Medical Biology - Theses
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ItemMolecular Study of LUBAC and its subunit HOIL-1( 2024-01)Ubiquitination is a form of eukaryote specific post-translational modification, which involves attachment of the small protein modifier ubiquitin (Ub) to substrates through enzymatic cascade of E1, E2 and E3 enzymes. Ub can be ubiquitinated on multiple sites, giving rise to a diverse arsenal of poly-Ub chains carrying various information. The linear ubiquitin assembly complex (LUBAC) is the only known mammalian protein complex synthesising “linear” or M1-linked poly-Ub chains where the next Ub is conjugated to the previous Ub’s Met1 residue. M1 chains are responsible for inflammation and innate immune responses against a broad range of stimulation including inflammatory cytokines and pathogen infection. The overall molecular assembly and catalytic activity of LUBAC is not fully understood. LUBAC consists of HOIP, HOIL-1 and SHARPIN in unknown stoichiometry. The presence of all three subunits is critical for the integrity and activation of LUBAC. However, the full molecular picture of LUBAC assembly is lacking. HOIP and HOIL-1 are RING-between-RING (RBR) E3s which characteristically catalyse ubiquitin transfer from E2 to substrate in two-step fashion. While it is well understood that HOIP independently synthesises M1 chains, HOIL-1’s E3 activity remains enigmatic. Latest evidence suggests that HOIL-1 may unconventionally ubiquitinate protein and non-protein substrates. My thesis investigates the molecular assembly of LUBAC, HOIL-1’s catalytic mechanism and specificity for noncanonical substrates to untangle LUBAC’s activity on the molecular level. Chapter 3 presents my successful reconstitution of active full-length human LUBAC in vitro. The recombinant LUBAC exists as HOIP/HOIL-1/SHARPIN heterotrimer and dimer of the heterotrimer. My initial structural characterisation reveals that the complex adopts an elongated shape. Chapter 4 addresses the catalytic mechanism of HOIL-1. I identified the mechanism by which specific Ub species allosterically activate HOIL-1. By solving the structure of HOIL-1 in complex with E2 and ubiquitin, I show how HOIL-1 enables ubiquitin transfer from E2 in the first catalytic step. The structure unveils a unique fold in the HOIL-1 C-terminus where an unconventional catalytic triad resides. Chapter 4 delves deeper into how HOIL-1’s catalytic triad enables Ub transfer to substrates in the second catalytic step, compares HOIL-1’s specificity among various noncanonical substrates and provides initial structural evidence that the HOIL-1 unique C-terminus may be involved in sugar binding. Taken together, my thesis precisely determines the stoichiometry of full-length LUBAC, the molecular mechanism of HOIL-1 catalysed two-step ubiquitin transfer and lays foundation for future investigation of how HOIL-1’s noncanonical activity contributes to LUBAC function.
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ItemIdentifying novel oncogenes and tumour suppressor genes in cancer( 2024-02)Cancer is a complex group of diseases driven by genetic and epigenetic alterations in tumour suppressor genes and oncogenes. There are unmet needs to identify and understand which aberrations drive cancer pathology. This knowledge will be harnessed to improve early diagnosis, screening and prevention, to put patients on the most effective therapies according to their cancer dependencies, and to develop new targeted therapies to improve patient outcomes and quality of life. Deep sequencing of human cancer genomes aims to catalogue all somatic mutations, yet identifying which of these constitute the driver mutations that contribute to tumorigenesis, and which are silent passenger mutations necessitates complementary functional genomic approaches. Since the emergence of the first gene editing technologies, including viral and DNA transposon mutagenesis, they have been deployed to interrogate which genetic aberrations are critical drivers of cancer pathology and how they do this. The adaptation of CRISPR/Cas9 technology for gene editing in mammalian cells proved ground-breaking, enabling identification of tumour suppressor genes in cancer through robust, high precision gene deletion. The versatility of CRISPR applications was extended by developing modifications of this technology that up-regulate transcription, with CRISPR activation (CRISPRa) exploited to explore pro-tumorigenic signalling pathways in cancer. Rather than systematically testing individual or small subsets of putative tumour suppressor genes or oncogenes as with older RNAi tools, CRISPR technology can be readily deployed in high throughput genome-wide screens both in vitro and in vivo, with the latter more accurately modelling cancer initiation and progression as it occurs in human patients. To date, very few unbiased genome-wide in vivo CRISPR screens have been performed to identify tumour suppressors and oncogenes in models of cancer. Our laboratory performed a genome-wide in vivo CRISPR/Cas9 knockout screen to identify suppressors of c-MYC-driven pre-B/B cell lymphoma development, identifying novel tumour suppressor genes