Medical Biology - Theses
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ItemThe roles of Hhex and Ikzf1 in murine NK cell biology( 2018)Natural killer (NK) cells are effector lymphocytes of the innate immune system that are known for their ability to kill transformed and virus-infected cells. NK cells originate from haematopoietic stem cells in the bone marrow and studies on mouse models have revealed that NK cell development is a complex and tightly regulated process. The development of NK cells can be broadly categorised into two phases: lineage commitment and maturation. Whilst the transcription factors Hhex and Ikzf1 have previously been implicated in NK cell lineage commitment, their importance in NK cell maturation has remained enigmatic until now. Using mouse models that have recently become available, the expression of these transcription factors was specifically inactivated in lineage-committed NK cells to determine the role of these transcription factors in maturing NK cells. The role of Hhex and Ikzf1 in NK cell effector function was also examined, since NK cell maturation is accompanied by the acquisition of cytotoxic functions. This study identifies Hhex and Ikzf1 as novel regulators of NK cell homeostasis, with the loss of either Hhex or Ikzf1 conferring defects in NK cell survival. Through comprehensive phenotyping and transcriptomic profiling, Hhex was determined to be required for NK cell survival by blocking accumulation of the pro-apoptotic factor BIM, while Ikzf1 promoted NK cell survival by maintaining IL-15 responsiveness. Overall, Ikzf1 appears to play a greater role in NK cell biology than Hhex, since Ikzf1-deficient NK cells presented additional anomalies in maturation and function. Interestingly, emerging data suggests that both transcription factors are required to maintain the appropriate rates of cell metabolism that underpins many aspects of NK cell biology. Finally, RNA-sequencing of Hhex- and Ikzf1-deficient NK cells has revealed new directions for future studies that will improve understanding of how these two transcription factors regulate NK cell biology.
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ItemInsights into mechanisms of resistance to apoptosis in Epstein-Barr virus-associated T and NK cell lymphomas( 2018)Ectopic Epstein-Barr virus (EBV) infection of T or NK cells is associated with several aggressive diseases including extra-nodal NK/T cell lymphoma (ENKTL) and chronic active EBV (CAEBV). Resistance to standard chemotherapy results in extremely poor patient prognosis. This thesis aimed to uncover the cellular and viral mechanisms behind such resistance, and to identify vulnerabilities for therapeutic targeting. An extensive collection of patient-derived cell lines demonstrated an EBV latency II phenotype and exhibited resistance to clinically administered genotoxic compounds. Importantly, apoptotic cell death could be consistently induced following treatment with the BH3 mimetic, A-1331852, that inhibits the BCL-2 pro-survival protein, BCL-XL. Furthermore, A 1331852 proved effective in 1 of 3 newly developed xenograft NSG mouse models. In the two non-responder cell lines, it was shown that BCL-XL dependency was potentially maintained by viral LMP1 in an IL-2-dependent manner. IL-2 withdrawal in vitro, emulating the in vivo environment, led to a survival dependency switch from BCL-XL to MCL-1, and ultimately a reduced response to A-1331852. Finally, to identify novel cytotoxic drugs for ENKTL and CAEBV, a high-throughput screen of 4079 compounds was performed. This identified a shortlist of effective hits for further study in these severe diseases where targeted treatment options are unexplored.
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ItemRegulation of human T-cell function by short chain fatty acids( 2018)Short chain fatty acids (SCFAs) are important bacterial metabolites produced by the fermentation of complex carbohydrates in the large gut. Luminal SCFAs are act locally to maintain the integrity of the gut epithelium and are also absorbed into the portal blood circulation to potentially have systemic effects. Studies of the immune modulatory functions of SCFAs have largely been confined to mice. There is a growing appreciation of the importance of SCFAs in human health and, in particular, on human immune function. The present studies examine the effects of SCFAs on human T cells and the mechanisms underlying these effects. I found, NaBu and NaPo significantly increased expression of IFN-g and IL-10 in human CD4+ and CD8++ IL-2 T cells. Despite expressing IFN-g, NaBu- or NaPo-exposed CD4+ and CD8++ IL-2 T cells exhibited regulatory (suppressive) function on T cell proliferation, which was partially reversedby antibody blockade of IFN-g or/and IL-10 signaling. Inhibition of glycolysis decreased the ability of NaBu to increase IFN-g expression. However, glycolytic activity was not affected by NaBu, suggesting that glycolysis is a necessary but not sufficient requirement for induction of IFN-g by NaBu. On the other hand, fatty acid oxidation (FAO) was activated in NaBu-treated cells and blockage of FAO partially reversed IFN-g expression induced by butyrate, indicating the involvement of FAO in regulation of IFN-g expression by NaBu. The intermediate metabolite of NaBu metabolism in mitochondria, citrate, which can be converted to acetyl-CoA, was found to be increased in NaBu-exposed T cells. NaBu exposure was associated with increased acetylation of histone and non-histone proteins. Further studies are required to determine whether butyrate promotes acetylation by activating citrate-acetyl-CoA-HAT pathway. Induction of IL-10 expression was not affected by blockage of glycolysis or FAO, indicating that induction of IFN-g and IL-10 by NaBu is by different mechanisms. Acetylation of H3K9 and H3K14 (H3K9ac and H3K14ac) was increased in NaBu-treated cells and H3K9ac and H3K14ac were enriched at the promoter region of the IL-10 gene. Interestingly, the IFN-g promoter was only enriched for H3K14ac. In addition, NaBu may modulate IFN-g expression post-transcriptionally. I found that NaBu increased acetylation of proteins, including GAPDH, in T cells and that IFN-g mRNA was bound to acetylated GAPDH, which increased its stability. Thus, butyrate promoted GAPDH acetylation, which in turn stabilized IFN-g mRNA by direct association. My findings underscore the complexity of immune modulation by short chain fatty acids and are relevant to understanding the relationship between diet and health.
