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Item Embargo Mechanisms Underpinning Functional Resident Humoral Lung ImmunityGuillaume, StephaneTertiary lymphoid organs form de novo in response to allergy or infection, in the inflamed sites of autoimmune diseases and even adjacent to solid cancers. Whether or not these structures are functionally competent and can support antibody affinity maturation is unknown. This project aims to elucidate key differences between classic and ectopic germinal centres which may inform how we can improve medical interventions to enhance humoral protection against autoimmunity and infectious disease. B cells undergo selection in secondary lymphoid tissue germinal centres which can improve the way their B cell receptors bind antigen and generate antibodies that provide protection against infections. Due to their de novo formation and less stringent assembly, it is unknown whether tertiary lymphoid organs are functionally competent and can support antibody affinity maturation. Evolutionarily, an identical GC reaction in secondary and tertiary lymphoid organs may represent an energetically expensive duplication of efforts, and therefore I hypothesise that the spontaneously formed tertiary lymphoid organs fail to support efficient somatic hypermutation, thereby producing more broadly reactive antibodies. CD69 is an early-activation marker which is understood to be upregulated rapidly upon lymphocyte activation and can promote tissue-retention of T cells. I hypothesise that CD69 has a role in ectopic germinal centre function and tissue-resident memory B cell retention. The function and mechanism of CD69 for germinal centre B cells has yet to be elucidated, as well as its role in the formation and maintenance of ectopic germinal centres as distinct from those in secondary lymphoid organs. Here, I reveal fundamental differences and similarities in the humoral biology that underpins germinal centres in the lung and lymph nodes. Lung ectopic germinal centres were capable of supporting affinity maturation and seeding memory B cells, despite their de novo formation. While CD69 on T follicular helper cells was dispensable to maintain functional lymph node germinal centres, CD69 expression on B cells was important for their participation in both the lung and lymph node germinal centre reactions, suggesting a role for CD69 on B cells. This research contributes to current understandings of ectopic and conventional germinal centre biology and may have implications in improving vaccine design and medical interventions.Item Embargo Investigation of IGF1R/IRS1 Signalling Mechanisms in B Cell DevelopmentFrench, Elise CatherineThe adaptive immune system declines as we age. This includes reduction in B cell number and function, contributing to increased vulnerability to infection in the elderly. Current evidence is consistent with age-associated defects emerging during early B cell development in the bone marrow (BM). Here, B cells develop by transition through distinct progenitor stages as the immunoglobulin heavy and light chain variable (V), diversity (D) and joining (J) genes are differentially rearranged in VDJ recombination. This assembles a complete B cell receptor (BCR) and is the primary step in producing a diverse BCR repertoire; an essential requirement for a robust adaptive immune response. The roles of interleukin-7 receptor, pre-BCR and chemokine receptor type 4 signalling are well established in this process. There is now growing evidence supporting a role for Insulin-like Growth Factor 1 Receptor (IGF1R) signalling in progenitor B cell development. Studies report marked reduction of B cell progenitor numbers and antibody diversity in aged mice and humans. The underlying mechanisms and causes are unclear. Genome-wide analysis of aged mouse BM B cell progenitors revealed that the major defect was multi-layered transcriptional downregulation of components of IGF1R signalling. This was most significant for the IGF1R adaptor protein, Insulin Receptor Substrate 1 (IRS1), and suggests multi-faceted dysregulation of IGF1R/IRS1 signalling in ageing B cell progenitors. IGF1R signalling functions in cellular growth and proliferation in numerous mammalian tissues, but its role in normal B cell development is poorly understood. Its impairment in ageing progenitor B cells, coincident with reduced cell numbers and antibody diversity, points to a key role of IGF1R signalling in B cell development. I therefore hypothesise a non-redundant role for IGF1R/IRS1 signalling in progenitor B cell development in the BM, and therefore that this signalling is critical to support the generation of adaptive immune cells. In this thesis, I aim to characterise the impact of IGF1R and IRS1 deletion in B cells during their development in the BM. To achieve this, I established two progenitor B cell-specific conditional knockout (KO) reporter mouse models. The first targets IRS1, to interrogate intracellular signalling events downstream of the IGF1R. The second targets the IGF1R, to investigate its response to the extracellular environment. Since IRS1 was the most significantly downregulated component of IGF1R signalling in ageing progenitor B cells, the hypothesis for a role of IRS1 in BM B cell development was tested first. Flow cytometry analysis of the IRS1 KO BM indicated a block at the pro- to pre-B cell progenitor transition. The model was then employed in functional assays to investigate the underlying mechanisms of this dysregulation. A mixed BM chimera setup was developed to evaluate the intrinsic ability of IRS1 KO B cells to progress through development in competition with cells without defect. Optimisation of culture methods for *in vitro* IRS1 KO B cells facilitated interrogation of the impact of IRS1 deletion on the pro- to pre-B transition *in vitro*. VDJ-seq analysis of the BCR repertoire provided insight into the putative role of IRS1 signalling in VDJ recombination and repertoire selection. Finally, the IGF1R KO was analysed to investigate the impact of IGF1R deletion during B cell development. Findings from this research establish a key role for IGF1R/IRS1 signalling in B cell development