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(2020)ThesisContinuous-flow left ventricular assist devices (cf-LVADs) now form a cornerstone of the treatment of advanced heart failure. With their increasing use, improving our understanding of the mechanical, histopathological and physiological interactions between these devices and the patients in whom they are implanted is paramount. Initially, through non-invasive assessment of the impact of dynamic manoeuvres on the pump flow waveform and left ventricular dimensions, I demonstrate that changes in afterload pressure, posture and intrathoracic pressure have significant and highly variable effects on pump flow. The relationship between the intrathoracic pressure changes, loading conditions and pump flow is then assessed invasively, with low preload, low arterial resistance and increased ventricular-arterial coupling predictive of pulsatility loss and suction events during modified Valsalva manoeuvre. Using a pulsatile mock loop circulation, I assess the contribution of the outflow conduit to pump afterload. Haemodynamically significant gradients can be generated across an unobstructed HVAD outflow graft, and their magnitude predicted using an empirically derived model incorporating conduit diameter, mean pump flow, systolic dQdt and conduit length. I then demonstrate in vivo that some degree of tissue ingrowth into the cf-LVAD outflow graft due to acute inflammatory, chronic inflammatory, fibrotic or neointimal reaction is a near-universal phenomenon and is associated with a small but measurable decrease in pump flow and flow pulsatility over time. In order to enable an integrated assessment of left ventricular contractility, energetics and loading conditions, I describe a method to derive pressure-volume loops using non-invasive inputs that are readily available in the clinic setting. This method is validated invasively by assessing its ability to detect predictable pharmacodynamic effects of intravenous Milrinone. Finally, I utilise this pressure-volume derivation in order to assess the haemodynamic effects of exercise, revealing increased left ventricular contraction that is likely driven by increased preload, and profoundly diminished unloading effect of increased pump speed during exercise. Overall, this thesis sheds light on the complex interplay between ventricular contractility, loading conditions and cf-LVAD performance under ‘real-world’ conditions, with significant implications for both clinical practice and future research in this area.
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(2021)ThesisMesenchymal cells in solid tumours have multiple roles in supporting carcinogenesis. They have emerged as important mediators of immune function and as such, are regarded as promising candidates for therapeutic targeting and immunotherapy. However, conflicting preclinical results necessitate further investigation of their phenotypic diversity, beyond that described using model experimental systems or small panels of markers. This thesis applies advanced multi-omics technologies to resolve the cellular heterogeneity of mesenchymal cells inside human breast cancers. To overcome the logistical and technical challenges of studying clinical tissues using single-cell sequencing, I first present a benchmarking of tissue cryopreservation methods. Analysis of cryopreserved cell suspensions and solid tissue fragments conserved the heterogeneity of tumours and the integrity of transcriptomes. These methods were applied to subsequent parts of this thesis and importantly, increases the opportunities for sample collection in future clinical studies. I next performed single-cell analysis on a small cohort of five patients of the aggressive triple-negative subtype (TNBC). This body of work presents the first single-cell analysis of mesenchymal cells in human TNBC. I identified functionally distinct subclasses of cancer-associated fibroblasts (CAFs) and perivascular-like cells that showed strong associations with immune evasion. In light of this, a comprehensive review of how the recent wave of single cell studies have shaped our understandings of stromal-immune interactions in cancer is presented. Lastly, to expand our findings to a larger cohort of patients from all clinical subtypes, I led a study that established a large single-cell and spatial atlas of primary breast cancers. This revealed new dynamics of mesenchymal heterogeneity, where cells fell into a spectrum of cell differentiation rather than discrete subsets, supporting future strategies to manipulate cell differentiation for therapeutic benefit. Spatial transcriptomics revealed that subsets of CAFs were spatially segregated, with unique subclasses colocalising with immune cells. Using both modalities, I identified a suite of potential signalling molecules between CAFs and T-cells that show spatial proximity, offering a list of candidates to investigate in future functional studies. I envisage the findings described here to help guide the development of effective stromal directed therapies for breast cancer.
