Minerva Elements Records

Permanent URI for this collection

http://hdl.handle.net/11343/251382

Search Results

Now showing 1 - 10 of 23

A Quantitative Analysis of Natural Killer Cell Homeostasis, Competition, and Collaboration

Hennessy, Robert John ( 2022-12)

Contemporary Immunology views Natural Killer (NK) cells as critical facilitators of immune protection in various pathological settings. Still, this has not always been the case; a somewhat challenging history of NK cell research has delayed full scientific appreciation of their importance and modus operandi, which rendered NK cells a mysterious and misunderstood immune cell subset for several decades. In more recent years, NK cells are receiving a resurgence in clinical attention owing to characterisation of their potent anti-tumour and immunomodulatory properties; however, as modern Immunology remains in the aftermath of an uncertain era for NK cells, harnessing this revolutionary therapeutic potential has proven difficult. NK cells are key inducers of early inflammation and systemic immune activation, as well as expert decision makers in the destruction of harmful cells versus protection of healthy tissue. As may be expected, catastrophic consequences can occur to a host if these processes are not properly regulated. There is growing appreciation in the research community regarding the sheer complexity and redundancy in regulatory processes that maintain NK cell homeostasis and functions, as well as the plethora of cytokines and cell-cell interactions that govern this regulated behaviour. As a means of dissecting these complex processes, we have applied a reductionist approach to study how various individual signals are integrated into the internal machinery of an NK cell to produce different outcomes. To this end, we applied quantitative methods previously established in adaptive T and B lymphocytes to delineate and quantify parameters relating to survival and proliferation. In this work, we uncovered that stimulatory proliferative signals from the cytokines IL-15, IL-18, and IL-12 are offset by enhanced propensity for NK cell death, which limits the overall efficiency of their expansion during stimulation. These responses were largely dependent on direct interactions between NK cells via Fas and FasL, which induce fratricidal killing of each other. These competitive relationships between fellow NK cells were heavily dependent on the type and dose of cytokine present. Further, our investigation of NK cell interactions led us to identify that NK cells also facilitate advantageous interactions with other NK cells in more homeostatic contexts, which were dependent on IL-15. We discovered that these homotypic collaborative interactions are the result of complex interactions and bidirectional signalling events between SLAM family receptors 2B4 and CD48, which together facilitate IL-15 responsiveness and education events, thereby enhancing NK cell fitness and function, respectively. This work offers valuable insights to improve in vitro culture protocols in the clinical cultivation of NK cells for immunotherapies, such as Adoptive Cell Therapy, as well as indicating broader and nuanced roles of immune and target cell interactions in the stimulation and regulation of NK cell fitness, function, and homeostasis.
The Role of Hyaluronic Acid in the Prevention of Neoplastic Therapy-induced Oral Mucositis

Mohammed, Ali Ibrahim Mohammed ( 2022)

