Medical Biology - Theses

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    In vivo imaging of plasma cell dynamics in the bone marrow niche
    Rimes, Joel Scott ( 2022-12)
    Plasma cells are critical effector cells that form part of the adaptive immune system. They produce antibodies that can bind to and flag foreign pathogens for clearance by other components of the immune system. Plasma cells are long-lived immune cells that are preferentially found in bone marrow, where they home to and reside after generation in peripheral immune sites. However, much remains unknown regarding the specifics of their behaviour. The duration plasma cells can survive in the bone marrow, the factors required for their maintenance, the potential existence and composition of a ‘plasma cell niche’, as well as their kinetic behavioural profile are still debated. In this thesis, I explore the role of the bone marrow in the regulation of plasma cell maintenance in the context of autoimmunity and immunisation. To achieve this, I combined in situ imaging techniques in live mice with a novel B6.MRL-Fas/lpr and B6.AIDcre confetti mouse model that allows visualisation of plasma cells in the bone marrow. Using multiphoton in vivo microscopy, plasma cells of the bone marrow were revealed to exist in dense clusters in a highly non-migratory state. These clusters consisted of largely plasma cells of single founder origin and thus, likely generated via plasma cell proliferation in the bone marrow. Additionally, plasma cell clusters appeared to manifest uniquely in bone marrow environments and not in peripheral tissues such as spleen, lymph node, or kidney. Investigation of dysregulated and pathogenic plasma cell biology was performed in murine models of systemic lupus erythematosus (SLE). These systems demonstrated a highly dysregulated phenotype, exhibiting significantly expanded bone marrow plasma cell clusters. Additionally, standard glucocorticoid therapy failed to significantly reduce plasma cell populations in the bone marrow of diseased mice, suggesting the need for identification for new drug targets for the clearance of potentially pathogenic plasma cell clusters. A novel intravital imaging and RNA-sequencing paired analysis was established, termed high-resolution imaging of the direct environment with single cell RNA-sequencing (HIDE-n-seq). This approach suggested that altered proliferation, not survival, drove cluster formation in the bone marrow. Furthermore, we identified potential regulators of plasma cell clustering that could be candidates for design of new therapies for SLE patients.
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    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.
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    The Parasite Genetic and Host Immunological Determinants of Immune Escape in Plasmodium falciparum Malaria
    Naung, Myo ( 2022-12)
    Abstract Human malaria remains a major global public health problem with an estimated 241 million clinical cases and 627,000 deaths in 2020, expected to increase in future years. Highly effective vaccines are urgently needed to progress the control and elimination of the disease. There are dozens of candidates in development, however only one vaccine (RTS, S) targeting the most virulent human malaria parasite, Plasmodium falciparum, has reached Phase 4 implementation trials with 50% efficacy that is short-lived and strain specific. As WHO has outlined a goal for malaria vaccines with a 75% efficacy against clinical malaria in all malaria-endemic countries by 2030, novel approaches are needed to increase efficacy. The limited efficacy of malaria vaccines to date has been in part attributed to the extreme diversity of parasite antigens being developed as ‘subunit’ vaccines, with only one or two randomly selected allelic variants as the basis for inducing immune responses. Antigen diversity has evolved as a means for malaria parasites to evade host immune responses - a process known as an immune escape. Pinpointing specific antigen polymorphisms that drive immune escape would help to prioritise antigens and alleles for inclusion in vaccine formulations. In my Ph.D. project, I investigated the hypothesis that specific polymorphisms in leading P. falciparum vaccine candidates are associated with immune escape. To test this hypothesis, I first analysed the publicly available MalariaGEN genome sequence data to catalogue the global genetic diversity of the genes encoding 25 leading P. falciparum vaccine candidate antigens. Predicted regions of immune selection were identified on both the linear gene sequence and the 3-dimensional protein structure. We then focused on two cohorts of children from malaria endemic regions of PNG conducted during moderate and high transmission periods. We analysed samples from 758 children, conducting multiplexed high-throughput assays on serum samples to measure IgG responses against 27 antigens, and targeted amplicon sequencing of 38 parasite antigen genes in sequentially collected samples from each child to measure the rate of allelic turnover for each antigen. The analysis identified critical immune escape genes and their specific polymorphisms that contribute to immune escape. The relationship between measures of genetic diversity and immune selection in the global data, and the antibody response in the children identifies antigens driving immune escape and those where diversity did not appear to contribute to immune escape. This research provides a vital framework for the prioritization of vaccine candidate antigens and a ‘serotype classification system’ to identify immune escape polymorphisms and for evaluating strain specific efficacy during vaccine trials.
