Medical Biology - Theses

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    Structural Studies of Type-I Haematopoietic cytokine receptors
    Sarson-Lawrence, Kaiseal Tane Garvey ( 2023-09)
    Haematopoiesis is a complex process by which the full complement of mature blood cells is produced from a small population of hematopoietic stem cells in the bone marrow. Cytokine signalling plays a crucial role in haematopoiesis and at least 14 different cytokines are involved in determining the fate of hematopoietic stem cells. Cytokines act on cells by binding and oligomerising cytokine receptors on the cell surface. This oligomerisation activates intracellular JAK kinases that kick off a signalling cascade resulting in a cellular response. As cytokine binding is the critical first step in receptor activation, understanding how different cytokines bind to their receptors and how this extracellular binding event translates into an intracellular signal is fundamental to understanding hematopoietic diseases and designing therapeutic molecules. Thrombopoietin (TPO) and granulocyte colony stimulating factor (GCSF) are haematopoietic cytokines that regulate the production of platelets and neutrophils, respectively, from their precursor cells. The thrombopoietin receptor (TPOR) belongs to the short family of cytokine receptors and contains a unique duplication of its ligand binding domain not usually seen in other cytokine receptors. While the structure of TPO has previously been determined by X-ray crystallography, the structure of the receptor and receptor-cytokine complex have not. The GCSF receptor (GCSFR) belongs to the “tall” family of cytokine receptors. This family is characterised by three additional fibronectin type-III (FnIII) domains in the extracellular domain, which extends the receptor out from the cell membrane. Although a partial structure of the GCSFR-GCSF complex has been determined previously, this structure lacked the three membrane-proximal FnIII domains. I expressed and purified recombinant extracellular domains of both GCSFR and TPOR along with their respective cytokines. Cryogenic electron microscopy (cryo-EM) was then used to solve the structures of both receptor-ligand complexes and study the mechanism of cytokine binding and receptor activation of the two receptors. This research has resulted in the first experimentally determined structures of the entire extracellular domains of both the TPOR and GCSFR receptors in complex with their cytokines. These structures and the corresponding biophysical data have improved our understanding of how these two receptors are activated by their cytokines.
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    Structural studies of the mitochondrial import pathway
    Webb, Chaille Teresa (University of Melbourne, 2008)
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    Understanding epilepsy genetic risk: integrating common and rare genetic variation
    Oliver, Karen Louise ( 2023-10)
    The epilepsies are a group of neurological disorders affecting up to 4% of people during their lifetime. There are many different epilepsy syndromes. At the broadest level, syndromes can be classified into those that are focal, where there are focal-onset seizures, or generalised, where there are generalised-onset seizures; these represent the most common epilepsies. The developmental and/or epileptic encephalopathies (DEEs) are the most severe group of epilepsies where seizure activity is associated with developmental slowing or regression. The DEEs typically begin in infancy and childhood and have many rare single-gene causes. With both rare and common variants established contributors to epilepsy genetic risk, the over-arching aim of this thesis was to explore the potential interaction of these different variant types. First, I curated all reported single-gene monogenic causes. The clinically heterogeneous DEEs had >800 reported single-gene causes. In contrast, <50 single-gene causes for focal and generalised epilepsies are known. Clinical genetic studies suggest these epilepsy types are polygenic, with current evidence suggesting many contributions from both rare and common genetic variants. Common variants are detectable by genome-wide association studies (GWASs). I was Consortium Coordinator and one of the analysts for the third International League Against Epilepsy GWAS involving >29,000 patients with epilepsy. Here, I led an analysis to prioritise genes near GWAS signals that demonstrated a convergence between common and rare gene pathways, and a study of cross-trait genetic correlations that highlighted the pleiotropic nature of many epilepsy-associated loci. Next, to explore potential rare and common genetic variant interplay, I focused on families with epilepsy. This familial approach was motivated by the observation that, even when sharing the same familial rare pathogenic variant of major effect, the clinical presentation for relatives can be highly variable. This supports a role for, yet unidentified, epilepsy genetic modifiers. In other complex traits, like cancer and heart disease, studies have shown that common polygenic background, as captured by polygenic risk scores (PRSs), derived from large GWASs, can modify the penetrance of rare monogenic causes. To determine if common polygenic background plays a modifying role in the epilepsies, I next demonstrated that epilepsy PRSs are enriched in patients with a positive family history for epilepsy compared to those without. Whilst families with epilepsy have previously been targeted for rare variant discoveries, we provide the first support for common genetic variation playing a role in the familial aggregation of epilepsy. Furthermore, common risk variants for focal epilepsy were shown to be enriched in a specific familial focal epilepsy syndrome, despite no variants in the largest focal epilepsy GWAS reaching genome-wide significance. Finally, I explored whether the role played by common epilepsy risk variants is disease modifying. This was done by studying 58 families with the clinically heterogenous syndrome of genetic epilepsy with febrile seizures plus (GEFS+), many with a known rare variant of major effect. In these families, I showed that higher epilepsy PRSs correlated with more severe epilepsy phenotypes. This provides the first support for common genetic background modifying the clinical expression of rare pathogenic variants in the epilepsies.