ABSTRACT GENERAL SUMMARY Mucositis is a common and most debilitating complication associated with cancer therapy. Approximately 30–40% of cancer patients treated with chemotherapy develop mucositis; this percentage rises to 60–85% for patients receiving conditioning regimens before hematopoietic stem cell transplantation (HSCT) and can increase to almost 100% for head and neck cancer (HNC) patients receiving radiotherapy concomitant with chemotherapy. The condition affects the entire alimentary canal from the mouth to the anus, causing ulceration and severe pain in both the oral cavity and intestinal tract. The consequences of mucositis are far reaching and can lead to a delay or even cessation of otherwise durable cancer treatment and negatively impacting the benefits of chemoradiation. Mucositis is not only an important driver of patients’ symptoms and infection risk, but its onset is also associated with poor health outcomes, the use of health resources, and incremental costs. Given that mucositis is ultimately a predictable and potentially preventable condition, this project aimed at optimising a pre-clinical model of mucositis and testing the protective effect of a natural compound on chemotherapy-induced mucosal injury. Chapter 1 discusses the generalities of mucositis, its causative agents and risk factors, its pathophysiology diagnosis, and management. After recalling basic concepts of cancer treatment (causative factor) and mucosal structure (target tissue), in this chapter I explain the clinical relevance and economic implications of mucositis. A large section is devoted to the pathophysiology of mucositis as this represents the starting point for the development of novel mechanism-based preventative and therapeutic strategies. The chapter then focuses on oral mucositis and, in this context, types of assessment and scoring scales are extensively discussed. Current management strategies of mucositis are scrutinised in the final part of Chapter 1. Despite its clinically devastating consequences, there is currently little to offer patients in the way of effective treatment to prevent or mitigate mucositis, and this provided the rationale for developing the aims of this project. General materials and methods are detailed in Chapter 2. The experimental design included in vitro experiments using an oxidative stress-induced model of human oral mucosal injury and an in vivo dual murine model of 5-FU-induced oral/intestinal mucositis. Formulations of hyaluronic acid (HA) tested in these models included Mucosamin, cross-linked (xl-), and non-crosslinked high molecular weight HA (H-MW-HA). Cell lines, culture conditions, morphological, functional and molecular assays (e.g. cell viability, cytotoxicity, and proliferation; intracellular ROS production, superoxide dismutase enzyme activity) are described. For the animal study, methodology for clinical, histopathological and morphometric assessment of oral and intestinal mucositis is reported in detail. Specific materials and methods are additionally described in the relevant chapters. In the experiments described in Chapter 3, we aimed to develop and characterise an in vitro model of oral mucosal injury that could mimic the initial events that trigger oral mucositis. Since the molecular mechanisms underlying chemotherapy and radiotherapy-induced oral mucositis involve the production of ROS at early stages and consequent activation of oxidative stress pathways, we established an oxidative stress model on human oral keratinocytes cultures whereby were able to identify optimum concentrations of hydrogen peroxide and incubation period for each of the cell line tested. Such a model allowed the screening of potentially xxx drugs reported later in this thesis. In Chapter 4, the biocompatibility of several hyaluronic acid derivatives in our in vitro cell model system was assessed through an extensive series of experiments. We defined the optimal concentrations of the following compounds: Mucosamin (1%, 5%, 7% and 10% v/v), native high molecular weight HA (H-MW-HA; 0.01%, 0.03%, 0.05%, 0.07% and 0.1% w/v), and cross-linked (xl-) HA (0.01% v/v of xl-HA 5/5, xl-HA 30/30, and xl-HA 100/100). Oral keratinocytes were incubated for 24, 48, and 72 hours in the presence of these HA products and a dose-response curve was developed. All HA compounds tested significantly promoted oral epithelial cell proliferation compared to control, except for Mucosamin; this commercial preparation did not affect cell growth at concentrations of up to 5% (v/v) and was cytotoxic at higher concentrations. Drawing on the results of the pilot experiments described in the previous chapters, in Chapter 5 we tested the protective effects of HA in a model of oxidative stress-induced oral mucosal injury. OKF6 cells were incubated with 400 uM (IC50 value) hydrogen peroxide (H2O2) and the effects of Mucosamin, H-MW-HA, Xl-HA 5/5, Xl-HA 30/30, and Xl-HA 100/100 were tested. While all HA compounds could attenuate to some extent the detrimental effects of ROS, the most marked effects were obtained with H-MW-HA. Specifically, pre- and then co-incubation of OKF6 cells with 0.01 % (w/v) H-MW-HA for 24 hours resulted in a sizeable increase in cell viability when compared to H2O2- treated cells. Remarkably, H-MW-HA also reduced the intracellular level of H2O2-induced ROS production, as measured by the ability of cells to oxidize CM-H2DCFDA. Hence, H-MW-HA at 0.01 % (w/v) was selected for the animal study. Chapter 6 describes a detailed set of experiments to show that HA prevents oral and intestinal mucositis induced by chemotherapy in vivo. To overcome the limitations of current models, whereby oral and intestinal mucositis are studied separately using different, organ-specific models, we first characterized a pre-clinical dual (oral and intestinal) murine model of mucositis by using intravenous 5-FU injections (50 mg/kg) every 48 hours for 2 weeks. In the test group, the mice were pre-treated one day prior to the initial 5-FU treatment and then daily thereafter with high molecular weight HA (H-MW-HA) (0.01 % w/v) in drinking water. Mice were monitored clinically for weight loss, diarrhea (as a surrogate of intestinal injury), and incidence and extent of oral mucositis. Microscopically, histomorphometric analyses of the tongue and intestinal tissues were conducted. The results strongly indicated that H-MW-HA prevented 5-FU-induced damage to the intestinal mucosa and tongue epithelium. We also demonstrated that H-MW-HA enhanced the activity of the SOD enzyme in the blood serum of 5-FU treated mice. This thesis comes to a conclusion with Chapter 7, where our results are discussed in light of current literature. These results reported in this thesis provide an experimental rationale to develop a novel HA-derived treatment as a therapeutic agent to protect against oral and intestinal mucositis associated with chemotherapy in cancer patients. We note that systemic administration of HA as a preventive or therapeutic tool in oral and intestinal mucositis may have profound clinical implications in patients, such as potential interference with cancer treatment, that require further elucidation. Future studies will address this important aspect of our research.
Characterising the consequences of heterozygous CASP3 deletion in colorectal cancer