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    Benchmarking long-read RNA-seq analysis methods
    Dong, Xueyi ( 2022-12)
    Application of Oxford Nanopore Technologies' long-read sequencing platform to transcriptomic analysis is increasing in popularity. To explore how much additional benefit it brings over short-read RNA-seq, as well as provide guidance in the choice of suitable long-read RNA-seq data analysis methods, I first created a long-read RNA-seq analysis pipeline using a mix of long-read specific preprocessing tools together with established short-read methods for differential expression analysis. Using this pipeline, I analyzed two nanopore long-read RNA-seq datasets and compared the results to short-read experiments with equivalent samples. The results showed that performing differential gene expression analysis using this pipeline can yield comparable results to those obtained using short-read data. Secondly, I designed a comprehensive long-read benchmarking experiment with ground-truth provided by sequin spike-ins and an in silico mixture design. Samples were deeply sequenced using both short-read and long-read technologies. By analyzing this dataset, I found that StringTie2 and bambu outperformed other tools from the 6 isoform detection tools tested, DESeq2, edgeR, and limma-voom were best amongst the 5 differential transcript expression tools tested, and there was no clear front-runner for performing differential transcript usage analysis between the 5 tools compared. Finally, I used the best-in-class methods identified in the benchmark study to detect aberrant minor class RNA splicing due to the knockdown of the RNPC3 gene in A549 cell line samples. Differentially expressed and differentially used transcripts detected on the long-read RNA-seq data were highly correlated with annotated minor intron-containing genes. I also visualized the long- and short-read coverage of the five top genes with differential transcript usage, which further highlighted the strength of long-reads in detecting and visualizing transcript splicing. The datasets generated and benchmarking results presented in this thesis provide useful resources and insights for long-read RNA-seq analysis to the research community.
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    Deconvolving gene expression changes associated with time and cell division following B cell activation
    Tarasova, Ilariya ( 2022-12)
    B cells, a type of lymphocyte, play a critical role in the adaptive immune system. They have a sophisticated decision-making mechanism of differentiation into multiple effector cell types. When stimulated, B cells divide and change levels of key regulatory molecules that alter and guide the differentiation path of each individual cell. However, the mechanisms governing this process and how cells transit to new cell types are still poorly understood at the molecular level. On the one hand, the timing of the division burst occurring during the proliferation phase of the B cells response is controlled by protein Myc, whose expression is shown to be dependent on time after activation. On the other hand, several studies have documented a connection between B cell differentiation, including isotype switching, and cell division progression. Thus, the question of globally assessing which gene changes can be affected by time or division arose. Therefore, the aim of the current study focused on quantifying the effect of time and division on the whole transcriptome. Here, we examine the transcription profiles of B cells in different conditions and investigate changes between two fundamental B cell activation programs: proliferation only and proliferation with further differentiation. As a result, we developed a method for quantifying time and division effects on the B cell transcriptome and highlighted the overall behaviour of genes. We also provided a high-level visualisation of the influence of these two variables on each gene transcript. Further investigation of the data revealed clustered and shared expression trends over time and division progression. Moreover, we interrogated common regulatory features and identified potential key regulators of underlying transcriptional programs in an unbiased manner. Together the findings presented here support the hypothesis of a time-dependent proliferation program and division-dependent differentiation. Overall, the statistical approach and analysis described in the current work could be applied to any cells that can evolve or transform into other cell types over time and during division progression.
