Medical Biology - Theses

Permanent URI for this collection

http://hdl.handle.net/11343/239

Search Results

Now showing 1 - 10 of 130

Investigating the function of the SOCS2-SH2 domain in targets recognition

Li, Kunlun ( 2024-01)

Src homology 2 (SH2) domains recognise protein targets containing specific phosphorylated tyrosine (pTyr) residues and regulate the transduction of various signalling cascades. Suppressor of Cytokine Signalling 2 (SOCS2) was discovered at WEHI in 1997 and contains a central SH2 domain that interacts with pTyr targets, and a C-terminal SOCS box motif that recruits an E3 ubiquitin ligase complex to direct SH2-bound targets for proteasomal degradation. SOCS2 is the key negative regulator of growth hormone (GH) signalling, as evidenced by the gigantic phenotype of Socs2-deficient mice. In addition, SOCS2 has been reported to be involved in diverse signal cascades, including those that regulate cancer development and immune cell activation, although the mechanism of action and phosphotyrosine targets for SOCS2 in these pathways remains enigmatic. The work presented in thesis investigated the biological and biochemical role of the SOCS2-SH2 domain and how it contributed to SOCS2 function. Mice bearing a germ-line mutation in the SOCS2-SH2 domain that abrogated binding to phosphotyrosine (Arg96 to Cys), displayed a similar gigantic phenotype to SOCS2 deficient mice, demonstrating the essential role of pTyr binding in SOCS2 function. Phage display screening identified an allosteric site or “exosite” on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhanced SH2 affinity for canonical phosphotyrosine (pTyr) ligands. The crystal structure of SOCS2 in complex with F3 peptide and the SOCS box adaptor proteins Elongin B and C, revealed F3 as an a-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3: exosite binding results in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. Moreover, affinity precipitation together with competition by F3 and pTyr peptides and mass spectrometry, identified a group of potential pTyr binding proteins and one exosite binding protein, a putative E3 ligase protein called RNF169. In support of this, residues 428-447 in RNF169 were shown to contribute to the interaction with SOCS2 and reciprocally enhanced SH2 binding to pTyr ligands. SOCS2-RNF169 interaction was further validated in intact cells using a proximity ligation assay. Analysis of online patient databases found that SOCS2 and RNF169 RNA levels were unregulated in AML samples, and that SOCS2 expression correlated with a poor patient prognosis. Further investigation of SOCS2 and RNF169 in AML cell lines indicated the potential roles in AML development and relapse after chemotherapy.
Defining the mechanics driving platelet formation via megakaryocyte membrane budding

Terreaux, Antoine Frederic ( 2024-02)

Over a hundred years after megakaryocytes were first described as the source of platelets, the exact mechanism by which megakaryocytes produce platelets remains incompletely understood. Currently, three theories exist to explain platelet biogenesis: proplatelet formation, cellular fragmentation, and plasma membrane budding. The aim of this research project was to investigate the mechanics of one of these theories, a novel model of platelet production termed megakaryocyte membrane budding, in hopes of filling this century-old gap in knowledge. This project aimed to explore membrane budding by examining the involvement of microtubules and the actin cytoskeleton at the plasma membrane during bud formation. Using immunofluorescence microscopy of the adult mouse bone marrow, I first investigated the relevance of budding to platelet production with a robust analysis of bud contents and morphology. Next, I investigated the involvement of key cytoskeletal proteins at the different stages of budding in healthy steady-state, and identified a pathway whereby components of the cytoskeleton become sequentially involved at each step. Finally, I was able to gain a more rigorous understanding of the mechanics and relevance of membrane budding by the study of multiple mutant mouse models. With these new insights, I aimed to guide fresh developments in the field of ex vivo platelet manufacture, having major impacts on platelet transfusions, personalised medicine and beyond.
Identifying vulnerabilities in treatment resistant lung adenocarcinoma

McDonald, Jackson Aloysius ( 2024-01)