Elliott, Meg Jillian ( 2022)

Caspase-3 is a cysteine protease predominately known for its role in apoptosis. Emerging evidence is highlighting alternative roles of caspase-3 in processes such as cell cycle, differentiation and migration. Genome sequencing projects have revealed the genomic landscape of human colorectal cancer (CRC) and identified recurrent heterozygous CASP3 deletions in ~10% of patients with non-hypermutated CRC. However, the contribution of CASP3 loss to CRC development or progression is unclear. This thesis aimed to define outcomes and significance of heterozygous CASP3 in CRC. Heterozygous CASP3 gene deletion in CRC patients was associated with late-stage disease and poor overall and recurrence free survival of stage II/III patients. Heterozygous CASP3 knock-out in CRC cell lines resulted in increased tumour cell viability and proliferation, but not significant or consistent changes in dead cell populations. Heterozygous Casp3 in the ApcMin/+ mouse model caused an increase in polyp and ACF number in the colon and decreased overall survival. RNA sequencing analysis of CASP3+/+ and CASP3+/- CRC cell lines revealed robust changes in gene expression, with CASP3 loss leading to upregulation of genes related to ribosome biogenesis. Ribosome biogenesis is a limiting factor for cell growth suggesting a mechanistic basis for the role of CASP3 loss in promoting intestinal tumour growth.
The role of IL-11 signaling in glioblastoma progression

Stuart, Sarah Florence ( 2022)

Glioblastoma is the most common and lethal brain tumour in adults with a mean survival rate of only 12-15 months with current treatment. The microenvironment of a tumour is becoming increasingly more important to current research, with many findings suggesting that the transcription factors driving oncogenic processes are more often due to cytokine stimulation than gene mutation. There have been multiple signalling molecules and corresponding receptors identified as key role-players in the development of glioblastoma, its severity and ability to evade treatment. Cytokines are molecules that initiate and mediate a range of cellular activities essential to the homeostasis of a heathy person but also to tumour growth, invasion and survival. This includes the critical growth factors and cytokines that activate signalling pathways controlling many pro-oncogenic cellular functions. The interleukin-11 (IL-11) cytokine has become increasingly recognised as a driver of the pathogenesis of a wide range of cancers, however, very little is known regarding its role in glioblastoma. Considering this, we hypothesized that IL-11 would contribute to glioblastoma cell viability, migration, invasion and overall tumour progression. We initially identified that IL-11 and its receptor (IL-11RA) inversely correlate with tumour grade and glioblastoma survival. To study the role of IL-11 in glioblastoma, we next determined the expression of endogenous IL-11RA in a range of cell lines and transfected those expressing very little of the gene with the IL-11RA (cell lines #20 and #28). Proteomic analysis was conducted to reveal changes in protein expression after transfection. A large number of proteins involved in proliferation, migration and invasion were seen to be upregulated in the IL-11RA transfected cells. Indeed, the IL-11RA transfected cells displayed significantly greater growth, migration and invasion in proliferation, wound healing, transwell and spheroid invasion assays. This was reversed with IL-11RA knockdown. The proteomic analysis also highlighted the upregulation of proteins involved in metabolism, particularly glutaminolysis and inhibition of apoptosis. Metabolomic analysis revealed the IL-11RA transfected cells displayed increased levels of glutamine oxidation, as well as increased proliferation and survival of these cells in conditions of depleted glucose or glutamine. Similarly, IL-11RA transfected cells displayed no significant difference in invasion rate in the presence or absence of glucose, when glutamine was available. Alternatively, blocking both glucose and glutamine metabolism with a number of drugs significantly reduced the proliferation, migration and invasion of these cells. Our findings suggest that the IL-11RA transfected cells are able to utilise alternative metabolites such as glutamine, in the absence of glucose, in order to proliferate, migrate and survive. Overall, the results of this thesis suggest that the IL-11RA plays an important role in proliferation, migration, invasion, survival and metabolism in glioblastoma.
Identification of compounds to inhibit the stress transcription factor, Heat Shock Factor 1, as novel anti-cancer agents