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ItemInhibitor of APoptosis proteins (IAPs) and SHARPIN regulate the immune response in the skin to limit inflammation and maintain homeostasis( 2018)The skin is a remarkable organ, a barricade between our vulnerable insides and a constantly changing environment full of physical, chemical, and biological aggressors. Maintenance of barrier integrity, immune surveillance, and rapid response are fundamental, and this multifaceted protection is orchestrated by the epithelial barrier and immune cells. Acute and chronic inflammatory skin diseases can arise due to abnormal over-reactions to the changing environment. A number of these diseases have been associated with genetic aberrations of the TNF super family and innate receptors signalling. My PhD studies have focused on the role of particular E3 ligases in regulating inflammatory signalling in skin homeostasis and inflammation. Inhibitor of APoptosis proteins (IAPs) and the Linear Ubiquitin-chain Assembly Complex (LUBAC) are E3 ubiquitin ligases that play crucial roles in innate immunity by regulating cell death and survival pathways from the TNF and pattern recognition receptor families. Genetic or pharmacological disruption of the IAPs or LUBAC member SHARPIN induce dermatological phenotypes with interesting parallels to a variety of human skin diseases. To investigate the contribution of immune cells to the Sharpincpdm cutaneous phenotype I utilised the transgenic Diphtheria Toxin Receptor (DTR) system to specifically ablate particular immune cell subsets in-vivo. I have found that Langerhans cells play a pivotal role in the cell death mediated skin disease that arises in Sharpin mutant mice, placing them as a potential cellular source of pathogenic TNF in the Sharpincpdm skin, and highlighting a T-cell independent role for Langerhans cells in driving skin inflammation. Epidermal specific genetic deletion of the cellular IAPs (cIAPs) resulted in early post-natal lethality due to widespread dermatoses. Pharmacological loss of cIAP1, cIAP2 and XIAP by subcutaneous injection of an IAP antagonist drug (smac-mimetic; SM) into mice induced a Toxic Epidermal Necrolysis (TEN) like local inflammatory lesion characterised by keratinocyte cell death, immune cell infiltration, and increased production of pro-inflammatory cytokines. Both the genetic and pharmacological phenotypes can be ameliorated by the loss of a single allele of RIPK1. I have conducted a screen injecting SM into a panel of knock-out and mutant mouse strains in order dissect the complex set of interactions initiated by injection of SM and leading to the TEN like lesional response. I found that disruption of IAPs leads to a breakdown in immune tolerance to commensal microorganisms, which can then initiate inflammatory responses in the skin. A full response to SM depends on interactions between innate immune signalling pathways, immune cells, and the microbiota, nicely highlighting the multifaceted processes involved in skin inflammation and cell death.