and further our understanding of the impact of its impairment in ageing. This study also provides opportunities for developing therapeutic approaches for healthy ageing; a pertinent issue for today’s ageing population.Item Open Access Towards elucidating the function of the oocyte Subcortical Maternal Complex: A multi-omic analysis of an Nlrp5-null murine modelNic Aodha, LeahThe subcortical maternal complex (SCMC) is composed of some of the most abundant proteins in the oocyte. SCMC mutations have been linked to early embryo arrest, recurrent molar pregnancies and imprinting disorders. Genomic imprinting depends upon correct patterning of DNA methylation over imprinted domains during oogenesis. It was previously shown that oocytes deficient in the human SCMC gene *KHDC3L* had globally impaired methylation, thus suggesting a role for the SCMC in the establishment or maintenance of DNA methylation marks at imprinted regions. This thesis aims to identify the functional role of the most abundant SCMC protein NALP5 and, by extension, the SCMC, in oocyte developmental competence. This study focuses primarily on elucidating the role of NALP5 in the context of DNA methylation patterning in the oocyte. This was done by establishing a null mouse line for *Nlrp5*, the gene encoding NALP5. A phenotypic analysis of the *Nlrp5* mutant confirmed that embryos derived from *Nlrp5*-null oocytes arrest before or at the 2-cell stage of development. Proteomic analysis of *Nlrp5*-null oocytes shows significantly reduced abundances of other SCMC proteins, demonstrating that the *Nlrp5* knockout model can effectively interrogate the function of the complex at large. GDF9 and BMP15, regulators of ovarian follicle development via paracrine signalling, were also notably reduced in *Nlrp5*-null oocytes. Other proteins altered in *Nlrp5*-null oocytes include epigenetic modifiers DNMT3L and UHRF1, both of which are reduced in the mutant, the former by over 75%, and the latter by 40%. Immunofluorescence analysis of *Nlrp5*-null oocytes indicates an altered DNMT3L localisation, but no change in DNMT1 localisation. Single-cell transcriptomic analysis of *Nlrp5*-null oocytes detected over 580 significant differentially expressed genes when compared with wild type oocytes, but no robust enrichment in any molecular pathways. Parallel single-cell methylation analysis indicates a slight global reduction in DNA methylation, which could reflect the strong reduction in the *de novo* methyltransferase DNMT3L. Some methylation differences overlapped with imprinted genes. Methylation profiling of *Nlrp5*-null murine oocytes does not indicate an impairment in methylation to the same degree as that of the human *KHDC3L* mutant but is still at a level that may cause transcriptomic dysregulation.Item Embargo The role of DEAD-box helicase 1 in humoral immune responsesKimber, RachaelA key characteristic of humoral immune responses is the ability of B-cells to recognise an endless array of antigens through their immunoglobulin (Ig) genes and secret these as soluble antibodies to help combat invading pathogens. Primary diversification occurs through VDJ recombination during B-cell development, whereas secondary diversification occurs during ongoing immune responses through the introduction of affinity enhancing point mutations in Ig variable genes, a process termed Somatic Hypermutation (SHM). Additionally, to tackle the variety of pathogens we may encounter, B-cells can substitute their Ig constant regions by Class Switch Recombination (CSR). CSR is a DNA deletional and recombinational event, in which the RNA-binding protein DEAD-box helicase 1 (DDX1) is implicated in targeting the mutagenic enzyme Activation-Induced Cytidine Deaminase (AID) to Ig loci to initiate CSR. The aim of this project was to interrogate further the role of DDX1 during humoral immune responses, to understand whether DDX1’s impact on B-cell biology extends beyond its role in CSR. To this end, we characterised DDX1’s contribution by utilising multiple conditional knock-out models. We focused on characterising the formation and function of Germinal centres (GC); microanatomical structures where affinity enhancement and B-cell clonal expansion occurs, following T-cell dependent immunisation. We discovered that GC-specific ablation of DDX1 resulted in reduced GC size with a reduction in antigen-specific B-cells, affinity maturation and clonal expansion, despite sufficient T-cell mediated selection. Further interrogation at the single-cell level pointed to a defect in post-transcriptional control of gene expression in GC B-cells upon DDX1-depletion. Furthermore, alongside a reduction in CSR, depletion of DDX1 prior to B-cell activation resulted in diminished generation of GC and extrafollicular B-cell responses. Although DDX1-deficient B-cells show comparable activation in vitro, these B-cells were severely impaired in proliferation, with indication that efficient translation in activated B-cells requires DDX1 expression. Finally, we demonstrate the validation of a novel mouse model which will enable characterisation of the contribution of DDX1’s ATP-dependent helicase activity on the GC phenotypes we have observed. Together, we have demonstrated that DDX1’s role in humoral immunity extends much further than its characterised role in CSR, with the identification of a novel role for DDX1 in modulating mRNA translation in activated B-cells.Item Embargo PROTEINi screens identify ORP9 as a novel regulator of KRAS-driven signallingWeatherdon, Laura JaneKRAS is one of the most frequently mutated proteins in cancer, driving the excessive activation of the downstream RAF-MEK-ERK cascade, alongside additional pathways that promote cellular proliferation, survival and migration. KRAS traffics to the plasma membrane in order to be activated and bind to its effector proteins. Despite almost 40 years of research, there has been limited success in direct targeting of KRAS, as well as attempts to inhibit KRAS localisation to the plasma membrane. While inhibitors to downstream pathway components have been clinically approved, patients generally develop resistance to these within a few months, highlighting