warranting further investigation. I validated the top hit, transcription factor activator protein 4 (TFAP4), which is mutated in ~10% of human B cell lymphomas and found that loss of its gene leads to deregulation of transcription factors, thereby blocking B cell differentiation and increasing the pool of pre-leukemic pre-B cells that undergo malignant transformation. Additional novel candidate tumour suppressors identified from this screen were NPRL3 and DEPDC5, essential components of the GATOR1 complex that negatively regulates mTORC1. I discovered that GATOR1-deficient lymphoma cells display abnormally elevated mTORC1 signalling and consequently altered cellular metabolism, rendering these malignant cells highly sensitive to mTOR inhibitor therapy. I also identified that p53 regulates the expression of components of the GATOR1 complex, demonstrating for the first time a mechanism of p53 mediated tumour suppression through regulation of a metabolic process. Lastly, I performed in vivo CRISPRa screens using newly developed sgRNA libraries and I identified oncogenes that enhance lymphoma/leukemia development in c-MYC overexpressing or p53 deficient cells. Together, the findings I present from this body of work demonstrate the power of unbiased in vivo CRISPR screens to identify and functionally interrogate novel critical drivers and suppressors of cancer. These screens can be applied to study other types of cancer, such as breast, lung or even brain cancer. Mechanistic investigations into the hits from these screens have expanded our understanding of complex cancer driving cellular processes and revealed potential vulnerabilities for novel therapeutic targeting strategies. Combining the results from these pre-clinical functional genomic studies with data from deep sequencing analysis of human cancer patients can identify patient cohorts that could benefit from novel targeted therapeutic interventions.
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ItemManipulation of host defence mechanisms by Toxoplasma gondii( 2023-10)Toxoplasma gondii is ubiquitous parasite of medical and agricultural importance, chronically infecting around a third of the global human population. Acute infection, caused by a form of the parasite known as a tachyzoite, in immunocompromised patients can cause blindness and encephalopathy. Treatments are limited to activity in the acute stage of infection, which is responsible for these disease processes. Chronic infection, characterised by differentiation of tachyzoites into bradyzoites, which are encysted parasites that reside in organs like the brain and the retina, is not only refractory to drug- but also immune-clearance. As a result, chronic infection is thought to persist for the lifetime of the host, serving as a reservoir for reactivation of acute infection during immune compromise. Being an obligate intracellular parasite, T. gondii must modify host signalling to create a parasite-permissive environment. This remodelling is, in part, attributed to the effector proteins exported by the parasite into the host cell. These proteins manipulate host signalling to evade the immune system and facilitate parasite persistence. In contrast to its role in acute infection, the impact of effector protein export in chronic infection remains largely uncharacterised. To better understand how effector protein export in chronic infection impacts host signalling, transgenic parasites were generated where the ability to export effector proteins was limited to either acute or chronic infection. Investigation of these parasites demonstrated that there is a defect in the ability of bradyzoites to survive host immune stress in the absence of effector protein export; restoration of this process confers a survival advantage in vitro. These findings also suggest that acute stage effector protein export is not sufficient in preventing clearance of bradyzoites, reiterating the idea that bradyzoites are not latently persisting in the host. Rather, they are active, dynamic entities that carefully regulate host signalling to prevent death of infected cells. In addition to its role in clearing bradyzoite infected cells, host programmed cell death (PCD) has also been implicated in acute T. gondii infection. Various PCD pathways have been investigated for their role in controlling acute infection, however, these findings are mostly in vitro and cell- or tissue-specific. It remains unclear how PCD affects parasite dissemination during acute infection in vivo, or whether there is redundancy in the PCD-mediated host defence against T. gondii, given the ability of the parasite to block certain arms of PCD using exported effector proteins. Mouse models of acute toxoplasmosis revealed a caspase-8 as a major mediator of resistance to acute infection, its role so critical that neither necroptotic nor pyroptotic death could act as a backup defence mechanism. This dissertation explores multiple avenues of host-pathogen interactions during acute and chronic T. gondii infection and provides new insights into how parasites establish successful chronic infection, as well as how the host attempts to control parasites before they can do so.
