Medical Biology - Theses
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ItemStructural studies of the mitochondrial import pathway(University of Melbourne, 2008)
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ItemCD40L expression by dendritic cells : a key to helper-independent killer T cell independent(University of Melbourne, 2008)
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ItemModelling the multistep pathogenesis of T-cell acute lymphoblastic leukaemia( 2019)T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive T-cell malignancy that is frequently caused by the overexpression of oncogenic transcription factors. Like many cancers, T-ALL is a heterogeneous disease, with the acquisition of many genetic alterations resulting in multiple clones that contribute to cancer progression. This poses a challenge for therapeutic intervention, as different clones within a tumour can possess different genetic signature and thus possess different levels of sensitivity to therapeutic strategies. Therefore, gaining a better understanding of how these clones arise, is crucial to developing more targeted therapies aimed at these cells. LMO2 is a transcription factor that is overexpressed in approximately 9% of T-ALL cases. The CD2-Lmo2 transgenic mouse model overexpress Lmo2 in the thymus, resulting in a developmental block at the DN3 stage of T-cell development, and subsequent development into T-ALL after a long latency (approximately 10 months). Using these mice, our laboratory has shown Lmo2 confers self-renewal capacity to these developmentally blocked DN3 thymocytes many months before the overt presentation of T-ALL. These self-renewing DN3 thymocytes were termed pre-CSCs due to their ability to self-renew, their capacity to still develop into mature functional T-cells, and not initiate leukaemia for many months when transplanted into recipient mice. This thesis will focus on gaining a better understanding of the multi-step pathogenesis of Lmo2 induced murine T-ALL development. In Chapter Three, we explore the role cellular competition for thymic niche space and signals plays in the Lmo2 induced T-cell developmental block, and Lmo2 induced T-ALL. Using competitive bone marrow transplantation experiments we show that the presence of WT thymic progenitors in the thymus severely hinders the development of Lmo2 transgenic thymic progenitors past the DN2 stage of T-cell development, and their subsequent development into T-ALL. Interestingly, we found that overexpression of Bcl2 in Lmo2 transgenic thymocytes severely abrogated the self-renewal capacity of Lmo2 transgenic thymocytes, and hindered their development into T-ALL. Furthermore, we show that Lmo2 downregulates Il7r in DN2 thymocytes. In Chapter Four, we crossed CD2-Lmo2 transgenic mice with CD2-Il7r transgenic mice to create the CD2-Lmo2;CD2-Il7r double-transgenic mouse line, to investigate the role of Il7r overexpression in the Lmo2 induced developmental block. We found that overexpression of Il7r in Lmo2 transgenic thymocytes does not alleviate the Lmo2 induced DN2, or DN3 developmental block, but does increase the engraftment potential of Lmo2 transgenic DN3 thymocytes. Surprisingly, despite the increase in engraftment potential, Il7r overexpression in Lmo2 transgenic thymocytes resulted in a delay in T-ALL induced death, however Il7r overexpression promoted an immature T-ALL immunophenotype. In Chapter Five, we generated an inducible Lmo2 knockin mouse model in which Lmo2 expression can be inhibited by Dox administration. Using this mouse line, we show that while Lmo2 is still required for the self-renewal of Lmo2 transgenic DN3 thymocytes, Lmo2 is not required for T-ALL maintenance in the majority of Lmo2-Induced T-ALLs.