the need to develop novel strategies to inhibit KRAS-driven signalling. Here, we have characterised reporter cell lines where induction of KRASG12V or BRAFV600E with Doxycycline drives the ERK1/2-dependent expression of GFP. GFP expression can be used as a reliable and quantitative readout for ERK1/2 pathway activation, allowing these cell lines to be used to identify novel regulators of the pathway. In a collaboration between the Cook lab and PhoreMost, PROTEINi screens identified two novel peptides with high sequence similarity, OPX-385670 and HPX-119370, that interfered with KRAS-driven signalling. In this thesis, we show that cell penetrating peptides derived from these, TAT-OPX and TAT-HPX, significantly inhibited pathway activation and subsequent GFP expression driven by mutant KRAS, but not mutant BRAF. Yeast-2-hybrid analysis revealed ORP9 to be a potential interactor of OPX-385670, and VAP proteins to be potential interactors of HPX-119370. ORP9 is a lipid transfer protein, and interacts with VAPs in the ER to transfer newly synthesised lipids out of the ER and into target organelles, such as the Golgi and late endosomes. ORP9 lipid cargoes include phosphatidylserine and cholesterol, which can be transferred out of the ER membrane in exchange for PI4P, which is then dephosphorylated by the ER-resident Sac1 phosphatase. TAT-OPX and TAT-HPX caused accumulation of PI4P on Rab7-positive endosomes, suggesting inhibition of ORP9-mediated lipid transfer. ORP9 knockdown reduced MEK1/2 phosphorylation, ERK1/2 phosphorylation and GFP expression downstream of mutant KRAS, but not mutant BRAF, highlighting ORP9 as a regulator of KRAS-driven signalling. As well as PI4P, TAT-OPX and TAT-HPX caused actin to accumulate on Rab7-positive endosomes, suggesting defects in actin nucleation. These actin puncta also co-localised with YFP-VPS29, a component of the retromer. The retromer is a coat complex that interacts with specific internalised cargo, budding off from early/late endosomes to deliver cargo back to the plasma membrane. This recycling relies on WASH-mediated actin nucleation for membrane fission and vesicle trafficking. CPP-mediated defects in actin nucleation coincided with inhibition of the retromer complex, determined by missorting of the retromer cargo GLUT1 from the plasma membrane to lysosomes. TAT-OPX and TAT-HPX, as well as ORP9 knockdown, also caused mislocalisation of KRAS from the plasma membrane in multiple cell lines, suggesting that the retromer may be involved in KRAS trafficking. Knockdown of VPS35, a retromer subunit, significantly inhibited KRAS-driven activation of the downstream pathway and expression of GFP, suggesting KRAS is a potential cargo of the retromer. The results presented in this thesis suggest that ORP9 is a novel regulator of KRAS driven signalling, potentially promoting its recycling to the plasma membrane. This may occur through both retromer-dependent and -independent routes, and suggests that affecting ORP9-mediated endosomal lipid levels may be a viable approach to inhibit KRAS localisation to the plasma membrane, and subsequent activation of downstream signalling pathways.Item Open Access An Investigation into the Function of CDS2 in Phosphoinositide Synthesis in Murine MacrophagesCollins, DanielPhosphoinositides (PIPn) are a branch of membrane phospholipids involved in regulating critical cellular functions, such as Phosphoinositide 3-kinase (PI3K) and Phospholipase C (PLC) signalling pathways, the definition of sub-cellular membrane identity and the sorting of proteins and lipids within intracellular compartments. GPCR-stimulated PLC generates the second messengers inositol (1,4,5)-trisphosphate and diacylglycerol (DAG) from phosphatidylinositol (4,5)-bisphosphate, resulting in a net loss in the cellular PIPn pool which is replenished by the activation of phosphatidylinositol (PI) synthesis. A critical intermediate for PI synthesis is cytidine diphosphate diacylglycerol (CDP-DAG), synthesized from phosphatidic acid (PA) in the endoplasmic reticulum by CDP-DAG synthase 1 & 2 (CDS1 & 2). The aim of this work is to investigate whether CDS2-dependent PI synthesis plays an active role in PIPn signalling and homeostasis under basal and PLC-activated conditions. In CDS2-KO bone marrow derived macrophages (BMDMs), basal PIPn levels are maintained, albeit with a clear steady-state accumulation of certain molecular species of PA, DAG and triacylglycerol (TAG). To interrogate the changes in pathway activity that underlie these effects, mass spectrometry coupled with metabolic tracing techniques (13C-glucose and 18O-H2O) were used. The results point to significant increases in both *de novo* PA synthesis and the phosphorylation of DAG by diacylglycerol kinases in CDS2-KO cells. This suggests that increased levels of PA synthesis support normal rates of CDP-DAG production and ultimately PI synthesis by CDS1, in the absence of CDS2. The effects of CDS2 deletion during stimulation of PLC by purinergic receptors was also investigated. Under conditions of chronic stimulation, CDS2-KO cells were unable to maintain their PI and PIP pools. This was also accompanied by an increase in PA accumulation and a substantial reduction in new PI synthesis, suggesting that loss of CDS2 compromised the ability of macrophages to sustain their PIPn pool under these conditions. In contrast, PIP2 pools were maintained in CDS2-KO cells and stimulated inositol trisphosphate production was normal. Interestingly, Ca2+ re-uptake into ER pools after PLC stimulation in CDS2-KO cells was found to be reduced, but the origin of this effect is unknown. Overall, the work in this dissertation defines an important role for the CDS2 isoform in supporting increased PI synthesis under conditions of chronic PLC stimulation and also highlights new points of regulation in lipid biosynthetic pathways that are used to maintain homeostasis in PIPn function.Item Embargo Optimisation of TrAEL-seq to study DNA damage and replication in complex and dynamic mammalian cell systemsKara, NeeshaMaintenance of genome stability is critical for cell survival, and consequently cells have evolved a complex set of mechanisms