Polidano, Joseph ( 2022)

Heat Shock Factor 1 (HSF1) is the master stress transcription factor which enables cells to overcome and survive otherwise lethal stresses, including but not limited to heat, acidosis, hypoxia, genomic aberrations and oxidative stress. HSF1 orchestrates these survival processes primarily through activation of the heat shock response (HSR) pathway, which enables cells to upregulate the expression of heat shock proteins (HSPs) and other pro-survival targets. While central to several pathophysiological conditions, in cancer cells, HSF1 is intimately linked with cancer initiation, progression and metastasis. As cancer cells must overcome numerous stress insults and exhibit elevated biosynthetic demands when compared to non-cancer cells, HSF1 has been identified as an essential factor for the survival of advanced cancer cells. Consequently, HSF1 has emerged as a major therapeutic target, which has motivated numerous research groups to undertake programmes that seek to develop small molecule-based HSF1 inhibitors. To this end, these have been largely unsuccessful, as a compound capable of direct and selective HSF1 inhibition remains to be fully validated. While various compounds which can modulate the HSF1 pathway have been identified, these have been shown to impact the HSF1 pathway only in an indirect manner or via an uncharacterised mechanism of action. In this thesis, an alternative approach to targeting HSF1 has been taken through the development of rationally designed peptide inhibitors which mimic the protein-protein interactions that are essential for HSF1 functionality. Using biophysical approaches, six rationally designed novel peptide sequences were characterised for their ability to inhibit the activation and functionality of HSF1. Through this work, a lead peptide inhibitor (HiPe4) was shown to interact with HSF1 in a pulldown assay and disrupt HSF1 binding to HSE-DNA oligonucleotides (IC50 60.1 uM). Through trial and incorporation of protein transduction domains/cell permeability sequences, it was also demonstrated that HiPe4 blocks HSF1 activity in cells by reducing levels of HSF1 regulated HSPs. Further experiments also elucidated that HiPe4 impairs activation of the HSR in three cell lines and reduces the viability of cancer cells. In contrast, the viability of a non-oncogenic cell line model was shown to be unaffected following treatment with HiPe4, suggesting that cancer cells exhibiting elevated expression and activation of HSF1 face increased susceptibility to HiPe4 toxicity. HiPe4 was demonstrated to significantly deplete levels of HSF1 in three cancer cell lines, pointing to a novel joint mechanism of action not previously demonstrated by peptides. In summary, the findings of this study present a significant turning point in the search for HSF1 inhibitors for use as a cancer treatment strategy. It is the first study demonstrating inhibition of HSF1 functionality and activity using biophysical, biochemical and molecular approaches. Furthermore, it is one of only two inhibitory compounds which have been shown to inhibit HSF1 activity without activation of the HSR. Therefore, this work is likely to provide a viable starting point for the development of a therapeutic HSF1 inhibitor, while also providing significant insight into the design approaches which should be taken when targeting other ‘undruggable’ molecular targets in the future.
Investigating the contribution of the cysteine protease legumain to oral squamous cell carcinoma progression