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ItemInvestigating the Genetic Causes of Primary Immunodeficiency and Autoimmunity( 2019)Primary Immunodeficiencies (PIDs) are a heterogeneous collection of several hundred disorders, that have in common deficient or dysregulated immunity. This leads to an increased susceptibility to infections, autoimmune disease, or to uncontrolled inflammation; in some PIDs there may be features of all three components. Understanding the pathogenesis of PIDs has led to insights into the immune system broadly, and this knowledge has aided improvements in the treatment of many more common infective and inflammatory diseases. Many PIDs are monogenic, and advances in technologies that enable the interrogation of the genetic basis of these conditions, have led to a steep increase in the number of diseases now recognized as PIDs. In this project we aimed to establish a cohort of individuals with the most common form of PID, Predominantly Antibody Deficiency (PAD). We sought to characterize the diagnostic and clinical features of these individuals, and apply genomic sequencing methods for more precise diagnoses, as well as to identify new genetic etiologies of PAD. One of the most striking findings regarding the clinical features of the patient cohort, was the morbidity associated with PADs. In particular we observed very frequent complications of immune dysregulation, that manifest as autoimmune disease, particularly affecting the haematological system as cytopenias, or gastrointestinal tract as enteropathy, or malignancies. These complications are challenging to manage in the setting of PID, hence understanding the mechanism of disease is crucial to improving outcomes. In a group of PAD patients we identified monogenic causes of disease, that led to some individuals receiving precision treatments. In the realm of gene discovery, we identified several novel genetic variants, two in genes that had not previously been recognized as disease-causing in PAD. The first, NFKB1, is now recognized as the most common genetic etiology of PAD. The importance of NF-kappaB signaling and its regulation was further highlighted by the discovery of other disease-causing variants in the NF-kappaB pathway. The effect of NFKB2 mutations on the immune response was investigated in a kindred with two sisters affected by PAD, who demonstrated disparate clinical and immunological features. Further studies were performed in a mouse model of NF-kappaB2 deficiency, whereby B cell and CD8+ T cell behaviours were studied in a quantitative manner, and revealed the cell-type specific effect of loss of this component on the adaptive immune response. Finally, an entirely new monogenic PID was identified. In a young man with a clinical diagnosis of PAD, complicated by autoimmune disease, a homozygous, frameshift mutation in NFKBID was identified. The mutation was demonstrated to cause loss of expression of IkappaBNS, an inhibitor of the NF-kappaB pathway, and with that, dysregulated NF-kappaB signaling. The finding of a second patient with PAD and compound heterozygous variants in NFKBID has added further evidence that this is a bona-fide novel PID.
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ItemAspartic proteases and their potential for transmission blocking strategies( 2019)Sexual stage development in Plasmodium spp. is essential for transmission through the mosquito and to the human host. It represents objects to study a broad range of biological processes, including stage conversion and parasite/host co-adaptation. After the bloodmeal, male and female gametes emerge from intracellular gametocytes and zygote formation follows fertilization. Ookinetes develop from the zygote and traverse through the midgut epithelial cell layer to the basal lamina side of outer wall and develop into oocysts, the only parasite developmental stage that grows extracellularly and this growth and development creates thousands of sporozoites. Once fully developed and egressed, these sporozoites are released into the mosquito hemocoel and they migrate to the salivary gland ready to infect next mammalian host and continue their life cycle. This sexual stage also represents a major bottleneck during the life cycle of Plasmodium as, in mosquito midgut, parasites have to persevere for up to 24 hours outside host cell, exposed themselves to various risk factors such as components of human immune system included within bloodmeal, natural midgut microbial flora in mosquito midgut, and mosquito innate immune system. This exposure can lead up to an approximate 300-fold decrease in parasite survivability during the transmission to mosquito. Due to this unique feature, sexual stage is prime target for transmission blocking intervention strategies aimed to inhibit spread of the disease by the mosquito. Protease enzymes are essential during many steps of malaria parasite development in the blood and transmission stages and an important group of these enzymes are the plasmepsins, of which there are 10 in Plasmodium acting at various points through the life cycle. So far, only 4 plasmepsins are identified to be involved in critical processes and required for transmission. Firstly, plasmepsin VI is highly expressed during sexual stages and was previously shown to be involved in sporozoite development in P. berghei. Secondly, plasmepsin VIII is expressed in mature sporozoite and responsible for sporozoite motility in P. berghei. Finally, PMIX and X are found to be essential in both blood and mosquito stages, making them stand out as promising drug targets. In this study, we attempted to determine the biological functions of plasmepsin VI, IX, and X during transmission of malaria parasites. We found that plasmepsin VI is required for transmission of P. falciparum and might plays an important role in sporozoite egress process instead of sporozoite development as observed in P. berghei. We also found that our dual inhibitor that target both plasmepsin IX and X is able to block the transmission of P. falciparum to mosquito while another antimalaria compound that target only plasmepsin X is enough to block transmission of P. berghei from mouse to mosquito suggesting that both plasmepsin IX and X are essential for transmission. Taken together, our data has identified 3 plasmepsins that play important roles in sexual stage of malaria parasites and more works are needed in order to determine the mechanism of action of these 3 proteases.