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ItemCytokine signalling in haematopoietic cells( 2018)Cytokine receptor signalling is essential for cell survival, proliferation and subsequent differentiation of haematopoietic stem cells (HSCs). Cytokines control development of haematopoietic progenitors into cells of the myeloid, lymphoid and erythroid lineages by stimulating cell cycle progression, proliferation and differentiation as well as by inhibiting apoptosis. My work focusses on Interleukin-3 (IL-3) and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), two essential cytokines in haematopoiesis. Binding of a cytokine to its specific receptor leads to the activation of multiple kinase signalling pathways, including the JAK/STAT, Ras-MAP kinase (MAPK) and PI3-kinase/AKT pathways. In this signalling network, the IkappaB Kinase (IKK) complex plays an important role as a downstream signalling hub. My research investigates how IL-3 mediated IKK activation promotes the survival of myeloid cells and what role this process may play in the development of related diseases, such as myeloproliferative disorders. Using immortalised growth factor (IL-3 or GM-CSF) dependent myeloid progenitor cells (FDMs), as well as employing various in vivo mouse models of haematopoietic development, I was able to show that 1) IKK is a major signalling hub linking IL-3, TNFR1 and p53 signalling to control the survival in haematopoietic cells by describing for the first time a role for the E3 ubiquitin ligase MDM2 downstream of IL-3- or TNFalpha-mediated IKK2 activation, suggesting crosstalk between the NF-kB signalling and p53 signalling pathways; 2) IKK regulates cellular metabolism through activation of NF-kB- and p53-dependent metabolic target genes by showing that deletion of IKK2 but not IKK1 in hematopoietic cells significantly alters cellular metabolism, impairing oxidative phosphorylation and upregulating glycolysis due to altered expression of p53-dependent metabolic target genes; 3) IKK plays a crucial role during haematopoietic development, regulating myeloid cell proliferation, lineage commitment and survival, showing that deletion of IKK2 but not IKK1 in haematopoietic progenitor cells severely affects haematopoietic development by skewing lineage commitment in vivo, resulting in neutrophilia, elevated circulating interleukin-6 and lethality due to severe gastrointestinal inflammation. The work presented in this thesis provides new important insights into the role of IKK in haematopoietic cells and haematopoietic development and clearly demonstrate that IKK1 and IKK2, the two catalytic subunits of the IKK complex, have distinct functions depending on the context of activation. In the future, this fact could be exploited to develop novel targeted therapies to specifically target a subunit in disease settings such as haematopoietic malignancies where aberrant NF-kappaB activity is frequently observed.
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ItemUnderstanding the mechanisms of protein export in Plasmodium berghei liver infection( 2018)The malaria parasite exports a large repertoire of proteins into the host erythrocyte during blood stage infection. This essential process of host cell remodelling is largely dependent upon the cleavage of a conserved amino acid motif, termed the Plasmodium Export Element (PEXEL). The aspartyl protease plasmepsin V is the enzyme responsible for this cleavage event and previous attempts at genetic deletion have been unsuccessful. Although years of research have revealed the mechanisms behind protein export in erythrocytic infection, less is known about how the parasite exports proteins during the liver stage of malaria infection. This dissertation identifies the molecular mechanisms the parasite requires to facilitate export into the host hepatocyte. Using the Flp-FRT system to conditionally control the expression of plasmepsin V across the life cycle of P. berghei (rodent malaria), we achieved a complete deletion of the 3’UTR of the plasmepsin V gene, specifically in sporozoites. Analysis of this parasite line revealed a defect in liver stage development as the parasites arrested from 6 h following sporozoite infection in mice. Infected mice did not develop patent blood stage infections. Together this suggests an essential role for plasmepsin V during liver stage malaria infection. In order to understand how the parasite exports proteins during liver stage infection, the importance of the PEXEL motif needed to be addressed. We identified several PEXEL-containing protein candidates and examined their export during liver stages to show that their export is dependent upon the presence of a functional PEXEL motif or plasmepsin V. These important findings may help the further identification of exported proteins. It is ultimately important to also contextualise how parasitic infection may change the dynamics of hepatocyte homeostasis, possibly by the result of exported proteins. Recent studies have shown the parasite’s ability to inhibit host hepatocyte apoptotic mechanisms during infection. We examined this using a knockout of key apoptotic inhibitory regulators (cIAP1/2) and found that their absence in mice caused an increase in resistance to liver stage infection. Chemical anatagonism of these apoptotic regulators replicated this effect, highlighting the potential for new drug targets for liver stage malaria. This thesis explores the underlying mechanisms behind protein export in liver stage Plasmodium berghei infection. The results shed light on what is required for the parasite to export proteins to the hepatocyte, for the first time, confirming the importance of plasmepsin V and the PEXEL motif. These discoveries will lead to potential new drug targets and vaccines candidates, aiding the world’s fight to eradicate the debilitating disease burden of malaria.