Anderson, Bethany ( 2022)

Oral cancer is the eighth most common form of cancer worldwide. The mechanism behind oral carcinogenesis is poorly understood and current treatment strategies are ineffective. Survival rates drop significantly once metastasis develops, and predicting which cancers will go on to metastasise is currently not possible. Further mechanistic studies are thus required to improve patient outcomes. Legumain is a cysteine protease that has previously been reported to contribute to the carcinogenesis of several cancers. We have recently demonstrated that legumain contributes to orofacial pain in mouse models of oral cancer, via cleavage of protease-activated receptor-2 (PAR2). We hypothesised that legumain may also contribute to other features of oral carcinogenesis. Using the legumain-selective activity-based probe LE28, we measured legumain activity in human oral squamous cell carcinomas (OSCC) and patient-matched normal oral mucosa. We found that legumain activity was strongly upregulated in tumours of all patients examined, compared to normal oral mucosa. We further validated this finding using tumour samples from two murine models of oral cancer: orthotopic xenografts of HSC-3 human OSCC cells and 4NQO carcinogen-induced OSCC. Immunohistological analysis of samples from these mice revealed that legumain is expressed in both tumour and stromal cells and is predominantly located at the invasive edge of tumours. We examined legumain levels in a panel of human OSCC cell lines, as well as in macrophages primed by conditioned media (CM) from these cells. Compared to normal oral keratinocytes, cultured human oral cancer cells express higher levels of active and total legumain, as do macrophages exposed to tumour CM. We next analysed the polarisation of CM-exposed macrophages by flow cytometry and qPCR and found that these cells were polarised towards a classical M1 activation state. Using CRISPR-Cas9 technology, we generated legumain-deficient HSC-3 and SCC-9 cells and analysed them for features of carcinogenesis in vitro and in vivo. Compared to wild-type cells, legumain knockout cells exhibited reduced proliferation. When legumain-deficient HSC-3 cells were inoculated subcutaneously in mice, the resulting tumours exhibited >90% reduction in volume and less vascularisation compared to tumours generated from wild-type cells. Thus, it appears that tumour-derived legumain promotes oral cancer growth. We next used unbiased bottom-up proteomics analysis to identify changes in protein expression between wild-type and legumain knockout HSC-3 cells. Pathways involved in energy metabolism were highly dysregulated in legumain knockout cells compared to wild-type cells. A cancer signalling phospho protein array on subcutaneous xenograft HSC-3 tumours revealed changes in metabolic pathways in legumain knockout tumours compared to wild-type tumours. Collectively, these results demonstrate that legumain is highly activated in the oral cancer microenvironment and may contribute to tumour growth and invasion. Future studies aim to distinguish the proteolytic and non-proteolytic functions of legumain in the oral cancer microenvironment as well as its cell-specific roles in oral cancer progression. Ultimately, we aim to determine whether legumain inhibitors will have therapeutic value, resulting in improved outcomes for oral cancer patients.
A Longitudinal Feasibility Study Exploring Cancer-Related Cognitive Impairment in Patients with Newly Diagnosed Aggressive Lymphoma

Gates, Priscilla Ruth ( 2022)

