has provided strong evidence that the P2X7 receptor is involved in acrolein-induced bladder cystitis and indicates the possible therapeutic role for P2X7 receptor antagonists in this type of bladder cystitis. The broad spectrum P2 receptor antagonist PPADS showed partial protection against the effects of bacteria, but it was independent of the P2X7 receptor, suggesting that other purinergic P2 receptors may be involved in this protective action.
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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.