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ItemA conserved molecular mechanism of erythrocyte invasion by malaria parasites( 2023-10)Malaria is major global health burden causing over 240 million cases every year and leading to over 600,000 deaths mostly in pregnant women and children under the age of five. There is an urgent need to development novel therapeutic interventions for the control and elimination of malaria. During infection, malaria parasites must invade host erythrocytes in order to live within them. Invasion is a complex multi-step process that involves many molecular host-parasite interactions. In Plasmodium falciparum, the deadliest species of the parasite, the invasion protein PfRh5 assembles into a complex to bind its receptor basigin on the erythrocyte surface. Recent work has revealed two novel members of this complex, PfPTRAMP and PfCSS, form a heterodimeric platform for PfRipr, PfCyRPA, and PfRh5 binding. The PfPTRAMP-PfCSS-PfRipr-PfCyRPA-PfRh5 (PCRCR) complex, and its engagement with basigin, is essential for P. falciparum invasion. PfRh5 does not have an orthologue in all species of malaria, however PTRAMP, CSS and Ripr orthologues are present across the entire Plasmodium genus. This thesis sought to investigate these conserved proteins in other malaria species to further dissect the essentiality of these proteins for Plasmodium invasion more broadly. Orthologues of PTRAMP, CSS and Ripr from two important species of malaria, P. vivax and P. knowlesi, were investigated using recombinant expression and biophysical analysis. Assessment of complex formation shows a conserved assembly of the three proteins in both species, with similarities to P. falciparum. Structural determination of part of the complex revealed the basis of heterodimer formation between PTRAMP and CSS. Antibodies and nanobodies were produced and exhibit a high degree of cross-reactivity between species. A novel protein was identified that may bind to the complex and impart an erythrocyte binding function. The function of the complex and its components in invasion was confirmed using ex vivo invasion assays in Cambodian P. vivax field isolates. Taken together, this thesis shows that a three-membered complex consisting of PTRAMP, CSS and Ripr is conserved in three species of Plasmodium, likely forming a common invasion scaffold in all species of the genus, suggesting a conserved invasion mechanism with implications for cross-species vaccine development for the control of both P. vivax and P. falciparum malaria.