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(2021)ThesisThe heterogenous tumour microenvironment (TME) has been well established in the progression of cancer and patient outcome. In particular, the success of anticancer therapy is influenced by various constituents and intercommunicating networks within the TME. Drug development research often focuses on targeting cancer cells alone without considering all the cellular and non-cellular components of the TME, which plays a crucial role in chemoresistance and disease relapse. Thus, there is a necessity to understand the individual features of the TME in order to deliver a more personalised therapy that improves the rate of successful outcomes while reducing the recurrence of the disease. However, the mechanisms of acquired drug resistance and pharmacokinetics within the tumour still remains elusive. Currently, there is a lack of high-throughput methods suitable to study the functional effects of anticancer drugs on tumour samples where the interplay between cancer cells and the TME remains intact. This thesis embodies the work in the development of the ALTEN (Alginate-based Tissue Engineering) platform, a biomimetic hydrogel system for rapid functional testing of anticancer drugs in explanted tumours. The alginate hydrogels provides a 3D scaffold that resembles the native extracellular matrix (ECM) that preserve the original characteristics of the tumour to permit detailed assessment of the TME and the complex molecular interactions between cell species. Here, we use high-resolution single-cell RNA-seq technologies to analyse the molecular effects of anticancer treatments within the tumour to study the mechanisms of acquired drug resistance. The combination of ALTEN and scRNA-seq technology enable high-throughput and high-resolution screening of tumour explants exposed to cytotoxic agents and immunomodulators. The impact of drug therapies within an intact TME could be evaluated to 1) determine treatment efficacy and 2) characterise drug resistant cancer populations. In summary, the ALTEN platform provides an innovative avenue allowing engineering of patient tumour explants to investigate the efficacy of therapies in a high-throughput manner and provide preclinical information on optimising treatments. The integration of both the high-fidelity drug testing capacity of ALTEN and the high-resolution molecular phenotyping scRNA-seq aims to assist oncologists in providing personalised therapies to improve patient outcome.
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(2020)ThesisThe pathogenesis of autoimmune diseases is poorly understood - this is explicit in our failure to effectively cure them. This thesis had two primary, interdependent, aims. The first was conceptual - to test the 'common root approach', designed to provide clues as to the cell-intrinsic effects of gene mutations in autoimmune disease, based on a priori knowledge of their recurrence and functional consequences in lymphoid cancer. The second was specific to a small set of carefully selected genes that are mutated in the germline to cause immune dysregulation and mutated somatically in lymphoid cancer - to determine their molecular and cellular effects in autoimmune disease. These genes included STAT3, EGR2 and CARDI 1. Collectively, they are some of the most recurrently somatically mutated genes In lymphoid cancer, and their mutations in the gerrnline cause immune dysregulation. I studied these genes in genome-edited mice to determine their roles in isolation from confounding genetic variation and environmental exposures - followed where possible by validation of my findings in humans patients with corresponding gerrnline mutations. The study of STAT3 revealed that gerrnline gain-of-function STAT3 mutations are su!tcient to cause pathology in mice, in isolation from additional genetic variation or environmental triggers. These mutations cause dramatic polyclonal expansion of effector COB T cells in mice and humans - cells in mice that are sensitive to inhibition of some signalling pathways but not of others. This study further revealed that STAT3 is a transcriptional regulator of co21tow CD23I0" age-associated B cells - such that STAT3 gain-of-function results in the accumulation, and qualitative change in mRNA and protein expression, of CD21 tow CD23·0w B cells. The study of EGR2 and EGR3 revealed that these transcription factors function in a cell-intrinsic and partially redundant manner to suppress the accumulation of two B cell populations enriched for self-reactive BCR specificities and aberrantly expanded in autoimmune and/or lympoproliferative disease - B1a cells and CD21I0"' CD2310"' age-associated B cells. Finally, the study of overactive CARD11 revealed that the gain-of-function Cardi 1M31JSK mutation has no significant cell-intrinsic effect in mouse B cells, but provides a cell-intrinsic advantage to activated CD4 and Treg cells. These findings demonstrate that the common root approach can be useful in the investigation of gene mutation-driven immune dysregulation.