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ItemRegulation of the apoptotic machinery by the E3 ubiquitin ligase Parkin( 2018)The cyto-protective molecule Parkin is a criticial player in autosomal-recessive Parkinson’s disease, with approximately half of all patients with this condition carrying a mutant allele of PRKN/PARK2. The E3 ubiquitin ligase Parkin acts in concert with its upstream kinase, PINK1, to survey the mitochondrial network and rapidly induce mitophagy in response to damage. Parkin-mediated ubiquitination of mitochondrial substrates leads to the selective tagging and quarantine of damaged mitochondria. The accumulation of ubiquitinated mitochondrial outer membrane proteins allows autophagy receptors to bind and subsequently recruit and extend the autophagophore. This leads to the specific autolysosomal degradation of damaged mitochondria and the maintainence of mitochondrial homeostasis. The motor-deficit associated with Parkinson’s disease is caused by the selective cell death of dopaminergic neurons in the brain and is associated with homozygous loss of function mutations in either PINK1 or Parkin in early onset-disease. However, the link between PINK1/Parkin-mediated mitophagy and cell death remains unclear. Here I present evidence that Parkin modulates intrinsic apoptosis by slowing the kinetics of both BAK- and BAX-mediated cell death. I have characterised BAK as a novel substrate of Parkin and identified K113 as the key site of ubiquitination (Chapter 3). This modification in the hydrophobic groove slows BAK activation, dimerisation and oligomerisation on model membranes and on mitochondria. I hypothesise that BAK inhibition by Parkin stalls mitochondrial outer membrane permeabilisation, allowing for the effective autophagic clearance of damaged mitochondria to prevent errant apoptosis. I also identified a potential new mechanism by which Parkin signalling can inhibit BAX-mediated apoptosis (Chapter 4). The mechanism by which Parkin inhibits BAX is distinct from that of BAK however, as I observed no direct ubiquitination of BAX. Instead, my data suggests that VDAC2, a key BAX interacting protein and predominant Parkin substrate, is the key determinant of Parkin-mediated inhibition of BAX. Furthermore I confirmed the lysine residues in VDAC2 that are required for its ubiquitination during mitophagy and postulate that abrogating VDAC2 ubiquitination will restore BAX-mediated apoptosis during Parkin signalling. Furthermore, this thesis interrogated the role of PINK1/Parkin signalling following apoptotic damage to mitochondria and discusses implications for inflammatory signalling in the context of Parkinson’s disease (Chapter 5). Here I propose a mechanism for Parkin signalling in silencing inflammation caused by persistent apoptotic mitochondria. This provides a link between defective autophagic clearance of damaged mitochondria and the induction of inflammatory signalling commonly observed in Parkinson’s disease. I postulate that mitochondrial dysfunction and BAK/BAX-dependent release of mitochondrial DNA are the key drivers of neuroinflammation in Parkinson’s disease and characterise murine models that will prove invaluable in interrogating this hypothesis. This thesis provides a comprehensive interrogation of the interplay between PINK1/Parkin signalling and the intrinsic apoptotic machinery. Here I describe a multi-faceted coordination of apoptosis and inflammation by Parkin and provide an in depth model for how Parkin acts as a master regulator in the context of Parkinson’s disease.
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ItemUnderstanding RNA transport via exosomes and SIDT2( 2018)The discovery that RNA not only functions as an intermediate between a gene and a protein but also possesses regulatory functions has brought significant interest in its potential role in development and disease, and it is now apparent that regulatory RNAs are important for the regulation of multiple biological processes. What is less clear, however, is the role of RNAs as intercellular signaling molecules and the mechanisms by which RNAs are transported between cells. One mechanism that has received significant attention involves exosomes, which are nanoscale vesicles released by multiple cell types. While exosomes have been shown to contain regulatory RNAs and to mediate intercellular communication, how RNAs are transferred into exosomes remains poorly understood. One protein that has been recently implicated in RNA transfer is the endo-lysosomal protein SIDT2. While it appears to be important in the transport of viral RNAs for innate immune recognition, SIDT2’s role in trafficking endogenous RNAs remains to be investigated. In this thesis, after providing a general introduction and outlining my relevant methods in the first two chapters, I describe in Chapter 3 how I profiled exosomal RNAs from mouse dendritic cells (DCs) using RNA-Seq and identified a long noncoding RNA, VL30, which is highly enriched in exosomes. Having observed that VL30 lncRNA can be transferred to recipient cells both in vitro and in vivo, my bioinformatic analysis revealed that exosome-enriched isoforms of VL30 lncRNA contain a repetitive motif. Subsequent experiments showed that the motif itself is efficiently incorporated into exosomes, suggesting the possibility that it might directly promote exosomal loading and be useful in future efforts to selectively load therapeutic RNAs into