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    Investigating molecular interactions in necroptosis and MLKL-mediated cell death
    Jacobsen, Annette Vivi ( 2022)
    Necroptosis is a lytic, caspase-independent form of regulated cell death that involves the swelling of cells and organelles, the breakdown of cellular membranes, and the release of damage associated molecular patterns and other cellular contents. From an evolutionary perspective, necroptosis is thought to have arisen as a defence against infection, particularly in the case of intracellular bacteria and viruses. Additionally, there is a growing body of evidence demonstrating that dysregulated necroptosis contributes to the pathology of a broad range of noninfectious diseases, including those involving neurodegeneration, inflammation, autoimmunity, and ischaemic tissue death, as well as having a role in the progression of some types of cancer. As such, having a clear understanding of the molecular processes involved in necroptosis is critical for both increasing our understanding of its role disease and determining the most effective strategies for pharmacological manipulation of the pathway. Although necroptosis can be activated by a range of intracellular and extracellular receptors, necroptotic signalling through tumour necrosis factor receptor 1 (TNFR1) is the most well-studied, as it has relevance to a broad range of disease states. For necroptosis to proceed through TNFR1, there are two critical signalling events that need to occur. Firstly, receptor interacting protein kinase 1 (RIPK1) needs to disengage from the membrane-bound TNFR1 receptor complex to form a death-promoting cytoplasmic complex which, importantly, includes caspase 8 (CASP8). Secondly, proteolytic activity of CASP8 needs to be restricted to allow RIPK1 to associate with the related kinase RIPK3, through their RIP homotypic interaction motifs. This association enables RIPK3-mediated phosphorylation and activation of the mixed lineage kinase domain-like (MLKL) pseudokinase, enabling MLKL to oligomerise and translocate to cell membranes, where it can initiate cell death. However, although this core signalling axis is well defined, the peripheral signalling events that control necroptotic cell death are not well established, particularly relating to events that occur at the level, or downstream, of MLKL activation. This thesis uses two different strategies to increase our understanding of factors that influence MLKL activation and subsequent cell death. Firstly, in Chapter 3, I developed a method for generating reconstitutable MLKL-/- human cell lines using CRISPR-Cas9 delivered by integrase-deficient lentivirus, which enabled me to test the functional consequences of mutations to MLKL without the complicating influence of endogenous MLKL. The results of these reconstitution studies, which are documented in Chapter 4, helped identify several novel residues that play an important role in MLKL function, including some that have previously been found to be mutated in human cancer. In Chapter 5, I explored the mechanism of action of AMG-47a, a small molecule identified in a phenotypic screen focussed on drugs that inhibit necroptosis downstream of MLKL activation. The key findings of this investigation include confirming RIPK1 and RIPK3 as the relevant targets of AMG-47a during TNFR1-dependent necroptosis and uncovering a potential role for RIPK1 downstream of MLKL activation. Together, my research has contributed to our understanding of how MLKL activation is controlled and expanded our knowledge of the molecular processes downstream of MLKL activation.
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    Identifying and characterising novel regulators of TRAIL-induced cell death and cholangitis-like liver injury
    Gabrielyan, Anna ( 2022)
    Primary sclerosing cholangitis (PSC) is a progressive, idiopathic cholangiopathy characterised by chronic inflammation of the biliary epithelium and cholestasis. PSC promotes fibrotic scarring of the intrahepatic and extrahepatic bile ducts often leading to premature death due to irreversible liver damage. Chronic persistent inflammation in the biliary tree further predisposes to the development of malignant cholangiocarcinoma (CCA). Tumour necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL)/TRAIL-receptor-mediated signalling was shown to play a substantial role in the pathogenesis of human sclerosing cholangitis-like disease in mice with TRAIL- mediated apoptosis contributing to the disease. However, the etiology and exact pathogenic mechanisms of TRAIL-dependent PSC, or inflammation-associated cholangiocarcinogenesis are largely unclear. Various genetic and environmental factors have been reported to play role in the pathogenesis of PSC. Recently, mutations in ZFYVE19 gene (protein name: ANCHR) were described as a novel cause of neonatal sclerosing cholangitis and hepatic fibrosis termed ZFYVE19 disease. However, the mechanism by which ZFYVE19/ANCHR is involved in the pathogenesis of sclerosing cholangiopathy in these patients has not been yet explored. In Chapters 3 & 4 of my thesis, I identify and characterise two novel regulators of TRAIL-induced cell death, the Abscission/NoCut Checkpoint Regulator (ANCHR/ZFYVE19) and its interacting protein E3 ligase Mind Bomb 2 (MIB2) and show that the loss of ANCHR or MIB2 sensitises TRAIL-resistant cancer cells to caspase-8- dependent death. Moreover, loss of ANCHR alone sensitises CCA cells to death in vitro. Given that the loss of ZFYVE19/ANCHR in a cohort of patients was associated with PSC and cholestasis, and TRAIL/TRAIL-R-mediated apoptosis has been suggested to play an essential role in a PSC-like disease in mice, I further interrogate the physiological consequences of ANCHR loss