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(2020)ThesisThe extracellular matrix (ECM) provides cancer cells with a 3D structural and biochemical scaffold that regulates cellular function and behaviour. Pancreatic Ductal Adenocarcinoma (PDAC) is characterised by its aggressive metastatic nature and a dense, desmoplastic ECM, which has been identified to both promote and prevent PDAC development. Together with late diagnosis the rapid and highly metastatic nature of the disease contributes to the poor survival rates in PDAC. The multifunctional protein, focal adhesion kinase (FAK), is at the intersection of various pathways often hijacked in cancer and is a known regulator of ECM stiffness and mechano-signalling in both stromal and epithelial compartments. In a highly metastatic mouse model of PDAC, we observe enhanced ECM deposition and remodelling throughout disease progression, which occurs in parallel with increased Focal Adhesion Kinase (FAK) expression and activity. Intravital imaging allowed the visualisation of fine-tuned epithelial and stromal manipulation via FAK inhibition to improve systemic chemotherapeutic efficiency whilst reducing metastatic spread. Here, intravital imaging of the FUCCI cell cycle reporter, in parallel with Second Harmonic Generation imaging of collagen fibres, was used to dynamically monitor tumour cell response to gemcitabine/Abraxane and FAK driven ECM manipulation, respectively, at both primary and secondary sites. Complementary in vitro 3-Dimenstional techniques were used to deconstruct the specific mechanism by which short-term FAK inhibition reduced PDAC metastasis and enhanced chemotherapeutic efficiency. Furthermore, stratification of PDAC patient samples suggest a subset of patients with high FAK activity are likely to respond to FAK treatment regimes, where ECM manipulation and epithelial FAK inhibition prior to chemotherapy may improve patient outcome. This fine-tuned stromal manipulation may allow us to maximise gemcitabine/Abraxane therapy whilst reducing drug toxicity associated with long-term combinational treatment and potentially reducing further metastatic spread in PDAC patients.
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(2021)ThesisGenomic instability (GIN), a genomic state facilitating large scale chromosomal rearrangements, is a hallmark of cancer. GIN can contribute to oncogenesis by disrupting genes, and leading to copy number aberrations (CNAs), the gain or loss of genomic segments. In this thesis I describe two projects linked by the overarching theme of GIN, outlined below: Project 1: Copy-number aberrations (CNAs) contribute to clonal diversity within cancer, with clinical implications. Breast cancer is one such example, but the effect of CNAs on gene expression in intra-tumour subclonal populations has not been properly characterised. Due to sequencing technology limits and lack of computational methods, it is difficult to assess CNAs at a subclonal level. Here, I have benchmarked the ‘InferCNV’ computational method and used it to infer single cell CNA profiles from 14 primary breast cancer single cell RNA-sequencing (scRNA-seq) datasets. I reveal diverse intratumoural heterogeneity involving at least four subclonal populations per tumour. Finally, I identify subclones with expression/CNA profiles indicative of metastatic potential, involving differential regulation of metastasis associated genes such as MUCL1, BST2 and IGFBP5. Project 2: High-grade serous ovarian cancer (HGSOC) is characterised by widespread GIN. Drivers of GIN include deficient DNA repair and amplification of Cyclin E1, however no major cause is known for one third of tumours. Deregulation of repetitive elements may contribute to GIN in HGSOC. It is difficult to investigate repetitive elements from sequencing data as they map to multiple places within the genome. I have quantified repetitive RNA in 99 high-grade serous ovarian cancer (HGSOC) and matched control RNA-seq datasets to determine their potential contribution to GIN. I identified retrotransposons which are deregulated in HGSOC, which may have been active during cancer development. Some of these retrotransposons were enriched at structural variant breakpoints, indicating potential causality. Finally, I identified retrotransposon-associated structural variants in proximity to deregulated oncogenes implicated in homologous DNA repair, which may have modulated their expression and contributed to cancer development. In summary, I have explored both a cause (retrotransposons) and consequence (CNA-based heterogeneity) of GIN in cancer, and shown how GIN can contribute to the modulation of cancer-associated genes which influence cancer development and outcomes.