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ItemControl of the Intrinsic Pathway of Apoptosis( 2019)Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.
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ItemIntracellular competition regulates B lymphocyte differentiation( 2019)The production of antibodies, with their potential to recognise unique targets and prevent repeat infections, is an important aspect of immune health. In order to generate free antibodies, the cells responsible, B cells, must undergo a differentiation step to transform from lymphoblast to antibody secreting cell (ASC). This differentiation step prevents further antibody modifications and hence the timing for optimal immunity requires a delicate balance between expanding useful clones and providing early protection. How differentiation is controlled to achieve this balance for an effective immune response is of great interest. In this study, the progression from naive B cells to ASC was investigated in the context of an emerging model for competing cell fates. By this model, alternate cell fates, such as division, death and differentiation, are pursued independently in individual cells but are in competition such that events which occur earlier prevent those that require more time from being observed. Evaluation and testing of this model requires careful measurement of distributions of times to fates which is only possible with single cell fate tracking. Here I have developed and applied methods for live cell imaging and analysis for assessing and evaluating cell fate changes over time. Using these methods, several modes of regulating differentiation times were revealed. Low levels of stimulation through CD40 produced a greater proportion of antibody secreting cells per generation as division is slowed and more time is allowed for differentiation, consistent with competing cell fates. A second mechanism was found where increasing division numbers directly reduced the amount of time required for cells to differentiate, without modulating division times, ensuring the natural development of ASC during the ongoing immune response. A direct method of uncensoring was explored where cell cycle inhibitors were used to prevent division, with the hypothesis that more cells would go on to differentiate in the absence of competition. Various inhibitors were assessed for their suitability to this task, and a panel of compounds were found to be suitable for uncensoring underlying differentiation and cell death times. Findings from this study are consistent with the model of independent and competing cell fates, and significantly advance our understanding of how antibody responses are controlled and can be modelled at the cell population level.
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ItemDiagnostics and impacts of soil-transmitted helminth infections among populations from South-East Asia( 2019)Gastrointestinal parasitic infections, in particular soil-transmitted helminths (STHs), are a major cause of morbidity in tropical developing countries worldwide. Globally, infections are estimated to cause disease-related morbidity of approximately 3.4 million disability adjusted life years and an infection prevalence of around 1.5 billion people. More than 3.5 million people manifest acute clinical symptoms, although the majority of infections represent asymptomatic cases. Severe infections cause acute clinical symptoms including malnutrition, abdominal pain, diarrhoea, fever, skin conditions and iron deficiency anemia. Most importantly, long-term sequelae in pre-school and school-aged children and pregnant women can lead to stunting, wasting and an impaired cognitive development. Inadequate sanitation, impoverished conditions, poor public health systems and population overcrowding all contribute to high infection prevalence levels. STH infections occur predominantly in sub-tropical to tropical regions worldwide, such as South America, Sub-Saharan Africa and South-East Asia. Diagnosis is confirmed by microscopy-based tools (Kato Katz Thick Smear method), which is both, cost-effective and applicable in remote settings, but has significant drawbacks in terms of labour intensity and diagnostic accuracy. The World Health Organization recommends annual or biannual mass drug administration of the front-line benzimidazole drugs, albendazole and mebendazole, to reduce overall infection prevalence and disease-related morbidity. However, there is an increasing concern that ongoing mass drug administration may lead to the emergence of drug resistance. There is an urgent need for accurate and streamlined diagnosis of STH infections, in particular among subpopulations harbouring low-intensity