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    3D Imaging and Cellular Barcoding: Novel Tools for Exploring Cancer Heterogeneity
    Lewis, Sabrina Milly ( 2023-09)
    Breast cancer affects 1 in 7 Australian women, and the risk of death from metastatic (stage 4) disease remains high. Progression to advanced disease is difficult to treat, especially when the availability of targeted treatments is limited for some cancer subtypes. Metastases form when cancer cells shed from the primary tumour, enter the blood and lymphatic vessels, exit and proliferate in distant organs. Understanding the interactions between these heterogeneous lesions and the vessels that facilitate their spread, will enable a better understanding of this process and potentially lead to improved cancer treatments. Not all tumour cells have the same ability to generate metastases. Specific clones (defined as cancer cells that have derived from the same ancestral cell) differ in terms of the organs they target, their behaviour in specific microenvironments, and how they cooperate with other clones. To date, the methods used to study the clonality and heterogeneity of cancer metastases often involve tissue dissociation or 2D imaging. Consequently, the spatial resolution of clones in their native microenvironment is lost. New methodologies and technologies are required to facilitate spatial discoveries, to advance our understanding of cancer heterogeneity, metastasis, and the tumour microenvironment. Here, I developed a novel pipeline for three-dimensional whole organ imaging of human-in-mouse models of metastatic breast cancer. Light-sheet microscopy was used to capture large volumetric datasets, reducing the information loss observed in 2D tissue sections. I used lentiviral gene ontology (LeGO) vectors, an optical barcoding method, to identify seven individual clones. In combination with vessel casting (a perfusion-based method that enables vasculature imaging), tissue clearing, and an analysis pipeline, I reveal the relationship of aggressive breast cancer clones and the blood vasculature in murine lungs and brain. This represents a method with unprecedented detail and clonal resolution at large volume scales. My results indicate that large vessels may be correlated with enhanced metastatic growth. I also show that metastases that wrap around blood vessels are more likely to be polyclonal (containing multiple clonal populations), which are more aggressive than monoclonal (single clone) metastases, with potential implications for treatment targets. Underlying the clones’ specific behaviours, are differences in gene expression. Based on these results, I propose that transcriptional information, in combination with clonal identity and spatial tissue context, is required to reveal the molecular pathways that are responsible for these clonal behaviours, which may represent novel therapeutic targets. To simultaneously track a higher number of cancer clones (i.e. thousands) and their gene expression in situ, I developed a novel smFISH and RNA barcoding method (FISHcodes). I show that the detection of hundreds of transcripts alongside dozens of clones (scalable to thousands) is feasible and will enable novel insights regarding clonal behaviour in cancer biology. Together, the results presented throughout this thesis have demonstrated that novel methodologies enabling the study of cancer cell heterogeneity and its interplay with the microenvironment, can address new questions about in situ cancer clone metastasis and growth.
