Medicine & Health

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  • (2022) Sloane, Jennifer
    Thesis
    From a child interrupting a conversation between her parents to ask "What's for dinner?" to a nurse interrupting a physician in the middle of a complex procedure with an urgent message, interruptions are an inevitable part of our daily lives no matter who we are, where we live, or what we do. Interruptions can have a variety of affects on people's performance and behavior. While interruptions may sometimes facilitate performance, often interruptions have negative consequences. For example, interruptions may result in people making more errors or forgetting to complete a prior task altogether. This thesis examines existing strategies to help mitigate interruption costs and explores the effects of interruptions within different decision environments. Chapter I introduces the topic by discussing a few theoretical frameworks of interruptions and reviewing prior research on what makes interruptions disruptive. One strategy to minimize interruption costs is to use what is called an interruption lag, which can be thought of as taking time to prepare for a pending interruption. Chapter II presents a novel experiment to systematically explore the potential benefits of interruptions lags and an alternative intervention (i.e. providing feedback) when interruption lags are not possible. Chapters III and IV discuss the results from three experiments and a final replication study that all focus on how interruptions affect people's decision making in unique environments. The environments consist of easy problems (i.e. basic arithmetic problems) and trick problems, designed in such a way to lead the reader down an incorrect path. Results from these studies were mixed. While there was some evidence that interruptions may make people more susceptible to falling for the trick answer, this finding was inconsistent across all the experiments. Chapter V applies the findings from the previous chapters to a medical context. This chapter presents novel medical cases that were developed with the help of a medical expert. These cases consisted of easy, hard, and trick cases designed for medical students. The goals of this chapter were to validate the cases and to investigate the effects of interruptions within the different case types. The final chapter (Chapter VI) concludes with a general discussion of the experimental findings, the theoretical implications of the results, and the broader implications of this research for the field of medicine.

  • (2022) Cao, Jun
    Thesis
    This thesis focuses on the development and applications of magnetic resonance electrical properties tomography (MREPT), which is an emerging imaging modality to noninvasively obtain the electrical properties of tissues, such as conductivity and permittivity. Chapter 2 describes the general information about human research ethics, MRI scanner, MR sequence and the method of phase-based MREPT implemented in this thesis. Chapter 3 examines the repeatability of phase-based MREPT in the brain conductivity measurement using balanced fast field echo (bFFE) and turbo spin echo (TSE) sequences, and investigate the effects of compressed SENSE, whole-head B_1 shimming and video watching during scan on the measurement precision. Chapter 4 investigates the conductivity signal in response to short-duration visual stimulus, compares the signal and functional activation pathway with that of BOLD, and tests the consistency of functional conductivity imaging (funCI) with visual stimulation across participants. Chapter 5 extends the use of functional conductivity imaging to somatosensory stimulation and trigeminal nerve stimulation to evaluate the consistency of functional conductivity activation across different types of stimuli. In addition, visual adaptation experiment is performed to test if the repetition suppression effect can be observed using funCI. Chapter 6 explores if resting state conductivity networks can be reliably constructed using resting state funCI, evaluates the consistency of persistent homology architectures, and compares the links between nodes in the whole brain. Chapter 7 investigates the feasibility of prostate conductivity imaging using MREPT, and distinctive features in the conductivity distribution between healthy participants and participants with suspected abnormalities.