to ensure DNA is repaired and correctly replicated before cell division. Deficiencies in these repair pathways are heavily associated with the development of cancer and ageing, and it is therefore of interest to be able to detect and monitor the distribution of DNA damage and replication events across the genome. This work describes the development and optimisation of TrAEL-seq (Transferase-Activated End Ligation Sequencing), a novel sequencing method for genome-wide detection of DNA double strand breaks, replication fork stalling, and replication fork movement. Our method captures single-stranded 3’ DNA ends, which can be mapped across the genome with base pair resolution. TrAEL-seq requires no labelling, synchronisation or sorting of cells, making it very flexible and simple to implement. In this work I refined the protocol to suit studies of mammalian cell systems; I developed a multiplexing protocol for high-throughput sample processing and comparative quantitation, and optimised themethod’s compatibility with fixed cells. Through collaboration with Artios Pharma, I then used TrAEL-seq to investigate the effect of different DNA damage response inhibitors in cancer cells. Comparison of DNA replication between different therapies and time points provided interesting insights on the mechanism of action of such treatments. Surprisingly, this TrAEL-seq data did not reveal distinct genomic locations vulnerable to replication fork stalling or double-strand breaks, supporting the notion that such events occur in a more random distribution pattern across the genome. I then applied TrAEL-seq to study an alternative model of DNA replication stress and damage; oncogene-induced senescence (OIS) resulting from *HRASG12V* overexpression. Analysis of TrAELseq replication fork movement across a time course of OIS revealed global changes in the levels of DNA replication across the genome. Interestingly, the data revealed an accumulation of TrAEL-seq signal around a subset of R-loops in senescent cells, which were localised in subtelomeric regions. Follow-up investigations which characterised the nature and origin of these peak sites offer new perspectives on cellular senescence. Taken together, this work demonstrates TrAEL-seq as a novel and exciting tool to investigate a range of dynamic and complex mammalian cell systems.Item Open Access Investigating the role of ACSS2-mediated acetyl-CoA metabolism on EGFR-PI3K-PKB signallingCragoe, BethanyThe epidermal growth factor receptor (EGFR) signalling network regulates growth, survival and differentiation in mammalian cells. There are multiple signalling pathways downstream of the EGFR, including the phosphoinositide-3-kinase (PI3K)-protein kinase B (PKB) pathway. This pathway is not only essential for cellular function, but has also been commonly implicated in the diseased state, including cancer and immunological deficiencies, therefore understanding its regulation is fundamental in revealing its role in both physiological processes, and diseased states. Recently in the field, there has been a large emphasis on how cells utilise metabolites as nutrient sensors, to coordinate nutrient availability with cellular proliferation. Acetyl-CoA is an essential metabolite that regulates key cellular processes, including *de novo* lipid biosynthesis, energy production and protein acetylation. Whilst there are multiple routes of acetyl-CoA synthesis, acetyl-CoA synthetase short chain family member 2 (ACSS2) is responsible for acetate-dependent synthesis of acetyl-CoA. The objective of this study was to investigate the potential impact of ACSS2-mediated acetyl-CoA on EGFR-PI3K signalling, shedding light on the regulatory roles of metabolism in MCF10a cells' response to external growth factors. A combination of phenotypic assays and mechanistic studies, including the use of the ACSS2 inhibitor VY-3-249, initially suggested that ACSS2 activity directly influences PI3K-PKB signalling. However, the generation of CRISPR/Cas9 ACSS2 knockout cells provided the crucial model required to confirm that the effects observed with VY-3-249 were occurring off-target. This finding is significant for the field, given the widespread citation and general acceptance of the inhibitor. With this in mind, I decided to phenotype ACSS2-KO cells independently of PI3K signalling, to further understand the breadth of ACSS2 activity and importance. Very interestingly, through the use of mass spectrometry and bioenergetic analysis, I have preliminary evidence for the novel role of ACSS2 in the regulation of succinyl-CoA levels, and subsequent succinylation and mitochondrial function. Overall, the work in this dissertation has identified an important insight into off-target effects of a widely used ACSS2 inhibitor, VY-3-249, which should be carefully considered when used in the field. Additionally, I have optimised and created useful tools to study other functions of ACSS2. Our work also highlights an exciting, previously unidentified role of ACSS2 on succinyl-CoA levels and mitochondrial capacity, opening up a new avenue of exploration between metabolism and mitochondrial function.Item Embargo Characterising FAM117 proteins as novel targets of DYRK1BCassidy, Megan AliceCompared to other CMGC kinases the dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) represent an understudied family of protein kinases. Whilst DYRK1A has garnered more attention due to its links to Downs’ Syndrome and Alzheimer’s Disease, the other class I DYRK, DYRK1B, remains comparatively unexplored. Prior work in our lab has explored potential new DYRK1B substrates by utilising a phospho-SILAC screen. From this screen we identified two proteins of the same family, FAM117A and FAM117B, which along with the third family member (FAM117C/GLCCI1) have previously been identified as binding partners of class I DYRKs. FAM117 proteins have no clearly established biological function and so understanding interactions between DYRK1B and FAM117 proteins may help to elucidate further functions of DYRK1B and also identify roles of FAM117 proteins. Here I establish that FAM117 proteins represent DYRK1B inducible phosphoproteins that rely on kinase