Background Cancer-related cognitive impairment (CRCI) is a recognised adverse consequence of cancer and its treatment. Published evidence speaks to the challenges of prospective, longitudinal CRCI studies, and, to date, the feasibility of collecting data on cognition in this patient cohort has not been evaluated. This study assessed the feasibility of collecting longitudinal data on cognition in patients with newly diagnosed, aggressive lymphoma undergoing standard therapy with curative intent via self-report, neuropsychological assessment, peripheral markers of inflammation and neuroimaging. An exploration and description of patterns of cancer-related cognitive impairment over the course of treatment and recovery was also undertaken. Methods Eligible participants completed comprehensive assessments pre-chemotherapy, mid-chemotherapy, and six to eight weeks post-chemotherapy completion. Participants completed neuropsychological tests and self-report measures including cognitive and other patient-reported outcome measures. Full blood examination counts were used to calculate blood cell-based inflammatory markers at all three time points. Neuroimaging was undertaken in a subset of patients; 18F-FDG-PET/CT scans were undertaken at all three time points; MRI scans were undertaken pre-chemotherapy and six to eight weeks post-chemotherapy completion. Results 30 of 33 eligible participants (91%, 95% CI: 76%, 97%) were recruited over 10 months. The recruitment rate was 3 patients/month (95% CI: 2.0, 4.3 patients/month). Mean age was 57 years (SD=17 years) and 16/30 (53%) were male. The neuroimaging sub-study was optional; 11/30 participants (37%) were eligible to take part, and all agreed. Retention and compliance with all assessments was high at all time points. Only one patient was withdrawn from the study due to disease progression. On average, the study sample performed worse on neuropsychological tests compared with population norms and healthy controls pre-chemotherapy. Notable changes from pre-chemotherapy at follow-up were observed on some neuropsychological tests and self-report measures including improvements in verbal fluency as assessed by the Controlled Oral Word Association Test and verbal memory as assessed by the Hopkins Verbal Learning Test; deterioration in perceived cognitive impairment and abilities as assessed by the Functional Assessment of Cancer Therapy-Cognitive Function measure; improvements in emotional wellbeing as assessed by the Functional Assessment of Cancer Therapy-General measure; worsening of fatigue as assessed by the Functional Assessment of Chronic Illness Therapy-Fatigue measure; and improvements in anxious symptomatology as assessed by the Patient-Reported Outcome Measurement Information System Anxiety 7a measure. Most associations between a global deficit score calculated based on responses to neuropsychological tests and self-report measures were trivial. Most associations between perceived cognitive impairment and abilities, and other self-report measures were small- to large-sized. Conclusion Findings from this PhD demonstrate that it is feasible to longitudinally assess cognitive status and impairment in people with newly diagnosed aggressive lymphoma during their initial treatment and recovery. Compared with population norms and healthy controls, mean neuropsychological test scores pre-chemotherapy indicate cognitive impairments may precede treatment for aggressive lymphoma. Estimates of change in cognitive function based on neuropsychological tests provided evidence of improvement in verbal fluency and memory. Conversely, estimates of change in cognitive function based on self-report measures provided evidence of deterioration in perceived cognitive impairment and abilities. Most associations between objective tests and subjective measures were trivial. This PhD has generated knowledge to inform the development of future research to test efficacy of novel interventions to improve patient experiences and cognitive outcomes.
Unplanned Emergency Department Presentations by Cancer Patients Receiving Systemic Anti-Cancer Therapy

Dufton, Polly Hypatia ( 2021)