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ItemThe role of programmed cell death and inflammation in the pathogenesis of SARS-CoV-2 disease( 2023-12)The impact of the SARS-CoV-2 pandemic was mitigated by an unprecedented scientific response that culminated in the implementation of vaccines and several other interventions. However, many questions remain which are relevant not only to SARS-CoV-2 but many other respiratory and pandemic viruses. Vaccines will continue to fall short of preventing infections as the virus evolves and antivirals cannot be distributed indiscriminately to mitigate mortality. There remains a pressing need to identify host factors that contribute to severe COVID-19 so patients can be stratified, and therapies delivered to those that would benefit the most. Severe COVID-19 is linked to a dysregulated hyperinflammatory immune response characterized by the release of pro-inflammatory cytokines. While cell death pathways have been postulated as central drivers of pathology, the molecular intricacies underlying these events remain elusive. To address this, we developed unique pre-clinical in vivo models that reproduce aspects of mild, severe and fatal COVID-19. Through serially passaging a clinical SARS-CoV-2 isolate in mice, we generated a mouse adapted strain that causes weight loss, inflammation and lung pathology in adult and is deadly in aged mice, reflecting key aspects of COVID-19. Our approach diverges from prevailing correlative or in vitro studies, offering a unique opportunity to delineate pathways causative of severe inflammation in vivo. Using gene targeted animals and transcriptomic analysis, we showed that the pro-inflammatory cytokines TNF and IL-1b drive severe disease. Interestingly, inflammasome pathways upstream of canonical IL-1b release do not influence disease outcomes, as loss of inflammasome components NLRP3 and ACS did not affect disease or viral burdens in vivo. Deletion of downstream targets of inflammasome signalling, such as pyroptosis initiators caspase-1/-11/-12 or effectors GasderminA/C/D/E did not ameliorate disease or viral burdens. Infection of animals lacking RIPK3 or MLKL showed that the lytic process of necroptosis, which lies downstream of TNF, did not contribute to disease. Collectively, these results suggested that lytic cell death did not contribute to SARS-CoV-2 disease, instead, the central determinant of severe disease outcome was caspase-8, a protein essential for the activation of apoptosis. Remarkably, instead of triggering cell death, infection drives caspase-8 to activate survival/inflammatory pathways and increase IL-1b levels. Combined deficiency of pyroptosis, necroptosis, and apoptosis mediators (caspase1/11/12/8/Ripk3-/-) provided no added benefit in ameliorating disease outcomes compared to caspase-8 deficient mice. In contrast, loss of caspase-1/-11/-12/Ripk3 caused a worsening of disease. Transcriptional profiling and comparison of lung tissues from compound mutant animals further confirmed that disease outcomes were not associated with differences in cell death pathways but rather with pro-inflammatory responses. The expression of full-length caspase-8 protein was upregulated in the lungs of SARS-CoV-2 infected mice and this was not accompanied by any increase in the fully cleaved, apoptotic form of caspase-8. The data collectively showed a pivotal role for caspase-8 in driving the pathogenesis of severe SARS-CoV-2 infection through the modulation of pro-inflammatory cytokines but not through the induction of apoptosis. This critical new understanding provides valuable insights for targeted therapeutic interventions and emphasizes the need for continued exploration of host-pathogen interactions in the context of COVID-19.
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ItemUnderstanding protein variants with high-throughput mutagenesis and machine learning( 2023-10)Genetic variations in protein-coding genes may cause amino acid substitutions in the matured proteins. These variants can potentially change the properties and functions of a protein. To evaluate the effects of these protein variants, multiple experimental and computational approaches have been utilised. Within these approaches, deep mutational scanning (DMS), a recently developed high-throughput mutagenesis method, enables the measurement of thousands of protein variant effects in a single experiment. To fully investigate the rich information in DMS results and have a better understanding of protein variant effects, here, I leveraged machine learning algorithms to build advanced computational models for DMS data. First, I reviewed that there are missing variant effect data in most DMS results, and I developed imputation models to fill in the missing values. I started by investigating the correlations between the variant effects measured within a DMS experiment and used these correlations to build imputation models. To understand the strengths and weaknesses of these models, I benchmarked them with previously published DMS imputation methods. At the end of this study, I built an ensemble imputation model by combining these novel and previously published methods to further improve the imputation accuracy. Many of the state-of-the-art variant effect predictors are built with DMS data, and I then managed to improve these predictors by further integrating variant effect data from alanine scanning (AS), a low-throughput mutagenesis approach. In this study, I established a rule-based classification tree to evaluate the compatibility between DMS and AS studies according to the similarity of their experimental assays. I showed that an improved variant effect predictor could be built only by modelling with high compatibility DMS and AS data. Finally, experimental measurements of protein variant effects may conflate protein stability and function. Here, I explored this relationship using DMS-measured variant effects and computed variant stability. I demonstrated that the correlation between variant effect and stability data differed on distinct protein regions and properties measured. Analysing these data with a dimensional reduction algorithm, I was able to automatically distinguish protein residues with different scales of fitness–stability association. Further investigation showed that this approach might be applied to discover protein functional sites and explain the mechanisms of loss-of-function variants.