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    Characterising the molecular regulation of erythroferrone
    Moir-Meyer, Gemma Louise ( 2023-10)
    ERFE encodes the hormone erythroferrone which is secreted from erythroblasts in response to increased erythroid drive. ERFE protein suppresses hepcidin, the master iron regulator, which allows iron to be released from body-iron stores and used for red blood cell production. In erythropoietic disorders such as thalassaemia, ineffective red blood cell production results in reduced tissue-oxygen levels, increased erythroid drive, and chronic hepcidin suppression. However, despite representing a possible drug target, the regulation of ERFE has not been well studied. This work has identified a putative ERFE control locus comprising an enhancer and several key transcription factors using in vitro differentiated Human Umbilical Cord Blood-derived Erythroid Progenitor (HUDEP-2) cells. ERFE transcription and chromatin accessibility were tracked during four stages of terminal erythroblast maturation using quantitative PCR and Assay for Transposase-Accessible Chromatin-sequencing (ATAC-seq). These data demonstrated a dynamic chromatin accessibility landscape with distinct erythroid maturation stages and an expression profile that peaked in intermediate erythroblasts (p<0.001). Capture-C then demonstrated contact between ERFE’s 5’ promoter and a putative enhancer that also aligns with trimethylation of lysine 4 and acetylation of lysine 4 on histone 3 (promoter marks), monomethylation of lysine 4 and aceytylation of lysine 27 on histone 3 (enhancer marks) Cleavage Under Targets & Release Using Nuclease (CUT&RUN). Moreover, when ERFE expression is at its highest, CUT&RUN showed that response elements in the enhancer are bound by master erythroid regulators GATA1, KLF1 and TAL1, and the stress erythroid response factor, STAT5, suggesting a role for multiple signalling pathways in ERFE activation. These pathways, and ERFE’s place within them, were further explored using weighted gene correlation network analysis on RNA sequencing from the four progenitor stages, where gene set enrichment analysis demonstrated that ERFE is co-expressed alongside genes that are highly associated with haem metabolism (p=3.65x10-30). Overall, this data provides new insights into the regulation of erythroferrone and may contribute valuable details for identifying therapeutic targets in iron-loading anaemias.
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    Sequencing and validation of variants causing autoinflammatory diseases
    Reygaerts, Thomas Jean F. ( 2023-09)
    Aberrant activation of the innate immune system leads to systemic and organ specific inflammation in the absence of pathogens or autoimmunity. This system is genetically encoded and mutations stimulating those pathways or impacting cellular homeostasis cause auto-inflammatory diseases (AIDs). Monogenic and complex (for which other genetic factors and the environment play a role) AIDs share phenotypic features and physiopathologic mechanisms. Only about 15-40% of patients suspected of monogenic AIDs will receive a specific molecular diagnosis. The Australian Autoinflammatory Diseases RegistrY (AADRY) gathers undiagnosed patients and provides whole exome sequencing to patients and families in search of new mechanisms explaining their diseases. In this thesis, we functionally validate the polymorphism E148Q in pyrin showing its functional effect in vitro. In patients with familial Mediterranean fever (FMF), it potentiates pathogenic mutations in cis and therefore could have a role in disease presentation and severity. I also assess a new mutation in CDC42 found in a family with members presenting an AID over three generation. A phenotypic and mechanistic hypothesis based approach shows that this variant promotes the pyrin inflammasome. Finally, I investigated somatic mosaicism which could explain around 15-20% of undiagnosed AID cases. I developed a cost effective ultra deep amplicon sequencing of the third exon of NLRP3, a part of the gene in which somatic mosaicism is particularly well described. These three projects intend to broaden our knowledge about the mechanism of autoinflammation.