infections in order to effectively treat populations at risk. Additionally, an improved evaluation of drug treatment efficacy including a subsequent monitoring of potentially emerging drug resistance is crucial for future studies. A global multi-factorial intervention approach is required to effectively move towards transmission interruption and ultimately eradication of STH infections. The here presented thesis has resulted in the successful development of a molecular diagnostic tool for qualitative and quantitative estimation of STH infections. A novel, semi-automated qPCR-based tool (multiplexed-tandem qPCR (MT-PCR)) was developed and validated using two cross-sectional cohorts from South-East Asia (Timor-Leste, Cambodia). The MT-PCR subsequently was used to investigate the infection prevalence and intensity in a cross-sectional cohort study of Karen ethnic pre-school and school-aged children from the Tha Song Yang district, Tak province, in Thailand. Furthermore, the effectiveness of three different preservation methods for faecal samples was compared in terms of soil-transmitted helminth infection recovery and gut microbiome characterisation. The impacts of infection on overall and gut microbial health were investigated. Thirdly, faecal DNA samples from three cohorts (Cambodia, Vietnam, Thailand) were screened for single nucleotide polymorphisms at codons Phe167Tyr, Glu198Ala and Phe200Tyr, that may be associated with the emergence of benzimidazole drug resistance. Using this amplicon sequencing approach, the variant allele frequency of the beta tubulin isotype 1 gene was analysed.
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ItemInvestigation of mammary gland development and resident macrophages by 3D and intravital imaging( 2019)The mammary gland is a fascinating organ that develops after birth and is capable of remodelling through multiple rounds of reproduction. The behaviour of mammary epithelial cells and how these interact dynamically with their environment are poorly understood. Cell morphology and arrangement can be addressed by three-dimensional (3D) confocal imaging to provide large-scale, subcellular resolution views of tissue architecture. Further insight can be gained from intravital imaging that allows direct observation of cell behaviour in vivo, but this has rarely been implemented for the normal mammary gland. Mammary ducts are embedded in adipose tissue, making in vivo imaging of mammary ducts extremely challenging. Chapter 3 provides a detailed protocol for an intravital imaging method that was adapted and optimised for the mouse mammary gland. This technique enables high-resolution, 3D intravital imaging of the mammary gland for up to twelve hours. The skin flap surgical technique was modified to expose the entire inguinal mammary gland, allowing rare accessible epithelial structures to be identified. Additional fine microdissection of connective tissue maximised the resolution of imaging. Significant measures were taken to achieve as near to physiological conditions as possible, including creating a sealed environment over the exposed tissue. Strategies used for image analysis are then discussed, including image stabilisation, cell tracking and 3D visualisation. This technique advances our ability to observe mammary cell behaviour in vivo and will enable future investigation of rare events that are spatially and temporally regulated, such as stem cell behaviour, tumour initiation and microenvironment interactions. Mammary gland morphogenesis occurs by migration of terminal end buds through the mammary fat pad. Terminal end buds are large, club-like structures comprising a cap layer and a multi-layered body that give rise to bilayered ducts. Epithelial progenitors within terminal end buds generate mature cells of ducts but how these behave and cooperate to generate the bilayer is not well understood. Chapter 4 describes the lineage-specific behaviours of terminal end bud progenitors as observed by intravital microscopy. Cap cell migration into the body was recorded at high resolution in vivo for the first time. High-dimensional image quantification of cap cell behaviour showed that most cap cells that migrate into the body die rapidly but a small proportion survive long term. Progenitors for the luminal lineages were observed to have contrasting behaviours, with hormone-sensing progenitors being highly migratory. Single cell transcriptomic analysis of terminal end buds is described, providing possible molecular drivers of the distinctive behaviour of hormone-sensing progenitors. This work provides an unprecedented view of mammary stem cell behaviour, making an important contribution to our understanding of how cellular behaviour