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ItemNo Preview AvailableStatistical models for pre-processing and simulating single cell RNA-seq data( 2023-10)Since the first protocol published in the year of 2009, single-cell RNA sequencing (scRNA-seq) has become one of the most popular technologies in the omics world. scRNA-seq has been broadly applied in different areas, including understanding tumor microenvironment, inferring embryonic development, and discovering regulation pathways involved in plant seeding. The diverse applications facilitate the development of the scRNA-seq, and new protocols tend to sequence numerous of cells at low cost. With rapid growth of scRNA-seq technology, many issues arise and wait to be resolved when processing the data, such as removing batch effects, addressing dropout events, and annotation of cell populations. However, the requirements to tackle the above issues bring opportunities to apply statistical methods and devise new computational tools for analysing the data. In this thesis, we focus on pre-processing and simulation of single cell data. In the first part of the thesis, we mainly discuss different demultiplexing methods in the pre-processing step. The first method, CMDdemux, utilizes Mahalanobis distance to distinguish cells from different samples. This method performs well in low-quality data with hashtag contaminations during the cell hashing experiment. The second method is LCADemux, which uses latent class analysis (LCA) to combine cell hashing- and single nucleotide polymorphism (SNP)-based demultiplexing results. This hybrid framework is advantageous for analysing low input cell hashing data. The third method, LCAdoublet, applies the LCA method to combine doublet information from cell hashing and VDJ data. LCAdoublet is better at identifying inter-sample doublets and T-cell doublets than methods solely using transcriptomics data. In summary, our three pre-processing methods enable the accurate identification of doublets and assigning cells to their samples of origins, which contributes to cleaner data for downstream analysis. In the second part of the thesis, we talk about a novel single cell simulation method and its applications for data analysis. Our method is named GLMsim, and it applies a generalized linear model to simulate the batch and biological effects simultaneously. Compared to other methods, our method is able to simulate single cell data resembling the original data, especially for the data collected under complex conditions. Our single cell simulators have multiple applications, such as benchmarking single cell integration methods, providing guidance for differential expression analysis and checking the assumptions of models. In short, our simulation method should help researchers develop better tools for downstream analysis. Overall, we applied multiple statistical methods to fill gaps existing in dealing with single cell RNA-seq data. Our methods have been demonstrated to overcome challenges when analysing single cell RNA-seq data. We hope that they can be applied in the medical field to extend our knowledge of human health and disease.
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ItemStructural and Pharmacological Investigation of the JAK/SOCS1 Interaction( 2023-09)Cytokines are important signalling molecules which can cause inflammation, regulate haematopoiesis and modulate the immune response. Over 50 cytokines achieve their biological functions on cells via the JAK/STAT pathway. SOCS (Suppressors of Cytokine Signalling) family are induced by the cytokine-JAK-STAT cascade and act as negative feedback regulators to specific cytokine pathway. The existence of the SOCS family prevents cells from persistent activation of cytokine signalling but can also play a role in cytokine resistance and tumour progression. SOCS1, a member of SOCS family, is specifically induced by IFN-gamma. And the SH2 domain and the KIR domain are critical for SOCS1’s function. The SH2 domain is responsible for protein-protein interactions by specifically targeting pTyr-containing substrates. However, the structural details of the SOCS1 SH2 domain interacting with its targets and how this interaction happens in vivo remained unclear. Our work determined the crystal structures of SOCS1 SH2 domain bound to various ligands, which uncovered the molecular interactions within this domain. Additionally, small molecules with potentially high affinity for targeting the SOCS1 SH2 domain were identified through structure-guided drug design. The KIR domain of SOCS1 can insert into the substrate-binding pocket and directly inhibit the catalytic activity of JAK1. Our research also attempted to develop novel type of JAK inhibitors based on the binding mechanism of KIR domain. These findings provided insights into the interactions between JAK and SOCS1 and also highlighted the potential of developing small molecules to disrupt these interactions.
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ItemStructural Studies of Type-I Haematopoietic cytokine receptors( 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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ItemStructural studies of the mitochondrial import pathway(University of Melbourne, 2008)