activity of DYRK1B for their phosphorylation and shown that FAM117B represents a direct substrate of DYRK1B. Furthermore, I demonstrate that both class I and class II DYRKs are capable of inducing post-translational modifications, that likely represent phosphorylation, of FAM117 proteins. This is not a characteristic shared by all CMGC kinases, with only the closely related CLK3 driving similar FAM117 phosphorylation. Whilst no 3D structures exist for FAM117 proteins, prediction software suggests they are highly disordered with many predicted phosphorylation sites. This high level of disorder suggests that stable expression of FAM117 proteins may rely on binding partners and/or modifications such as phosphorylation. Indeed, I show that phosphorylation of FAM117 proteins by DYRKs is associated with their increased expression in both short & long term overexpression systems as well as the endogenous proteins. Using FAM117B as a model FAM117 protein, I identify a high number of DYRK1B inducible phosphorylation sites, many of which are located in the PFAM domain which is conserved between FAM117 proteins. Additionally, the scaffold protein DCAF7, which mediates the interactions of class I DYRKs with their substrates, also binds FAM117 proteins and I demonstrate that FAM117 proteins, DYRK1B & DCAF7 form ternary complexes in cells. Understanding FAM117 function and how their interaction with DYRK1B affects the functions of both proteins may help us to better understand their functions. I demonstrate that expression of FAM117 proteins disrupt the normal distribution of DYRK1B, each in different ways, and that all are capable of perturbing a normal DYRK1B function, in this case the promotion of processing body formation. From observing FAM117 localisation I also show that they partially co-localise with cytoskeletal proteins, such as tubulin, and show strong co-localisation with focal adhesion proteins. FAM117 proteins are reported to be capable of binding the microtubule protein DYNLL1 and work from our lab has identified the intermediate filament protein KANK2 as a possible DYRK1B interactor. Both of these have been suggested to link microtubules to focal adhesions. Here I demonstrate that both DYNLL1 and KANK2 immunoprecipitate with FAM117 proteins and DYRK1B, suggesting a role for DYRK1B and FAM117 proteins in the regulation of focal adhesions which in turn may affect cell adhesion and migration. Given the role of microtubules in regulating membrane organelle dynamics this binding to DYNLL1 may also provide an explanation for the dysregulation of processing bodies caused by FAM117 proteins. These new insights into DYRKs, specifically DYRK1B, and FAM117 proteins are discussed further and used to suggest hypotheses for future investigation. Furthermore, the FAM117 proteins may serve as useful biomarkers for DYRK activation or inhibition.Item Open Access Investigating the immunosuppressive functions of the GPCR adapter protein NorbinChetwynd, StephenThis project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (NcdnΔmye) to investigate its role in these cells. They showed that NcdnΔmye mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli.
I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of NcdnΔmye mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of NcdnΔmye mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of NcdnΔmye mice during septic peritonitis. I showed that NcdnΔmye neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in NcdnΔmye neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk.
Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in NcdnΔmye neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of NcdnΔmye neutrophils.Item Embargo The Role of Autotaxin in CancerMaggs, LaurenAutotaxin (ATX/ENPP2) is a secreted phospholipase D enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA is the agonist for at least six G protein coupled receptors whose activation stimulates cell proliferation, survival and migration. Overexpression of ATX and increased levels of LPA have been linked to fibrotic and inflammatory diseases and to several cancers. ATX is a therapeutic target and the ATX inhibitor IOA-289 is currently in Phase 1B clinical trials for the treatment of patients with metastatic pancreatic cancer, in combination with gemcitabine. ENPP2 is overexpressed in both pancreatic adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC). In HCC, ATX is produced by both the tumour microenvironment (TME) and tumour cells and our data show that ATX signalling can drive tumour cell growth in an autocrine manner. In contrast, in PDAC the TME primarily expresses ATX. The cell lines, PANC-1 and MIA PaCa-2 and the cancer associated fibroblast (CAF) derived cell line, 0082T, were used as a model to explore ATX signalling in PDAC. Consistent with publicly available mRNA expression data, 0082T cells, but not the PDAC cancer cells, secrete ATX and its substrate LPC. Conditioned media from 0082T cells increased the growth of both PANC-1 and MIA PaCa-2 cells. Treatment of 0082T CAF cells with IOA-289 during media conditioning reduced PDAC cell growth, without impacting CAF cell numbers. Lipidomic analysis showed IOA-289 and PF8380, another ATX inhibitor, reduce the generation of LPA in 0082T CAF conditioned media. 