drives organogenesis. Chapter 5 describes a previously uncharacterised population of resident intra-epithelial macrophages that were revealed by 3D confocal imaging. These cells, termed mammary ductal macrophages, are regularly positioned over the entire mammary gland at all stages of development. They do not migrate but monitor the epithelium by dendrite movement, allowing them to rapidly sense and respond to epithelial damage. Ductal macrophages proliferate in pregnancy to maintain their density on the epithelium in lactation. During involution following weaning, they rapidly phagocytose dying alveolar cells to facilitate remodelling. Breast tumour-associated macrophages are pro-tumorigenic and strongly resemble ductal macrophages, not stromal macrophages. Macrophages are emerging as important targets for breast cancer treatment, therefore, better understanding of parallels between DM function in healthy and perturbed tissue may enable development of improved cancer therapies. Finally, in Chapter 6, the presented results are summarised and their context within the field, wider implications and possible future directions are discussed. Overall, this thesis presents original research that advances our technical ability to address questions of cell dynamics in the mammary gland, provides important insights into mammary stem cell behaviour during morphogenesis, and characterises a novel tissue-resident macrophage population, finding a key role for these in mammary gland remodelling.
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ItemEstablishing pre-clinical models for advanced colorectal cancer( 2019)Colorectal cancer (CRC) is the third most common cancer worldwide, and one of the most common cancers in Australia. When detected early, there are multiple treatment options for CRC; however, patients often relapse and ultimately succumb to metastatic disease. 5-Fluorouracil (5-FU)-based chemotherapy is a common first-line treatment for CRC. However, chemotherapy response rates remain low, often due to the development of resistance, which is one of the main limitations in the management of the disease. Our understanding of the progression of CRC, and the development of new therapeutics for CRC, has been facilitated in part by animal models. Unfortunately, many murine models of CRC are adenomas, with few patient-derived models, or models of metastatic disease available. As a result, the opportunity to improve our understanding of the pathogenesis of advanced disease, or test the efficacy of novel therapeutics for advanced disease, is limited. The overarching aim of this PhD thesis was to generate new models of CRC to facilitate the study of this devastating disease. A biobank of 16 CRC patient-derived xenografts (PDXs) was successfully established in immunocompromised mice. These PDX lines recapitulated the histopathological, molecular and genetic features of the original patient tumours. These PDXs represent new pre-clinical tool that will allow for testing the efficacy of potential new therapeutics. A selection of four PDX lines underwent serial 5-FU treatment to generate a library of resistant PDXs, and their matched non-resistant chemonaive controls. The 5-FU resistant PDX tumours underwent an upregulation of the IL-11R/STAT3/Bcl-2 pathway in response to 5-FU. This suggests that IL-11 signalling is elevated in response to 5-FU to promote tumour cell survival. Thus, targeting the IL-11/IL-11R signalling may be a promising strategy to overcome chemoresistance. Finally, four different genetically-engineered mouse models (GEMMs) were established to generate a reproducible model of metastatic CRC. It was found that mutations in Apc (or dysregulation of Wnt signalling) restricted to the colonic epithelium lead to the formation of adenomas, as did the addition of mutations in Tp53. The combination of Apc mutations with Tp53 and Kras mutations lead to an earlier tumour onset, but did not result in metastasis, contrary to previous reports in the literature. It was found however, that mutations in Tp53 and Kras in the stem cell compartment, combined with dysregulation of Wnt signalling, lead to potential metastasis to the liver. However, this did not occur in 100% of the animals, and is thus not amenable to therapeutic studies. Future studies will incorporate alterations to TGFb signalling, in an effort to increase the reproducibility of metastasis. These studies highlight the lack of our understanding of the drivers required for tumour cell metastatic potential. Taken together, the research described in this thesis has led to the generation of a number of new animal models of CRC, that may be of use to future studies of the pathogenesis and treatment of this disease.