With close to 15,000 new diagnoses in 2022, lung cancer is the 5th most diagnosed form of cancer and the leading cause of cancer-related death in Australia. Whilst clinical advancements in cancer treatment has improved survival outcomes for patients, the heterogeneity of the disease remains a clinical challenge. Particularly, mutations in KRAS are common, and historically, KRAS-mutant lung cancer has been difficult to treat with impaired responses to classical therapeutic strategies, such as chemotherapy and radiotherapy. With approximately 16-17% of all lung cancer cases harbouring mutations in KRAS, understanding the molecular and cellular mechanisms that govern tumourigenesis and progression are key to developing new treatment strategies to improve patient survival. Recently, the development of targeted therapies and immunotherapy have improved outcomes for KRAS-mutant lung adenocarcinoma (LUAD) patients. However, the stratification of tumour suppressor genes co-mutated with KRAS has highlighted distinct patterns of therapeutic response. The three most common tumour suppressor genes found co-mutated with KRAS are TP53, KEAP1 and STK11 (also known as Lkb1). Whilst loss-of-function mutations in either of these 3 tumour suppressor genes display aggressive forms of the disease, mutations in either KEAP1 and/or STK11 display impaired response to chemotherapy. Immune checkpoint blockers (ICBs) that target the PD-1/PD-L1 axis form a class of immunotherapy aimed to unleash the cytotoxic killing ability of CD8 effector T cells against tumours, are currently first-line treatment. However, patients who harbour mutations in both KRAS and STK11 (KL-mutant LUAD) or KEAP1 (KK-mutant LUAD) fail to respond to ICB. Critically, KK-mutant LUAD are also recalcitrant to newer KRAS inhibitors, such as Sotorasib (KRASG12C inhibitor). Therefore, a greater understanding of the tumour-intrinsic characteristics that govern underlying cancer processes for KL and KK mutant LUAD are required to improve therapeutic outcomes. Thus, the overarching goal of this body of work was to identify novel vulnerabilities distinct to these subtypes of KRAS-mutant LUAD and develop new personalised treatment modalities for patients. To investigate the impaired immunotherapeutic response of KL-mutant LUAD, the development and validation of top gene candidates from a whole-genome CRISPR/Cas9 screen was performed in a genotype specific manner. The use of pre-clinical genetically engineered mouse models (GEMMs) was also leveraged to replicate the disease in vivo. Impaired CD8 T cell function is a defining characteristic of KL-mutant LUAD. To replicate this feature of disease, I developed an unbiased in vitro CRISPR/Cas9 whole-genome knockout (KO) screening platform whereby murine KL-mutant lung tumour cells are co-cultured with cytotoxic CD8 OT-I T cells to mimic tumour-immune interactions in vivo. This strategy successfully identified components of the MHC-I antigen presentation (Tap2 and B2m) and Jak/Stat signalling (Jak1, Jak2, Stat1, Stat2, Ifngr1 and Ifngr2) pathways, readily reported to confer resistance to immunotherapy, validating my screening approach. Importantly, this platform also identified known immunotherapeutic candidates (Ptpn2 and Serpinb9) that sensitised KL-mutant tumour cells to killing by cytotoxic CD8 T cells. Excitingly, Kdm2b an epigenetic regulator of the variant polycomb repressive complex 1 (PRC1.1) was identified as a novel gene candidate that sensitised KL-mutant tumour cells to CD8 T cell killing. Furthermore, through in vitro co-culture assays and in vivo growth assays, Kdm2b inhibition sensitised the growth and proliferation of KL-mutant tumour cells following immune pressure. Critically, the KO of Kdm2b synergised with anti-PD-1 treatment in vivo, highlighting a novel immunotherapeutic strategy for the treatment of KL-mutant LUAD in the clinic. To investigate characteristics unique to KK-mutant LUAD, as a means to design personalised treatment approaches, an in depth investigation of commonly utilised models of Keap1-deficiency was undertaken. These studies focused on interrogating the cellular origin of KK-mutant LUAD. Integrative transcriptomic analysis through RNA-sequencing (RNA-seq) technologies identified the upregulation of ciliated cell markers in lung tumours that arose in multiple Keap1-deficient GEMMs. Whilst the use of cell specific adeno-Cre viruses did not define a putative cell-of-origin for Keap1-mutant LUAD, RNA-seq analysis revealed the upregulation of the Sonic hedgehog (Shh) pathway, a critical pathway implicated in ciliogenesis and cilia formation within the lung, was restricted to Keap1-mutant tumours. With multiple Shh pathway inhibitors being trialled in the clinic, these results suggest a much-needed, novel therapeutic strategy for the treatment of KK-mutant LUAD. Taken together, my work highlights novel treatment strategies within KL- and KK-mutant LUAD, providing robust investigative studies on the utility of pre-clinical models for KRAS-mutant LUAD. I have utilised sophisticated pre-clinical GEMMs to replicate the human disease in a living organism and interrogate underlying characteristics that define these subtypes of KRAS-mutant LUAD, with future results from these studies guiding new treatment approaches in the clinic. Moreover, my PhD studies have established a CRISPR screening platform that may serve as the foundation of future work to investigate and interrogate novel immunotherapeutic targets in other non-responsive subtypes of lung cancer, and other cancer types.
Identification of molecular pathways associated with susceptibility and immunity to severe dengue and malaria