22:6 LPA was shown to be generated by an ATX independent pathway, and production of 22:6 LPA was upregulated upon ATX inhibition. Additionally, 22:6 LPA accumulates in conditioned media when 0082T, PANC-1 and MIA PaCa-2 cells are present, but species of LPA generated by ATX are specifically degraded (16:0,16:1 18:0,18:1). Cell surface lipid phosphate phosphatases (LPPs) dephosphorylate LPA to produce monoacylglycerol. This presents the first evidence for LPA species substrate specificity of LPPs and suggests LPPs specifically regulate ATX generated species of LPA. RNA-seq analysis of ATX inhibitor and DMSO treated CAF cells revealed significantly altered expression of genes encoding extracellular proteins, such as CTGF. A combination of modulating the mitogenic CAF secretome and a reduction in LPA is proposed as the mechanism of ATX inhibition in regulating CAF-driven PDAC growth. ATX has non-catalytic functions that could be mediated by specific protein-protein interactions. An HRP-based proximity labelling method was optimised for investigating the extracellular protein interactors of ATX. Several potential interactors are identified, including fatty acid binding protein 5 (FABP5). Modelling with AlphaFold-Multimer indicates FABP5 may interact with ATX at the opening to ATX’s lipid binding active site. Further investigation will be required to validate this idea.Item Open Access Investigating PLEKHS1 adaptor function in PI3K signalling and human cancer biologyAnwar, ArqumThe Class-IA PI3K signalling pathway regulates key anabolic cellular processes including proliferation, survival, growth and metabolism. Oncogenic hyperactivation of the pathway occurs across tumour types, commonly via genetic alterations in key pathway genes (e.g., *PIK3CA*, *PTEN*). Despite extensive research, the primary drivers of healthy or oncogenic PI3K signalling *in vivo* are largely unknown and PI3K pathway inhibitors show limited clinical benefit in solid tumours. Identifying upstream regulators of oncogenic PI3K activation can reveal novel therapeutic targets and/or biomarkers. Previously generated *in vivo* interactomics data identified the RTK/IRS network as the primary driver of homeostatic PI3K activation in healthy mouse prostate, and PLEKHS1 as the primary driver of oncogenic PI3K activation in cancerous, PTEN-null mouse prostate. PTEN phosphatase is a tumour-suppressor and inactivated commonly in prostate cancers. PLEKHS1 is otherwise a poorly characterised protein. This thesis presents research investigating the relevance and mechanisms of PLEKHS1 adaptor protein function to oncogenic PI3K pathway remodelling in human cancers, with a focus on prostate cancer. TCGA pan-cancer analyses report *IRS1* downregulation and *PLEKHS1* upregulation in several primary carcinomas, compared to healthy tissue. In prostate, endometrial and breast cancers, *PLEKHS1* expression correlates with PI3K pathway activation and alterations in key pathway genes (*PTEN*, *PIK3CA*). In castration-resistant prostate cancer, *PLEKHS1* is under-expressed in neuroendocrine cancers, compared to adenocarcinomas. ChIP-Seq datasets report binding of key prostate cancer transcriptional regulators to the *PLEKHS1* locus (e.g., AR, FOXA1). To investigate PLEKHS1 adaptor protein function *in vitro*, LNCaP prostate cancer cell line is used due to its PTEN-null status and PI3K pathway hyperactivation. Transient over-expression of PLEKHS1 in LNCaP cells generates a robust PIP3 response via YxxM-dependent interactions with endogenous PI3K regulatory subunits (p85, p55γ). PLEKHS1 phosphorylation and PI3K interaction are driven by SFK activity and independent of serum growth factors. In this context and alike *in vivo* findings, the PLEKHS1 PH domain which specifically binds PIP3 and PI(3,4)P2 mediates positive feedback PI3K activation. PLEKHS1 adaptor function is mostly conserved across mouse and human protein isoforms but altered for human isoform 2 which has a truncated PH domain. Endogenous PLEKHS1 function is also investigated in LNCaP cells. CRISPR/Cas9 mediated PLEKHS1 knockout does not significantly affect PIP3 levels, Akt phosphorylation or cell viability in LNCaP cells cultured as monolayers or spheroids. Consistently, no endogenous PLEKHS1-PI3K interaction is detected using co-immunoprecipitation in LNCaP cells. However, other *in vitro* disease models did provide evidence of endogenous SFK-PLEKHS1-PI3K signalling axis. In the PTEN-null breast cancer cell line HCC70, endogenous PLEKHS1 is highly expressed and SFKs mediate YxxM phosphorylation and interaction with PI3K regulatory subunits (p85, p55γ). However, siRNA mediated PLEKHS1 knockdown does not affect PIP3 levels, Akt phosphorylation or cell viability. Mouse dorsolateral prostate derived organoids are used as an alternative *in vitro* model. PTEN-KO organoids show elevated PLEKHS1 protein expression and PIP3 levels compared to WT. PTEN/PLEKHS1-DKO organoids phenocopy *in vivo* data with reduced PIP3 levels and growth compared to PTEN-KO organoids. Collectively, this work substantially advances prior *in vivo* findings from mouse models by (1) demonstrating the relevance of PLEKHS1 adaptor function to PI3K signalling in human cancers using *in vitro* and clinical data, (2) elucidating the molecular mechanisms of the SFK-PLEKHS1-PI3K signalling axis, and (3) revealing highly context-specific utilisation of PLEKHS1 as a primary driver of oncogenic PI3K signalling.Item Open Access Understanding the Effects of Ageing on B cells Response to VaccinationLee, Jia LeAgeing is often associated with a reduction in antibody-secreting cell formation and vaccine-induced antibody titres, resulting in poor vaccine responses among the older population. Understanding the mechanisms underlying this age-related defect is imperative for guiding strategies to improve vaccine responses in older people. Here, I tested the hypothesis that B-cell intrinsic changes with age contribute to the impaired antibody response to vaccination during ageing. I show that B cells from older people do not have defects in their proliferation and differentiation into antibody-secreting cells *in vitro* compared to those from younger donors. Adoptive transfer of B cells from aged SWHEL mice into young adult recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the germinal centre (GC) during the early stages of GC formation, compared to those from young donors. Nevertheless, by the peak of the GC response, GC B cells derived from B cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age-related intrinsic B cell changes might delay the GC response but are not responsible for the impaired antibody-secreting response or smaller peak GC response in ageing. Using the B1-8i adoptive transfer system, I replicated the data showing that B cells from aged B1-8i mice were equally able to differentiate into extrafollicular plasma cells and mount a peak GC response, compared to their younger counterparts. Furthermore, GC B cells from aged donor mice had no defects in receiving positive selection signals and undergoing