  • (2022) Joshi, Nidhi
    Thesis
    RNA interference (RNAi) has emerged as a promising tool to silence any kind of gene expression from viral infection to genetic disease, especially in the field of cancer treatment. siRNA-based therapeutics offer an efficient and specific targeting of disease-causing genes. An aberrant expression of Wnt pathways and ROR receptors which are the transmembrane protein of tyrosine kinase family, have been reported in many cancers, including ovarian cancer. The upregulation of Wnt pathways and ROR receptors are known to be potential contributors in ovarian cancer progression and metastasis. Therefore, targeting these receptors could be a powerful approach towards designing and developing new therapeutic materials. In that regard, siRNA-based therapeutics offer an efficient and specific targeting of overexpressed disease-causing genes. However, the challenge remains for the effective and safe delivery of siRNA therapeutics while maintaining its efficacy and therapeutic integrity. Polymeric nanostructures hold great promise towards designing a compatible delivery vector for nucleic acid therapeutics, especially for siRNA drugs. Among polymeric systems, polycationic carriers based on PDMAEMA are widely explored as nucleic acid delivery vectors. The low toxicity and high transfection efficiency make them an excellent candidate for targeted siRNA therapy. There is an immense scope to study PDMAEMA based vectors as cancer therapeutic carriers for siRNA drugs in platinum-resistant and high-grade ovarian cancer. Therefore, this study aims to develop well-defined biocompatible polymeric nanocarriers for targeted siRNA (ROR2 siRNA) therapy in cisplatin resistance (A2780) and high grade serous ovarian cancer (HGSOC) cells. Polymeric systems containing PDMAEMA as siRNA condensing core have been employed with various structural modifications to investigate the delivery efficiency and therapeutic potential of siRNA drugs. The ability of polymeric nano-vector either generated from PEGylation or BSA modification of PDMAEMA to efficiently bind and release ROR2 siRNA in both 2D and 3D ovarian cancer cell model have been investigated. It was observed that the inhibitory effect of ROR2- siRNA encapsulated in the core of polyion complex (PIC) is strongly dependent on the polyPEGMEMA block length. Reduction of ROR2 expression in both transcription and translation levels was observed in A2780 cells. Alternatively, the BSA modified PDMAEMA nanoparticles present a biocompatible approach for targeted siRNA delivery in HGSOC cells. BSA decorated nanoparticles have shown potential to deliver ROR2-siRNA efficiently in the cytoplasm and hence displayed a significant reduction in migration and invasive features of HGSOC cells.

  • (2022) Amaldoss, Maria John Newton
    Thesis
    Nanocatalytic tumor therapies involve established strategies to increase the concentration of endogenous oxygen species (ROS) H2O2 to cytotoxic levels. These strategies are based on increasing the ROS levels through stimuli from drugs, the action of ROS-producing agents, and nanoparticulate catalysis. However, these techniques frequently are indiscriminatory, being cytotoxic to diseased cells and normal cells alike, leading to significant unwanted side-effects. The present work reports a new paradigm strategy based upon the catalytic action of a cell-discriminative, ROS-mediating, autophagy-suppressive nanoparticle, which is CePO4·H2O (rhabdophane). CePO4·H2O nanoparticles were synthesised using CeNO3·6H2O precipitated in an aqueous solution of sodium tripolyphosphate (STPP) at room temperature. The nanoparticles were well crystallised, equiaxed (~10-35 nm), of positive surface charge, and of general valence ratio 〖"Ce" 〗_"0.8" ^"3+" 〖"Ce" 〗_"0.2" ^"4+" 〖"PO" 〗_"4.1" . Materials characterisation involved particuological (hydrodynamic particle size, surface area, zeta potential), mineralogical (X-ray diffraction, laser Raman microspectroscopy), chemical (X-ray photoelectron spectroscopy), structural (Fourier transform infrared spectroscopy), and microstructural (transmission electron microscopy) analyses. Biological characterisation involved examination of the effects on HT-1080 fibrosarcoma cells and MRC-5 normal fibroblasts in terms of cellular interactions (cell viability by MTT assay), cellular uptake and trafficking (confocal laser scanning microscopy, biological transmission electron microscopy, flow cytometry), ROS generation (confocal laser scanning microscopy, flow cytometry), apoptosis (annexin V-FITC assay), gene expression (q-RT-PRC), and protein expression (western blot analyses). The key observations and conclusions from the biological evaluation are as follows: Discriminative Cytotoxicity: CePO4·H2O nanoparticles are the first to exhibit discriminative cytotoxicity: At 24 h, fibrosarcoma HT-1080 cell viability is ~10% but MRC-5 normal cell viability is ~45%. Discriminative Uptake: CePO4·H2O nanoparticles are the first, without the use of a targeting ligand, to be internalized readily by cancer cells but scarcely by normal cells. Self-Targeting: CePO4·H2O nanoparticles are trafficked toward the mitochondrial environment and possibly the converse trafficking. Mitochondrial Starvation: The preceding proximity between CePO4·H2O nanoparticles and cancer cell leads to increased phosphate concentration in the cellular environment, the concentration gradient of which effectively starves the mitochondria, leading to mitochondrial stress and dysfunction. Discriminative ROS Generation: CePO4·H2O nanoparticles are the first to demonstrate elevated cellular ROS in cancer cells by multiple mechanisms while normal cells exhibit only a low level of such elevation. Autophagy Suppression: CePO4·H2O nanoparticles suppress autophagy, thereby increasing cellular stress and suppressing cancer cell survival, thus offering a complement to mitochondrial starvation. Redox Switching: CePO4·H2O nanoparticles are the first nonmetallic nanoparticles to balance redox switching through simple electronic charge compensation rather than more complex ionic charge compensation. Biocompatibility: As hydrated phosphates, CePO4·H2O nanoparticles are more biocompatible than metals or oxides, suggesting greater feasibility of renal clearance. These advantages derive from the key role of the redox and defect equilibria arising from the oxidation reaction Ce3+ → Ce4+ + e′, which is induced by the acidic pH environment of the cancer call versus the stability of the Ce3+ valence in the basic pH environment of the normal cell. The former both elevates the ROS level and disrupts the electron transfer chain. Ultimately, the suppression of the proliferation of cancer cells derives from the cross-talk involving cellular ROS elevation, autophagy suppression, and their mitochondrial control.