Background In Australia, emergency department presentations have been found to be common for people affected by cancer, and even more so for patients receiving systemic anti-cancer therapy. These presentations are associated with lengthy admissions, treatment dose reductions, and treatment delays. The emergency department often provides cancer patients with access to unscheduled medical care and to specialist cancer advice for symptoms they may experience. At a health system level, there has been an increasing number of people presenting to the emergency department, and subsequently, a need to reduce emergency department presentations in people who are not critically unwell. The need to deliver safe and high-quality care to mitigate unplanned emergency department presentations has received considerable attention in the international health care community. However, substantial work is needed to develop models of care to reduce emergency department presentations that may be potentially avoidable. The aim of this thesis is to understand why patients who receive systemic anti-cancer therapy make unplanned emergency department presentations. This understanding can support the development of robust evidence to inform future research and strengthen the development of models of care to support patients receiving systemic anti-cancer therapy. Methods This study used a convergent, mixed-methods research design, underpinned by Andersen’s behavioural model of health service use, to explore patterns of health service use and contextual, individual and health behaviours that influence cancer patients’ decisions to engage with health services. Study 1 was a retrospective cohort study of unplanned emergency department presentations that occurred within 28 days of receiving systemic anti-cancer therapy in the outpatient setting between December 2014 and November 2017. This study explored patterns of, and risk factors associated with, making an emergency department presentation to one large public tertiary hospital in Melbourne, Australia. Study 2 was a systematic review that explored methods reported in published literature investigating the incidence of unplanned emergency department presentations by patients receiving systemic anti-cancer therapy. Study 3 was an explanatory sequential mixed methods study that explored the contextual, individual and health behaviours that influenced the decision to engage with health services during systemic anti-cancer therapy. Thematic analysis of textual data was undertaken using template analysis. Participants in Study 3 were recruited from October 2016 to March 2017. Results Study 1 identified that 45% (n = 1,182) of cancer patients made a total of 2,310 emergency department presentations within 28 days of receiving systemic anti-cancer therapy in the outpatient setting. More than half (58%, n = 1,341) of the emergency department presentations resulted in patient admission for further care. Patients born outside of Australia and those diagnosed with head and neck, upper gastrointestinal, colorectal, lung, skin or breast cancer were identified as having a significantly increased risk of making an emergency department presentation. Study 2 comprised a total of 21 papers, and overall, the risk of bias was assessed as moderate. There was substantial clinical and methodological heterogeneity in the studies included in the systematic review, such as the population and study setting, the period for which the participants were observed, and in the reporting of participants who were lost to follow-up. In Study 3, 16 of 58 (28%) participants presented to the emergency department over the 6-month study period. Four overarching themes were generated from the 19 semi-structured interviews involving: 1) missed opportunities for preparation and support; 2) the importance of symptoms and need for care; 3) navigating the system and preference for care provider; and 4) enablers and barriers to accessing care. An additional two integrative themes were generated from the semi-structured interviews: 1) the unknown and unpredictable; and 2) individual health beliefs and behaviours. Integrated findings found that perceived need was a stronger driver of emergency department presentation than socio-demographics factors. Participants experienced a combination of enablers and barriers to accessing other sources of health care that may have influenced their need to present to the emergency department; these included that the availability of appointments with their general practitioner was a barrier, and the presence of social supports was an enabler. Based on findings from the literature review and this PhD, a new conceptual framework for emergency department use by patients receiving systemic anti-cancer therapy is offered. Conclusions The knowledge generated in this PhD contributes novel and important insight as to why patients receiving systemic anti-cancer therapy present to the emergency department. Studies have shown that some emergency department presentations are avoidable; the findings from this PhD can assist in developing new strategies to mitigate these avoidable presentations and can inform future healthcare policy, organisational planning, and clinical practice. This PhD has provided robust evidence for future research and the need for sophisticated research designs that reflect the complexity of cancer patients and the health care system.
Structure and dynamic studies of AAA+ ATPase p97 molecular machine

Valimehr, Sepideh ( 2021)

The superfamily of AAA+ ATPases (ATPases Associated with diverse cellular Activities) is a conserved and vital family of proteins in all living organisms. AAA+ ATPases hydrolyse ATP and convert the chemical energy to mechanical force. These mechanochemical enzymes perform a variety of cellular functions. Members of this superfamily most commonly assemble as homohexamers, the functional form of these macromolecules. A prominent member of this family is the protein named p97, or VCP. In all eukaryotic cells, p97 is one of the most abundant cytosolic proteins where it plays a central role in organelle and protein homeostasis in ubiquitin-dependent and independent pathways. It acts as an unfoldase to unfold its substrate. To play a role as a multifunctional protein in the cell, p97 interacts with a vast number of cofactors. The monomer of the homohexameric AAA ATPase p97 contains the cofactor or substrate-binding domain (N) and two ATPase domains (D1 and D2), and a disordered C terminal region. A single amino acid substitution in this protein causes neurodegenerative diseases, and an increased expression level of p97 has been identified in several cancers. It also plays a role in viral infections. Therefore, it is an important therapeutic protein. The structure of p97 has been widely investigated, and the high-resolution X-ray and cryo-EM structural information of full-length p97 became available in recent decades. However, structural knowledge and detailed insight into the disease-associated mutant, and the interaction of p97 with cofactors have limited our understanding of how disease-associated mutation and cofactors regulate or modify the functions of p97. The work presented here attempts to characterise how one of the most clinically relevant mutations of p97 (R155P) and one of the best-characterized cofactors (p47) modify the dynamic properties of p97. My approach has been to combine a series of structural and biochemical techniques such as single-particle cryo-EM, HDX-MS, X-link MS, and 19F NMR. In chapter 2, I describe how I have determined the high-resolution cryo-EM structures of the R155P and wild-type p97 in interaction with ATPgS. In chapter 3, using a combination of the structural and dynamical approaches, I show that the mutant stabilizes the up conformation of the N domain and affects the intra and inter subunit communication of the p97. During these studies, I have captured the wild-type p97 protein in a novel conformation and identified that the conformation of the N domain is not solely determined by the nucleotide occupancy of the D1 and D2 domains. In chapter 4, I present evidence I collected using SEC-MALS, AUC, and SPR that support the fact that the cofactor p47 is a monomer in solution. This analysis addresses controversy in the field. Using HDX-MS and X-link MS, I have shown changes in the conformations and interacting domains of both p97 and p47 upon complex formation with and without adding ATPgS. In chapter 5, I present preliminary studies on how 19F NMR would be an appropriate approach to study the dynamic flexibility of the p97 N domain. Together, these studies provide additional knowledge to the molecular mechanisms that regulate p97 function, our work will lead to progress in using hybrid structural biology techniques to understand the mechanism of macromolecules’ function.
Developing novel methods of infection surveillance in haematology-oncology patients and implications for health policy