affinity maturation to produce high-affinity clones. Conversely, the transfer of B cells from young adult mice into aged recipient mice resulted in smaller numbers of antigen-specific GC B cells and impaired affinity maturation, compared to transfers into young recipient mice. Together, this work shows that B cells from older bodies are not intrinsically defective in responding to stimulation and becoming high-affinity clones. Rather, B cell-extrinsic factors contribute to the age-associated impairment in humoral immunity. To have an unbiased approach in identifying mechanisms that drive defects in antibody response among older people, I employed a systems immunology approach to investigate the molecular pathways and cellular populations associated with antibody responses to seasonal flu vaccination in individuals aged above 64 years old. Although bulk RNA sequencing analysis of pre-vaccination and day 1 post-vaccination blood samples did not yield differentially expressed genes between vaccine responders and nonresponders, responders exhibited a trend of enhanced upregulation of type I interferon (IFN-I) response genes at day 1 post-vaccination. Furthermore, flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) collected 6-10 days post-vaccination revealed that long-lasting antibody responses to H1N1 antigen positively correlated with higher proportions of HA-binding plasmablasts and circulating T follicular helper (cTfh) cells. These findings suggest that an effective response to seasonal flu vaccine among older individuals relies on a robust antiviral response via IFN-I signalling, coupled with the generation of plasmablasts and cTfh cells. Together, the results from this thesis contribute to the understanding of mechanisms underlying age-related defects in humoral response to vaccines, which can be used to guide the design of more effective vaccine strategies to protect vulnerable members of our population.Item Open Access Long-read transcriptome profiling of germinal centre B cellsGizlenci, ÖzgeAlternative splicing (AS) is a major regulatory process underpinning the development and function of the immune system. 29% of alternatively spliced genes are specific to immune cells, yet the regulation and function of AS during B cell activation remain largely unknown. The activation of naive B cells and the subsequent germinal centre (GC) reaction drive the cascade of reactions resulting in short- and long-term antibody responses, making the GC crucial for adaptive immunity. However, abnormal function of GC B cells contributes to autoimmune disease and the development of lymphomas. Hence, GC reaction needs to be tightly regulated. Previous studies have linked individual AS events in GC B cells to B cell malignancies using short-read sequencing; however, this methodology is limited in defining the complete sequence of transcript variants generated by AS. Therefore, many transcript variants remain undefined. During my PhD, I have developed a long-read sequencing methodology Oxford Nanopore Technologies (ONT) workflow to understand post-transcriptional regulation at both gene and isoform levels in human and mouse GC B cells. Because one of the challenges of ONT is the accurate computational analysis of isoforms, we developed the ‘Nexons’ pipeline to identify the differentially spliced transcript variants. Transcriptome characterisation using ONT allowed us to detect differentially expressed transcripts during antigen-mediated activation of GC B cells in human and mouse. Moreover, identification of individual isoforms with Nexons revealed differential splicing of transcripts, including potentially novel splice variants, as well as changes which were undetectable in short-read sequencing data. An in-depth analysis revealed the differential regulation of poison exons (PE) in serine/arginine-rich splicing factors (SRSF) (e.g., SRSF3 and SRSF7). Naive B cells preferentially expressed isoforms carrying PE, leading to nonsense-mediated mRNA decay, whilst the PE were preferentially removed in activated and GC B cells. Notably, we found this regulation of PE in splicing factors is conserved between human and mouse. We validate an ONT/Nexons workflow as a suitable method for the identification and quantification of transcript isoforms and highlight the SRSF family as important candidates for regulating the GC reaction.Item Open Access Defining the essential regulators of naive human pluripotent stem cell reprogrammingBendall, Adam; Bendall, Adam [0000-0002-6865-2625]Human pluripotent stem cells (PSCs) exist in two distinct states – naïve and primed, which are interconvertible. Naïve PSCs uniquely harbour several desirable properties that primed PSCs do not, including their ability to model human pre-implantation biology and developmentally-regulated epigenetic phenomena in vitro. Naïve PSCs also have an expanded differentiation potential to produce cell types with possible therapeutic applications. Naïve PSCs are predominantly generated by reprogramming primed PSCs. This project aimed to address a significant gap in our knowledge about the molecular events that underpin reprogramming, which could assist in unlocking the full potential of naïve PSCs. A genome- wide screen identified novel regulators of naïve cell reprogramming, unexpectedly highlighting the Polycomb Repressive Complex 1 (PRC1) subtype PRC1.3. In this project, I sought to understand the role of PRC1.3 in human pluripotency and reprogramming, and determine the PRC1.3-dependent mechanisms that modulate the acquisition of naïve pluripotency. I used CRISPR-Cas9 to delete the core PRC1.3 complex component PCGF3 in primed PSCs and ascertained that PRC1.3 is dispensable for human primed pluripotency but critical for naïve cell reprogramming. I used ChIP-sequencing to identify genes that gain PRC1.3 occupancy during reprogramming and these genes become transcriptionally repressed as cells successfully reprogramme, suggesting a key gene silencing role for PRC1.3. Deletion of PRC1.3 reduces the global abundance of the PRC1-deposited H2AK119ub1 modification, and levels of PRC2-deposited H3K27me3 also, in primed PSCs. Using a proteomics approach, I identified a