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    Defining the intranodal spatial requirements for the formation and maintenance of long-lived T cell memory
    Duckworth, Brigette Catherine ( 2023-10)
    Immune memory is critical for providing superior protection against infection. Current vaccine strategies exploit immune memory with varying success, with most relying on humoral immunity while failing to elicit and maintain durable CD8+ T cell responses. As such, vaccine outcomes for pathogens which require a strong T cell response are poor. The next generation of vaccines against infectious disease and cancer require robust T cell memory. However, we lack the fundamental understanding of how the longevity of T cell memory is regulated so that we may optimise this strategy. During my PhD, I have made a series of discoveries that offer new insight into this problem. Firstly, I have discovered that memory cell differentiation is imprinted in the centre of draining lymph nodes (LNs). Secondly, I have identified a distinct intranodal location where memory T cells reside long-term. Lastly, I shed light on the changes to this positioning that occur as we age. Following infection, the differentiation of T cells is driven via dynamic interactions with multiple, distinct cellular subsets. I developed and employed a novel platform to quantify cell location in 3D to examine the spatial requirements that instruct T cell fate in intact LNs. Following viral infection, I established that CD8+ effector T cell fate correlates with positioning at the LN periphery, instructed by CXCR3 signalling. In the absence of CXCR3, T cells were retained in the LN paracortex and alternatively formed stem-like memory cell precursors. I also showed that CXCR3 ligands, CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Finally, I demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I IFN signalling, to promote effector or stem-like memory fates. Increasing evidence suggests that, in the steady-state, CD8+ central memory T cells (TCM) are positioned strategically in LNs; however, the mechanisms that regulate this location remain unknown. I used 3D light-sheet fluorescence microscopy of intact LNs to identify the location of TCM cells following the resolution of viral infection. In steady-state LNs, I showed that a higher density of TCM cells occupy the cortical ridge and interfollicular regions than naive T cells, which primarily reside in the T cell paracortex. This distinct TCM location was observed following various infection challenges and mRNA-LNP vaccination. Furthermore, in the LNs of aged mice, this TCM niche was disrupted and TCM cells relocated to the T cell paracortex. To explore cell-cell contacts regulating TCM location, I employed high-resolution confocal microscopy to identify specific dendritic and stromal cells which interact with TCM within this niche. Together, these findings suggest that TCM occupy a conserved and precise LN memory niche. This provides a platform for further spatial interrogation to determine how this niche promotes cell-specific interactions and sustains long-term TCM maintenance. In identifying the key mechanisms regulating the intranodal TCM niche, my work may contribute to improved vaccine strategies grounded in robust, long-lived T cell memory.
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    Characterisation of dual-specific Chimeric Antigen Receptor T cells against heterogeneous tumours
    Hughes-Parry, Hannah Emily ( 2023-06)
    The heterogeneity of solid tumours is a significant obstacle to the response and long-term remission of patient malignancies following Chimeric Antigen Receptor (CAR) T cell immunotherapy in the clinic. While long-term remission has been achieved against haematological cancers, relapses have frequently occurred several months post-treatment due to antigen escape, and solid tumour responses have been less effective. Therefore, to improve solid tumour elimination and prevent relapse, CAR T cell immunotherapy may be improved by the targeting of multiple tumour-associated antigens through dual-specific CAR T cells, in which T cells are engineered to express CARs against multiple antigens. Existing studies have observed significant improvements over single-specific CAR T cells; however, few studies have interrogated the underlying biology in immune competent systems. In this thesis, I explored whether dual-targeting CAR T cells targeting the HER2 and EGFRvIII tumour antigens were able to effectively clear heterogeneous tumours both in vitro and in vivo. I assessed their cytotoxic function and cytokine secretion against different heterogeneous tumour targets in vitro. I found that dual-specific CAR T cells exhibit enhanced killing of heterogeneous tumour cells, but not elevated levels of cytokine secretion or exhaustion markers compared to single-specific and pooled single-specific CAR T cells. This enhanced ability for multi-antigen targeting T cells to eliminate heterogeneous tumours allows for more complete clearance of the entire tumour cell population and may subsequently mitigate opportunities for antigen escape. To explore the utility of multitargeting tumour antigens in vivo, we used CRISPR technology to generate an immunocompetent mouse model (RHEO), tolerant to human HER2, EGFRvIII and OVA, to evaluate dual-targeted CAR T cell immunotherapy approaches. I demonstrate that administration of dual CAR T cells results in improved survival in vivo in RHEO mice using a heterogeneous intracranial tumour model, and a combination therapy, by combining dual CAR T cells with anti-CD137 agonist, results in complete intracranial tumour clearance in all mice. These results highlight the importance of evaluating CAR T cell efficacy in an immunocompetent mouse model. While targeting the entirety of a heterogeneous tumour with multi-targeting CAR T cells is critical for tumour elimination, combining CAR T cell immunotherapy with other immune modulatory agents may be necessary to achieve complete tumour clearance.