Studniberg, Stephanie Irene ( 2023-12)

In this era of increasing globalisation, urbanisation, and worsening climate change, the geographical range of transmission-competent mosquito vectors is shifting. Mosquito-borne diseases such as malaria and dengue are gradually emerging in previously unaffected areas, and re-emerging in areas where they had once previously subsided. With alarming increases in dengue case incidence, and for the first time, a chapter addressing the influence of climate change on malaria transmission in the World Malaria Report, it is clear that these two important vector-borne diseases are of utmost global relevance. As per the World Health Organization (WHO) guidelines, individuals presenting with warning signs signifying progression to severe dengue are required to remain under hospital observation. However, these warning signs appear late in the disease course and are non-specific. Consequentially, hospitals become overwhelmed with patients admitted for in-patient observation, many of whom do not progress to severe dengue. Biomarkers to detect progression to severe dengue upon hospital presentation are much needed to improve patient triage and resource allocation. In malaria, despite the great achievement of the recommendation by the WHO for the use of the RTS,S and R21/Matrix-M vaccines in children living in endemic areas, reductions in malaria case incidence remain at a prolonged stall. It is clear that efficacious vaccines approved for children to adults are required to reduce the global malaria burden. Further elucidation of the molecular mechanisms underlying the immune response to dengue and malaria is imperative if these outcomes are to be achieved. To address these outstanding concerns, an integrative systems immunology approach was utilised to identify molecular pathways associated with susceptibility and immunity to severe dengue and malaria. The studies within this thesis have integrated data from single-cell mass cytometry, serology, and transcriptional profiling of peripheral blood mononuclear cells from individuals progressing to either dengue fever (DF) or dengue haemorrhagic fever (DHF), as well as individuals living in a malaria-endemic regions of Indonesia with either symptomatic or asymptomatic Plasmodium falciparum and Plasmodium vivax malaria. Integrative data analysis identified frequencies and transcriptional profiles of effector CD4+ and CD8+ T cells as important components of dengue immunity in individuals progressing to DF. Furthermore, high frequencies of defined populations of CD4+ non-classical monocytes were associated with increased odds of developing DHF. Our approach discovered a strong transcriptional phenotype of immunosuppression underlying asymptomatic P. falciparum malaria, suggesting that the carriage of these infections could preclude complete parasite clearance. Lastly, unlike symptomatic P. falciparum malaria that induced a highly inflammatory response, clinical P. vivax infection featured the upregulation of anti-inflammatory pathways and checkpoint receptors, providing a feedback loop to ameliorate symptomatic infection. Furthermore, gene set enrichment analysis revealed profound dysfunction of the blood monocyte compartment in both symptomatic and asymptomatic P. vivax malaria. Together, the findings in this thesis have critical implications for the deployment and efficacy of malaria vaccines, and for the development of diagnostic tools to predict disease outcomes for dengue patients at point-of-care.
Molecular Study of LUBAC and its subunit HOIL-1

Wang, Xiangyi ( 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.
Identifying novel oncogenes and tumour suppressor genes in cancer

Potts, Margaret Anne ( 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.
Manipulation of host defence mechanisms by Toxoplasma gondii

Ruparel, Ushma ( 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.
A conserved molecular mechanism of erythrocyte invasion by malaria parasites

Seager, Benjamin Andrew ( 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.
The role of programmed cell death and inflammation in the pathogenesis of SARS-CoV-2 disease

Mendes Bader, Stefanie ( 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.
Understanding protein variants with high-throughput mutagenesis and machine learning

Fu, Yunfan ( 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.

Medical Biology - Theses

Permanent URI for this collection

Filters

Date

Author

Subject

Type

Settings

Sort By

Results per page

Statistics

Citations

Search Results