  • (2021) Ewans, Lisa
    Thesis
    Mendelian disorders are rare, heritable conditions that cause medical, financial, psychological, and social burdens. Diagnosis is key for improving patient care but is challenging due to frequent genetic and phenotypic heterogeneity. The genome-wide next generation sequencing (NGS) technologies of whole exome sequencing (WES) and whole genome sequencing (WGS) have revolutionised Mendelian disorder diagnosis in little more than a decade. Yet, the rapid adoption and complexity of genomic technologies have resulted in a gap between implementation and understanding how to utilise their full potential. The main objective of this research was to establish the benefits of NGS to Mendelian disorder diagnosis for clinical translation. To achieve this, the diagnostic potential and utility of WES and WGS were assessed in a cohort of individuals with suspected, but undiagnosed Mendelian disorders. Both WES and WGS were able to dramatically improve diagnostic rates over traditional testing, with diagnostic yields of first-line WES of 52% and WGS, 61%. WES reanalysis with improved pipelines and scientific knowledge demonstrated gains in diagnostic yield at 12 months, and again 2 years later. A WES diagnosis of PLOD3-related disease, an ultra-rare Mendelian disorder, was extended with phenotype delineation, tissue expression, and protein modelling. The resulting disease description and proposed classification is expected to positively impact future patient diagnosis. To assess the economic implications of genomic testing pathways, comparisons of WES to traditional testing and to WGS were made. The early application of WES in intellectual disability saved AU$782 for each additional diagnosis compared to the traditional model. While WGS was shown to have a greater diagnostic yield than WES, when used as a first-line test each incremental additional WGS diagnosis cost AUD$29,000. Thus, from an economic perspective, first-line WES is preferred for routine testing, with WGS reserved for situations where a diagnosis would have a high chance of clinical intervention. Notwithstanding this, WGS additional diagnostic gains should not be underestimated given the increased potential for future diagnosis and the gap in understanding the potential downstream costs benefits of a diagnosis, which may dwarf the initial test cost. In conclusion, NGS technologies provide significant gains over traditional methods and should be adopted early in Mendelian disorder diagnosis to positively impact patient care.

  • (2023) Xie, Yike
    Thesis
    Single cell sequencing has emerged as one of the most powerful technologies to study cell diversity during the past decade. Beyond single cell RNA sequencing, new single cell omic approaches such as CITE-Seq and scATAC-Seq have been developed to study additional molecular fingerprints of cells. In parallel, fluidic force microscopy, patch-clamp and microfluidics have been combined with single cell sequencing to study cell phenotypes in great detail. In my thesis, I developed new computational and experimental approaches to study the molecular states and phenotypes of individual cells. First, I analysed single cell transcriptomic data on two biomedical problems, i.e. neonatal lung development and dengue virus infection. Second, I developed an integrated approach that measures morphology, autofluorescence intensity and transcriptomics from the same cell. In Chapter 3, I analysed cell-cell communications between endothelial cells (ECs) and mesenchymal cells in murine neonatal lung at different development stages. I observed that the number of interactions between distinct mesenchymal cells and ECs is highly dependent on EC subtypes. Less intense cell-cell communications between most pairs of cell types were observed at P7 than E18.5. Lymphatic and venous ECs are more active than other EC subtypes. I also found that distinct EC subtypes communicate with pericytes using specific ligand-receptor channels. This project showed the importance of studying cell-cell communication between cell types during development and disease. In Chapter 4, I measured the abundance of host and viral transcripts in about 200, 000 human peripheral blood mononuclear cells (PBMCs) derived from 19 dengue virus (DENV)-infected children and 4 healthy controls using virus-inclusive single-cell RNA sequencing 2 (viscRNA-Seq 2). I found that B cells contain the most DENV RNA among all cell types in the blood. I also used machine learning to predict infection severity using relative abundances of immune cell subtypes. I also identified differentially expressed genes (DEGs) and analysed cell-cell communications in severe versus uncomplicated infections. In Chapter 5, I developed HyperSeq, an experimental approach that combines hyperspectral autofluorescence imaging with transcriptomics from the same cell and scales to hundreds of cells in a single experiment. I correlated the morphological features of each cell with gene expression and exon usage. Additionally, I linked optical features, machine learning techniques and abundance of NAD(P)H with the expression of specific genes. HyperSeq can therefore be used to study cell state combining morphological, optical, metabolic, and transcriptomic features in a single, consistent approach. In summary, my work has advanced the frontier of single cell technologies with the goal to study cell phenotypes and omics within the same cell. By combining medicine, biology, engineering, and computer science, my research will benefit a large cross section of the research community, allowing a better understanding of complex biomedical problems.