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(2020)ThesisDNA cytosine methylation is an important epigenetic modification that plays a key role in gene expression. DNA methylation has been shown to be involved in numerous processes, including X-chromosome inactivation in mammals, retrotransposon silencing, genomic imprinting, carcinogenesis and the regulation of tissue specific gene expression during development. Gene expression is tightly regulated via DNA methylation (5mC) and the aberrant expression of meiotic genes in mitotic cells via CpG promoter hypomethylation has been proposed to cause cancer. Cancer/Testis Antigens (CTAs) are a group of genes that encode tumour specific antigens and are expressed in the testis, certain cancers but not in normal post-natal somatic tissues. CpG island methylation and histone modifications appear to play a role in the epigenetic regulation of CTA expression, however, very little is known about their functions in vivo. A widely studied but poorly understood question to date is the mechanisms behind aberrant CTA reactivation in cancer. Given that 5mC mediated gene repression has been found to exist in vertebrate genomes and CTAs have also been identified to be a subset of highly evolutionarily conserved genes, it is critical to understand the role of 5mC mediated CTA silencing in vertebrates. By gaining a deeper understanding into the mechanisms behind this highly conserved pattern of gene repression on a specific subset of genes, we would be able to identify methods to prevent aberrant gene expression. In this study, I analysed publicly available whole genome bisulfite sequencing (WGBS), RNA-seq and chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq) data of developing embryonic and adult somatic tissue of 3 vertebrate species to elucidate the evolutionary epigenetic regulation of CTAs in vertebrate genomes. Integrative WGBS, RNA-seq and ChIP-seq analysis revealed that CTAs are evolutionarily conserved in zebrafish, mice and humans and mechanisms of their epigenetic regulation are also conserved. I observed that histone modifications could potentially serve as an indicator of the methylation status of CTA gene promoters and that the expression of CTAs was inversely related to gene promoter 5mC levels. I demonstrate that CTAs when over-expressed cause embryonic lethality in zebrafish and the same genes are aberrantly hypomethylated at their CpG islands in a subset of human cancers. Overall, my work shows that CTAs are epigenetically regulated in an evolutionarily conserved manner and possibly via a conserved transcription factor, ETS1, that is expressed both in embryonic and cancerous tissue.
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(2022)Thesis3D computer generated biomedical animations can help audiences understand and contextualise scientific information that can be challenging to communicate due to resolution and complexity. Biomedical animators bring together multiple sources of authentic scientific data, to translate abstract information into a visual form through storytelling and visualisation. The field of biomedical animation has emerged from a long history of science visualisation and science-art endeavours, and despite there being rich discourse in the fields of data visualisation and science communication, the academic literature in the field of biomedical animation is limited, and focussed on the technical methods for visualisation, or the role these animations play in scientific research, rather than the processes through which they are created. However, as the field matures, there is a need for a deeper understanding of the creative process, and the field is now poised to expose and characterise these aspects, particularly from the perspective of the practitioner. This practice-based research project aims to expose and characterise both the visible and invisible factors that influence my personal process of creating a biomedical animation, and the tacit dimensions that influence orchestrated design choices. This research project employs a multi-method and reflective practice approach with disciplined capture and documentation of critical moments of self-reflection, that ultimately comprise the data for analysis. Thematic analysis was then used to analyse the data, and to identify themes that could contribute to frameworks that represent my personal process(es) in creating 3D biomedical animations. This has allowed me to identify and contextualise my creative process both in terms of my personal and professional position as well as within the field more broadly. I am now able to better advocate for the intangible and often undervalued aspects of my creative practice, and can articulate how a hierarchical decision matrix that considers multiple inputs contributes to my creative process. These insights will also be relevant to others in the field of biomedical animation and in the field of design more broadly, who may gain a deeper insight into their own processes of working and ways of exploring creative practice.