exosomes for clinical use. However, the repetitive motif is predicted to fold into a long double-stranded RNA (dsRNA) hairpin and, consistent with this, its overexpression was associated with induction of a type I interferon response and cell death. In Chapter 4, I explored the role of the dsRNA transporter SIDT2 in incorporating RNAs into exosomes. By confocal microscopy, I showed that SIDT2 co-localises with the exosomal marker CD63, suggesting the possibility that SIDT2 might promote exosomal RNA loading. However, my subsequent studies failed to demonstrate a role for SIDT2 in influencing either the microRNA or long RNA content of exosomes. In Chapter 5, motivated by a desire to understand the possible endogenous RNA substrates of SIDT2, I explored the role of SIDT2 in skeletal muscle. I observed that mice deficient in SIDT2 develop a skeletal muscle myopathy and die prematurely, and that loss of SIDT2 leads to impairment of autophagy, with an accumulation of autophagic vacuoles. If and how the RNA transport function of SIDT2 relates to the impairment of autophagy remains unclear, but interestingly I found that in the absence of SIDT2 endogenous dsRNA accumulates in muscle fibres, leading to the induction of a type I interferon response and apoptosis, with selective loss of fast 2B myofibers and a corresponding reduction in muscle force and grip strength. Finally, in chapter 6, I investigated the effects of SIDT2 in an RNA editing-deficient mouse model that carries the ADAR1 E861A loss-of-function allele. These mice accumulate endogenous dsRNA due to their inability to perform RNA editing of dsRNA substrates, and die during embryonic life due to a massive type I interferon response. Given that loss of SIDT2 has been found to modulate the interferon response by trafficking viral dsRNA, I hypothesised that loss of SIDT2 might modulate the phenotype of the ADAR1E861A/E861A mice, but found this not to be the case.
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ItemThe functional role of interferon regulatory factor 4 in plasma cells( 2018)Plasma cells are a critical component of the adaptive immune system being responsible for the production of large amounts of antibodies which can bind and remove pathogens. Plasma cell formation and function is regulated by the interactions between pro-plasma cell and pro-B cell transcription factors. Transcription factors which promote plasma cell fate include IRF4, Blimp-1 and XBP-1. In the absence of IRF4 plasma cells fail to differentiate from B cells and established plasma cells rapidly die. IRF4 is therefore known to be critical for plasma cell differentiation and survival, however, the mechanism by which it exerts its function remains unclear. The work in this thesis aims to define this mechanism. In this thesis, the deletion of IRF4 was identified to induce apoptosis in plasma cells. An in-vivo model was established which allowed the conditional deletion of IRF4 whilst over-expressing the anti-apoptotic protein Bcl-2 during which a proportion of plasma cells survived IRF4 deletion (Chapter 3). This in-vivo model allowed for a detailed interrogation into the transcriptional changes that occur on IRF4 deletion in plasma cells. RNA sequencing identified that IRF4 plays a critical role in plasma cell identity by transcriptionally regulating cell surface proteins such as CD138, transcription factors such as XBP-1 and immunoglobulin heavy and light chains (Chapter 4). Additionally, IRF4’s transcriptional network suppresses other lymphoid lineages, however, a loss of IRF4 does not cause plasma cells to de-differentiate into B cells (Chapter 4). A loss of IRF4 leads to structural changes in plasma cells predominantly manifesting as a reduction in the amount of endoplasmic reticulum; changes which are likely mediated by reduced XBP-1 protein levels. In addition, IRF4 plays a role in regulating plasma cell metabolism with deletion leading to increased metabolic capacity, mitochondrial numbers and increased reactive oxygen species (Chapter 5). In conclusion, this thesis defines the changes that occur within established plasma cells on IRF4 deletion and places IRF4 in a central and indispensable role for the cellular function and identity of plasma cells.
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ItemImproving differential expression analysis of single-cell RNA-seq data: method and application( 2018)Single-cell RNA sequencing (scRNA-seq) technology enables high-throughput transcriptome profiling at single-cell resolution, providing researchers with an unprecedented opportunity for dissecting heterogeneous biological systems. However, the distinct features of scRNA-seq data also present us a variety of analytical challenges. This thesis focuses on the bioinformatic analysis of scRNA-seq experiments. We first describe the design of a novel statistical framework for differential gene expression (DE) analysis. Through explicit modelling of the molecule capturing process, we are able to perform DE analysis on the inferred pre-dropout molecule counts. Benchmarking using simulated and real data showed improved performance compared with existing methods. Then we show a case study of a breast cancer tumour infiltrating T cell dataset. Leveraging several newly developed methods for scRNA-seq data, including our DE method, we identified and characterized a novel cell population, tissue-resident memory T cells, in the tumour infiltrating T cells.