in mice, particularly focusing on TRAIL-mediated cell death in the liver. In Chapters 3 & 4 I demonstrate a role for ANCHR in limiting TRAIL-induced cell death in vivo and show that loss of ANCHR in mice sensitises to TRAIL-mediated liver cell death. A significant increase in liver cell death in Zfyve19 knock-out mutant mice is observed compared to the wild-type mice after anti-TRAIL-R2 monoclonal antibody (MD5- 1) injection, with concurrent increase in cholangiocyte cell death, suggesting a role for ANCHR in limiting the TRAIL-mediated cholangitis in mice by limiting TRAIL-induced cell death in the liver. Lastly, in the Chapter 5 of this thesis, I demonstrate, as a proof-of-concept, that ANCHR and MIB2 can be efficiently targeted and degraded using the emerging degradation tag (dTAG) PROteolysis-TArgeting Chimera molecules (PROTACs). Our preliminary results serve as a basis for future research and suggest that anti-apoptotic ANCHR and MIB2 are feasible targets for target-specific protein degradation for development of future TRAIL therapeutics. Overall, this thesis expands our understanding on how TRAIL-signalling is regulated and provides a mechanism for the interplay between ZFYVE19/ANCHR loss and TRAIL- mediated PSC-like liver disease. Furthermore, our studies provide correlative evidence for the relationship between the PSC pathology seen in patients carrying bi-allelic nonsense mutations in the ZFYVE19 gene and an overactive TRAIL signalling or overactive liver sensitivity to endogenous TRAIL.
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    Interrogating the cells-of-origin of BRCA mutant cancers to identify therapeutic targets for cancer prevention
    Joyce, Rachel ( 2022)
    It is currently estimated that approximately one woman dies every minute of breast cancer across the globe. While the greatest risk factor for developing breast or ovarian cancer is merely having female reproductive organs, the cumulative life-time risk of developing breast and ovarian cancer for women who carry pathogenic mutations in their BRCA genes is significantly higher than non-carriers. Men who harbour mutations in their BRCA genes are also at increased risk of developing breast cancer within their lifetime. Currently there are no clinically approved strategies for breast or ovarian cancer prevention in BRCA mutation carriers beyond highly invasive and irreversible surgical procedures such as prophylactic mastectomy and bilateral salpingo-oophorectomy. Targeted therapeutic strategies for cancer prevention in BRCA mutation carriers are thus a sought-after alternative. Significant headway has been made by our research group and others in identifying RANK-ligand inhibition as a putative chemoprevention strategy for the onset of breast cancer in female BRCA1 mutation carriers; subsequently, a phase 3 international clinical trial BRCA-P (ClinicalTrials.gov Identifier: NCT04711109) is currently recruiting female BRCA1 mutation carriers to assess the efficacy of RANK-ligand inhibition in preventing breast cancer development using the FDA-approved drug denosumab. A portion of this thesis describes the functional and biological consequences of denosumab treatment on the putative cell-of-origin of BRCA1 mutant breast cancer, the RANK+ luminal progenitor, from patients enrolled in the Melbourne Health BRCA-D pre-operative window study; these patients received denosumab treatments prior to undergoing prophylactic mastectomies. This work indicated that BRCA1 mutation carriers who received 1 denosumab injection per month for 3 months had significantly reduced numbers of RANK+ luminal progenitors in their breast epithelium, and these cells also displayed decreased colony forming activity ex vivo, compared to cells from untreated BRCA1 mutation carriers. This thesis also seeks to shed light on the biological mechanisms driving ovarian cancer development in BRCA1 mutation carriers, and describes novel subsets of BRCA1 mutant fallopian tube secretory cells that are putative cancer cells-of-origin. To date, there have been no prospective studies or chemoprevention trials for breast cancer development in BRCA2 mutation carriers. As such, there is a pressing need for the identification of novel therapeutic pathways for breast cancer prevention in these patients; this thesis makes several promising developments in this effort. Using preneoplastic breast tissue samples from BRCA2 mutation carriers and wildtype patients, luminal cells, including a subset of ERBB3lo luminal progenitors and mature luminal cells, were found to be expanded in breast tissue epithelium of BRCA2 mutation carriers. ERBB3lo luminal progenitors from preneoplastic BRCA2mut/+ patients were found to have increased colony forming activity ex vivo, and exhibited upregulation of genes involved in mTORC1 signalling, protein synthesis and proteostasis. Indeed, a functional protein synthesis assay revealed increased protein translation in preneoplastic luminal cells from BRCA2 mutation carriers compared to wildtype patients ex vivo. A genetically engineered mouse model of BRCA2 mutant breast cancer was used to faithfully recapitulate the preneoplastic phenotype of luminal epithelium identified in BRCA2 mutation carriers, and showed a significant delay of BRCA2mut/+ mammary tumourigenesis upon short-term treatment with an mTORC1 inhibitor in vivo. In summary, the findings detailed in this thesis describe several developments in our understanding of the mechanisms of breast and ovarian cancer development in BRCA mutation carriers, and uncover mTORC1 inhibition as a putative strategy to delay or prevent the onset of breast cancer in BRCA2 mutation carriers. Cumulatively this work provides important insights of clinical significance for women harbouring mutations in their BRCA genes.