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    Studies of Plasmodium-Iron Interactions
    Clucas, Danielle Bridget ( 2023-09)
    Background Iron deficiency anaemia and malaria co-exist across sub-Saharan Africa where they disproportionately affect young children and pregnant women. Iron supplementation is recommended to treat anaemia, but there are concerns regarding its safety and the potential protection afforded by iron deficiency. An interaction between host iron status and malaria risk has been hypothesised for decades, but confounding factors mean that conclusions are difficult to draw from field studies alone. In this thesis the complex interaction between Plasmodium and iron is investigated using clinical, pre-clinical and in vitro studies. Methods The effect of host iron deficiency on the risk of Plasmodium falciparum parasitaemia by PCR was assessed in a cohort of 711 anaemic Malawian pregnant women, and the risk of intravenous iron supplementation on subsequent risk of parasitaemia explored. These findings were dissected using Plasmodium berghei infection in C57Bl/6 mouse models of iron deficiency (Tmprss6 knockout (Tmprss6-KO)) and iron overload (inducible hepcidin knockout (iHamp-KO)). Tmprss6 knockdown (Tmprss6-KD) was used to assess the phenotype in Tmprss6-KO mice and to explore Tmprss6 as a druggable target; achieved through treatment of wild-type C57Bl/6 mice with siTMP, a GalNAc conjugate targeting Tmprss6. The effect of iron restriction on the parasite was investigated through iron chelation of in vitro P. falciparum cultures, followed by transcriptomic and proteomic analysis. A role of post-transcriptional regulation in the response to iron chelation was further explored. In the setting of known post transcriptional regulation of cellular iron in other organisms via the iron responsive element (IRE)/Iron regulatory protein (IRP) system, and with an IRP-like protein described in P. falciparum, the role of PfIRP was explored through the generation and characterisation of PfIRP-KO parasites. Results Among anaemic Malawian pregnant women iron deficiency was associated with a 53% reduced risk of P. falciparum parasitaemia (Adjusted risk ratio 0.47, 95% confidence interval (0.34, 0.60), p<0.0001), with this finding robust to varied definitions of iron deficiency. Intravenous iron did not increase the subsequent risk of P. falciparum parasitaemia. These findings were supported by the mouse models. Tmprss6-KO mice had improved survival when infected with P. berghei. Conversely, iHamp-KO mice exhibited decreased liver stage infection but an unaltered course in the blood stage of infection. Tmprss6-KD did not replicate the KO phenotype; the disease course was not changed in siTMP treated mice. In vitro, iron chelation inhibited parasite growth and induced substantial changes in the transcriptome and proteome of P. falciparum. Differential expression of genes and proteins involved in key processes in the parasite’s lifecycle, and with plausible links to iron were identified, as was a possible role for post-transcriptional regulation. Investigating this showed PfIRP-KO parasites were more susceptible to iron chelation. Transcriptomic and proteomic analysis identified proteins that might be regulated in an IRE/IRP-like manner. Conclusions This work adds to the current understanding of the complex interaction between Plasmodium and iron. Taken together, these data support iron deficiency being protective against P. falciparum infection. In vitro studies highlighted genes of potential further interest in P. falciparum iron homeostasis and support an iron related role for PfIRP.