Valentine, Jake Christopher ( 2021)

Healthcare-associated and opportunistic infections are a leading cause of mortality, morbidity, and increased healthcare costs in haematology-oncology patients. Surveillance is recognised as the cornerstone of infection prevention to guide clinical decision making and to monitor quality improvement. The utility of current case ascertainment methods is poorly delineated in patients with underlying malignancy. Administrative data comprise a standardised ontology to classify disease with the potential to support large scale infection surveillance programmes; however, it is unclear if administrative data can support surveillance activities in high-risk settings. The overall aims of this thesis were to: (i) present and argue the case for novel case ascertainment methods; (ii) develop and apply a methodology using administrative data to identify infection in haematology-oncology patients, and determine the classification performance and healthcare funding implications of these data in line with current health policy; and (iii) establish and evaluate a hospital-wide linked dataset integrating multiple data sources, together with administrative data, to achieve maximal performance for automated surveillance in patients with haematological malignancies. Methods include a systematic review to describe the scope of existing surveillance methods among haematology-oncology units, analysis of continuous statewide surveillance datasets and hospital-level administrative data extracts, performance evaluation of administrative data to classify infection and simulation of pay-for-performance funding methodology in a cancer casemix, and development of a hospital-wide linked dataset to identify discrete data combination yielding highest performance for automated infection surveillance. The thesis findings demonstrate significant heterogeneity in existing infection monitoring methods among haematology-oncology patients. Estimates of the burden of disease in a predefined haematology-oncology population relative to a statewide cohort were determined, together with longitudinal trends in incidence over time. Administrative data show to be a feasible alternative to current surveillance data to enable standardised comparison of intra- and interhospital infection epidemiology in patients with underlying malignancy, however, at the expense of poor-to-moderate classification performance associated with significant shortfalls in hospital remuneration. Linkage of administrative data with microbiology, histopathology, and antimicrobial-dispensing data according to specific data combinations demonstrated improvements in classification performance for discrete opportunistic and healthcare-associated infections in patients with haematological malignancy. It was demonstrated that although administrative data enable standardised comparison of infection epidemiology, these data are an unreliable proxy for infection surveillance in Australian haematology-oncology units. Refinements to current pay-for-performance funding specifications are necessary before administrative data can reliably be used as quality improvement measures in a cancer casemix. This thesis posits data linkage as an efficient means to optimise the utility of administrative data, together with hospital-level datasets, to support an automated surveillance strategy in haematology-oncology patients. Future research agenda are outlined regarding the evaluation of electronic medical record data and other codified nomenclature to support electronic surveillance and quality improvement monitoring.

Minerva Elements Records

Permanent URI for this collection

Filters

Date

Author

Subject

Type

Settings

Sort By

Results per page

Statistics

Citations

Search Results