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    Intestinal Microfold Cells Orchestrate Microbe- Immune Interactions
    Cao, Wang ( 2022)
    Microbiota–immune cell interactions play a vital role in defenses against potentially harmful external organisms such as viruses and bacteria, and environmental agents including food. Microfold (M) cells are specialized cells within the epithelium of the intestines that sample the gut contents and pass them to the local guardians – a complex array of immune cells. Once harmful invaders are detected by immune cells, they swing into action to fight the infection. M cells provide the pivotal link between the gut lumen and the immune cell network, positioned to rapidly orchestrate appropriate immune responses. Exactly how M cells orchestrate these events, however, is not clear. Despite their critical function, to date few specific tools have existed to study intestinal M cells, the molecular mechanisms that regulate their generation, or how they drive mucosal immunity. To overcome this gap, we have generated novel gene modified mouse strains to allow us to visualize M cells and tease apart their behavior. I discovered that M cells are present along the entire intestinal tract, and not just localized to the Peyer’s patch as previously thought. Analysis of gut epithelial cells at different sites along the gut using single cell RNA sequencing revealed tissue-specific heterogeneity allowing us to define distinct gene signatures for M cells based on their location. These molecular blueprints identify distinct maturation programs that reflect local environmental cues shaped by the ingested material and the microbiota. Collectively, these features shape the delicate network of immune cells and show how the body can regulate gut region-specific diseases.
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    Exploring the ubiquitin proteasomal system in Toxoplasma gondii
    Khurana, Sachin ( 2022)
    The phylum Apicomplexa comprises a group of unicellular eukaryotic organisms including Plasmodium spp., Toxoplasma gondii and Cryptosporidium spp. Infection with Toxoplasma gondii causes severe congenital birth defects and disease in immunocompromised individuals and persists for life. Efforts to develop effective vaccines have been unsuccessful and emergence of drug resistance has reduced the utility of current antiparasitic compounds, highlighting the need for new therapeutics. My PhD focussed on two areas: Firstly, understanding degradative ubiquitination in apicomplexan parasites as a step towards developing first in class anti-apicomplexan PROTACS. PROTACS are heterobifunctional molecules which on one end bind an E3 ubiquitin ligase and force it to bind with a neo-substrate which is then degraded via the proteasome. This technique has shown great promise in developing new therapeutic approaches to treat disease like cancer but has never been tested against any apicomplexan parasite. The absence of conventionally used E3 ligases for PROTAC development became a challenge which I decided to overcome during my thesis. I developed a new system to screen for degradative parasite E3 ubiquitin ligases that can be used to enhance current therapies. The second part of my PhD focused on the role of ubiquitination during differentiation of Toxoplasma gondii into latent stages. We performed a whole genome CRISPR screen to identify genes required for differentiation into latent forms. The screen identified several genes involved in protein ubiquitination. These encompassed a complete multi protein E3 ubiquitin ligase complex that regulates differentiation from acute to latent forms and was the orthologue of the GID/CTLH E3 ligase complex which has been characterised in yeast and humans. Genetic removal of this complex resulted in markedly reduced cyst formation in vitro even in conditions favouring differentiation and reduced burden in the brain in vivo. Furthermore, we have identified potential substrates of this E3 complex and the likely mechanism by which it regulates differentiation. This work highlights the importance of ubiquitination during stage transition into latent forms and highlights the fascinating biological processes that ubiquitination regulates.