  • (2023) Browne, Kate
    Thesis
    Healthcare-associated infections are the most common adverse event affecting hospital patients. With the emergence of antibiotic-resistant bacterial strains, there is an increasing number of untreatable infections. Therefore, there is a clear need for the development of novel antibacterial strategies to both prevent, and treat, healthcare-associated infections. This thesis explores three key approaches to treat and prevent the transmission of healthcare-associated infections. First of all, a library of novel antibacterial compounds were assessed in vitro for their clinical potential. The compounds with the greatest clinical potential were then tested in various biomaterial applications (biocompatibility in an animal model, and their activity when covalently attached to biomaterial surfaces). Finally, this thesis explores antibacterial strategies to prevent the transmission of bacteria from clinical surfaces. The lead antimicrobial candidates, melimine and RK758, are peptidomimetics that address the drawbacks of conventional antibiotic therapy. They showed broad-spectrum antibacterial activity (including clinical antibiotic-resistant isolates), anti-biofilm activity and thermal stability. When peptidomimetics were used in combination with traditional antibiotics, bacteria were unable to develop resistance towards antibiotics or peptidomimetics. This combination antibiotic-peptidomimetic therapy also showed synergistic activity, which further increases the potential for these compounds to treat multidrug-resistant infections. When assessed in two clinical applications, peptidomimetics showed promise for their continued clinical development. Both melimine and RK758 were biocompatible in animal models. When peptidomimetics were loaded into bone allograft, there was no inhibition of bone healing in a critical-sized distal femur defect model. Moreover, when these peptidomimetics were covalently attached using a new polydopamine coating method to biomaterial surfaces, they retained their broad-spectrum antibacterial activity and prevented biofilm formation in Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. This thesis also explores the transmission of pathogens from clinical surfaces, and how ultraviolet-C disinfection could be used in healthcare-facilities to reduce the bacterial bioburden on various clinical surfaces. Overall, adjunct ultraviolet disinfection significantly reduced the bioburden on surfaces in operating theatres and clinical rooms. Overall, these three strategies work together to prevent healthcare-associated infections. Preventing the transmission of pathogens from the environment to patients, is known to reduce healthcare-associated infection rates. Furthermore, the development and application of novel-acting antibiotics offers a solution to the antibiotic-resistance crisis and addresses the urgent need for antimicrobials with broad-spectrum antibacterial activity. If commercialised, the strategies proposed in this thesis have the potential to increase activity of ineffective antibiotics and prolong their use in the clinic.

  • (2023) Doculara, Louise
    Thesis
    Despite improvements in the treatment and management of acute lymphoblastic leukaemia (ALL), the survival rate remains low for patients who either relapse or have chemotherapeutic resistance. High-risk subtypes including mixed lineage leukaemia-rearranged ALL (MLL-r ALL) occur in 80% of infants with ALL, where event-free survival is 30-40%. MLL-r ALL is characterised by MLL fusions which are known drivers of the disease. The strongest prognostic factor for ALL relapse is the persistence of minimal residual disease (MRD) throughout treatment in the bone marrow. However, there are critical challenges with MRD testing. The sensitivity of techniques is limited by the amount of bone marrow sample that can be collected at any one time. Also, MRD testing is invasive and cannot detect relapse in extramedullary sites. These limitations highlight the need for alternatives to bone marrow MRD testing. Circulating tumour DNA (ctDNA) as a minimally invasive tumour biomarker has rapidly drawn interest for MRD detection, assessment of treatment response and its representation of the total tumour burden in adult and solid tumours. This is not the case for childhood malignancies, in part due to the lack of preclinical models to study the variability of ctDNA levels and ctDNA kinetics at different disease stages. Also, the detection of levels of ctDNA at low disease burden stages (including remission before relapse) is a formidable challenge. Therefore, novel nanotechnology-based strategies including biosensors are emerging as potential improvements for ctDNA analysis. This study aimed to address these challenges of MRD testing in childhood ALL through developing patient-specific assays for ctDNA detection in preclinical models of MLL-r ALL. In vivo data demonstrated a significant concordance between ctDNA concentration and leukaemia burden in mice inoculated with a panel of MLL-r ALL PDXs. CtDNA levels reflected multiple organs of disease burden during remission and relapse in mice treated with a novel small-molecule inhibitor for MLL-r ALL. The ctDNA levels were then investigated in a pilot retrospective study in ALL patient plasma samples and a developed biosensor was used for the detection of patient-specific ctDNA in plasma. In summary, this study demonstrated the clinical potential of ctDNA for disease monitoring in high-risk ALL patients.