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(2021)ThesisTacrolimus is the key immunosuppressant used in most solid-organ transplant recipients, including heart transplants, to prevent graft rejection. However, tacrolimus dosing strategies are complicated by the narrow therapeutic window and considerable pharmacokinetic variability. Individualising lifelong tacrolimus therapy to avoid graft rejection and minimise adverse effects is essential for heart transplant recipients. This thesis aimed to investigate the individualisation of tacrolimus therapy in adult heart transplant recipients using the pharmacokinetic modelling approach. In Chapter 1, I present an overview of tacrolimus clinical pharmacology, including clinical factors influencing tacrolimus pharmacokinetics (e.g., concomitant azole antifungal therapy). In Chapter 2, I explore tacrolimus dosing and monitoring practices in heart transplant recipients (n=87) at St. Vincent’s Hospital Sydney, a major heart transplant centre in Australia. Additionally, I assess the ability of a Bayesian dosing software, approved by the Therapeutic Goods Administration to predict tacrolimus concentrations in heart transplant recipients. Tacrolimus dosing and monitoring practices were discordant with the hospital guidelines. The population pharmacokinetic model integrated within the software was suitable in guiding tacrolimus dosing only after 11 days of therapy. This finding necessitated the identification of other model(s) that might be more suitable for use in heart transplant recipients, particularly for the immediate post-transplantation phase. In Chapter 3, I conduct a systematic review summarising published population pharmacokinetic models of tacrolimus (n=69) developed from various organ transplant recipient populations. In Chapter 4, I select relevant tacrolimus models (n=17) from the systematic review and evaluated their predictive performance in heart transplant recipients (n=85). The evaluated models displayed poor predictive performances. This finding complements the work from Chapters 2 and 3 highlighting a tacrolimus model for heart transplant recipients is required. In Chapter 5, I successfully develop a tacrolimus population pharmacokinetic model for heart transplant recipients. The model incorporated the effects of concomitant azole antifungal use, haematocrit, and body weight on tacrolimus pharmacokinetics. Model evaluation in an independent heart transplant recipient cohort displayed good model performance. The model can be implemented in clinical practice to individualise tacrolimus dosing in heart transplant recipients. In Chapter 6, I discuss the clinical implication of this work and recommendations for future research.
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(2021)ThesisRNA modifications, collectively referred to as the ‘epitranscriptome’, are not mere decorations of RNA molecules, but can be dynamically regulated upon environmental queues and changes in cellular conditions. This dynamic behaviour is achieved through the RNA modification machinery, which comprises “writer”, “reader” and “eraser” proteins that modify, recognize and remove the modification, respectively. Chapter1 presents a comprehensive analysis of the RNA modification machinery (readers, writers and erasers) across species, tissues and cancer types, revealing gene duplications during eukaryotic evolution, changes in substrate specificity and tissue- and cancer-specific expression patterns. Chapters 2 and 3 presents the exploration and development of novel methods to map and analyze RNA modifications transcriptome-wide. Nanopore direct-RNA sequencing technology was used to provide RNA modification maps in full-length native RNA molecules. Firstly, it is shown that RNA modifications can be detected in the form of base-calling ‘errors’, thus allowing us to train Support Vector Machine models that can distinguish m6A-modified from unmodified sites, both in vitro and in vivo. Secondly, it is demonstrated that distinct RNA modification types have unique base-calling ‘error’ signatures, allowing us to exploit these signatures to distinguish different RNA modification types. It is found that pseudouridine has one of the most distinct signatures, appearing in the form of C-to-U mismatches. Finally, this information was used to predict novel pseudouridine sites on ncRNAs and mRNAs transcriptome-wide, as well as to obtain quantitative measurements of the stoichiometry of modified sites. Chapter 4 presents the development of a novel nanopore-based method, which is termed ‘Nano3P-seq’, to simultaneously quantify RNA abundance and tail length dynamics in individual molecules in both the coding and non-coding transcriptome, from cDNA reads. It is demonstrated that Nano3P-seq offers a simple approach to study the coding and non-coding transcriptome at single molecule resolution regardless of the tail ends. Together, this work provides a comprehensive framework for the study of RNA modifications and polyA tail dynamics using third generation sequencing technologies, opening novel avenues for future works that aim to characterize their dynamics and biological roles both in health and in disease.