  • (2023) Yam, Andrew
    Thesis
    Neutrophils are responsible for protection from microbial infections. But they can also infiltrate solid tumours, and their presence within tumours is linked with cancer growth and poorer prognosis. However, this view oversimplifies their relationship with tumours, as recent research suggests that neutrophils also have anti-tumour properties. In addition, neutrophils possess considerable functional plasticity, and physiological and pathological factors can alter their function. In this study, we examined how neutrophil plasticity could be exploited to shift the tumour immune microenvironment to favour the inhibition of cancer growth. We achieved this by injecting inactivated Staphylococcus aureus (S. aureus) bioparticles into tumours. As a result, the TME (TME) was altered by the rapid influx of activated neutrophils that had acquired a pathogen killing effector phenotype. When examined using two-photon microscopy, S. aureus bioparticle activated neutrophils within tumours had increased motility and interacted with tumour cells. Repeated administration of S. aureus bioparticle therapy maintained a neutrophil activating environment within tumours. This led to neutrophil-dependent inhibition of tumour growth, highlighting an important role for microbe activated neutrophils in achieving cancer control. S. aureus bioparticle treatment also enhanced CD8+ T cell responses within tumours and draining lymph nodes. Notably, mice treated with our microbial therapy were protected from cancer recurrence, which suggests that a long-lasting protective immune response was elicited by the treatment. In keeping with these results, we found that S. aureus bioparticle treatment had a synergistic tumour inhibiting effect when combined with systemic checkpoint inhibitor therapy. In conclusion, we demonstrated that neutrophils are critical for microbe based cancer immunotherapy. We leveraged neutrophil plasticity and their rapid tissue infiltration in response to S. aureus bioparticle induced inflammation to alter the TME and control tumour growth. This body of research supports a model for developing neutrophil based immunotherapy using microbial bioparticles and its application with existing clinical cancer therapies.

  • (2023) Jakovija, Arnolda
    Thesis
    Neutrophils are the most abundant white blood cells in humans and play an essential role in wound healing, inflammation and cancer. They are the first cells to arrive at injury sites and in the early phases of carcinogenesis. The current literature addresses the role of neutrophils in acute injury, but there is a lack of knowledge about their contribution to the post-acute phase of wound healing. Furthermore, neutrophils play a multifaceted role in tumour development, as they can both promote and inhibit tumour growth. Neutrophil function, whether they promote or kill tumours, are determined by factors in the TME and manipulating their plasticity could have therapeutic benefits. This project adopts cutting-edge methodologies, such as intravital microscopy and single-cell RNA-sequencing technologies, to investigate the role of immune cells in both regenerative wound healing and tumour development. Specifically, in vivo neutrophil imaging was leveraged to create a detailed picture of the temporal evolution of neutrophil functions during wound healing and cancer inflammation. The findings indicate that neutrophils survive in the skin for extended period of times after injury, and have a distinct phenotype, migration, and function. Furthermore, analysis of neutrophil function in tumours demonstrated that altering the tumour microenvironment from an aberrant wound healing response to acute microbe-triggered inflammation induced significant changes in tumour neutrophil transcription, migration, and function, ultimately suppressing tumour growth in a neutrophil-dependent manner. These data sheds new light on the complex interplay between neutrophils and the immune system in the context of both wound healing and cancer. A more comprehensive understanding of neutrophil transitional states from tissue-damaging to tissue-restoring is critical for the development of more effective therapeutic innovative strategies aimed at inhibiting pro-tumoral inflammation and enhancing neutrophil anti-tumoral capabilities.