novel, naïve-specific interaction between PRC1.3 and PRDM14, which cooperate in transcriptional repression of PRC1.3 target genes, I also discovered a developmentally-regulated composition shift in the PRC1.3 complex between human naïve and primed PSCs, which was corroborated in early human embryo datasets. This change in PRC1.3 composition does not appear to alter the enzymatic activity of PRC1.3 in vitro. Overall, I identified that the PRC1.3 complex plays a critical role in human naïve cell reprogramming. The PRC1.3 complex influences the human primed PSC epigenome and is associated with transcriptional repression, with the complex undergoing both intrinsic and exogenous regulation across the cell state transition. These findings advance our knowledge of molecular events that underpin successful naïve cell reprogramming, and this progress could optimise our ability to produce human naïve PSCs, as an in vitro developmental model and a cell type with considerable therapeutic potential.Item Open Access NANOGP1 as a Model to Study the Consequences of Gene Duplications on Human Pluripotency and DevelopmentMaskalenka, KatsiarynaGene duplication events play an important role in genome evolution; they can also create developmental strategies that differ between species. However, the functional contribution of duplicated genes in early human development and pluripotency is poorly understood. To address this knowledge gap, I investigated NANOGP1, which is a duplicated pseudogene of a key pluripotency factor called NANOG. NANOGP1 was chosen as a model for studying gene duplication in human pluripotency for several reasons. Firstly, NANOGP1 is an evolutionarily conserved duplicate in Hominidae that appears to have an intact coding sequence. The pseudogene is currently annotated as non-protein-coding, although no functional assays have been performed to test this. Secondly, upon investigating the expression of pseudogenes in human naïve pluripotent stem cells (PSCs), I found that NANOGP1 is among the top 1% of the highest expressed pseudogenes. Because high expression levels of NANOG are crucial for maintaining human pluripotency, I hypothesised that a duplicated copy of this important developmental regulator could have similar properties and might contribute to the regulation of human pluripotency. Gene expression profiling revealed that NANOG and NANOGP1 have overlapping but distinct expression patterns, both in human embryos and in PSC states. NANOGP1 is highly expressed in naïve pluripotent cells but is significantly downregulated in primed pluripotent cells, while NANOG expression levels do not differ to the same extent between the two pluripotent states. RNA splicing analysis predicted that NANOGP1 encodes a protein with an intact homeodomain and transactivation domain, but lacking part of the N-terminus. The divergent N-terminus is the main structural difference between NANOG and NANOGP1 and was therefore used in this study to distinguish between the two genes. Using CRISPR/Cas12a-mediated gene editing in naïve PSCs, I introduced an epitope tag at the start of the predicted protein sequence, and this enabled me to demonstrate for the first time that endogenous NANOGP1 encodes an expressed protein. The ability to be translated into the stable protein raised the possibility that NANOGP1 could have a functional role. To test this, I performed a series of assays and established that at least two key functional properties are conserved between NANOG and NANOGP1: gene autorepression, and the ability to promote primed-to-naïve PSC reprogramming. Alongside this, however, downregulating NANOGP1 expression using inducible CRISPRi in naïve PSCs did not lead to a differentiation phenotype, 4 which is in contrast to NANOG loss of function. Finally, using ChIP-seq, I showed that NANOGP1 shared a subset of chromatin binding sites with NANOG, and also, surprisingly, had a small number of unique, NANOG-independent sites particularly at the promoters of neural-associated genes. Overall, I conclude that NANOGP1, a previously overlooked duplicated copy of NANOG, is an expressed, protein-coding transcription factor in human naïve PSCs. Most of the CDS, and several of the functional properties, are conserved, implying that NANOGP1 could be supporting or cooperating with its ancestral gene copy in stabilising pluripotency. At the same time, differences in the N-terminal of the CDS, binding occupancy, and distinct expression patterns, could potentially contribute to functional diversification. These differences could have significant evolutionary consequences for creating species-specific developmental strategies, such as novel cell type-specific activity, expanded protein interaction networks and interplay with signalling pathways. Collectively, these potential new properties might extend functional potential and, hence, could encourage diversification of developmental mechanisms. Taken together, my work has demonstrated that NANOG/NANOGP1 duplication serves as a paradigm for exploring how pseudogenes could support their ancestral copies, as well as expand the evolutionary potential of conserved developmental programmes.Item Open Access Investigating the role of DPPA2 and DPPA4 in the Epigenetic Control of Lineage Programs in Human Embryonic Stem CellsMalcolm, Andrew; Malcolm, Andrew [0000-0001-6240-7701]The precise co-ordination of cell fate specification during human early development is a vital yet poorly understood process. To navigate the dynamic transcriptional and epigenetic changes associated with germ layer allocation, pluripotent cells maintain developmentally important genes and their regulatory regions in a poised but repressed chromatin state. The poised state has been proposed to allow for precise and coordinated activation or complete repression of gene expression depending on the instructive signals from the external and intrinsic environment. Poised promoters and enhancers adopt multivalent histone modification states, comprised of both active and repressive modifications, such as H3K27me3, H3K4me3 and H3K4me1. Combining opposing modifications may help to maintain robustness of genes to low levels of signal, whilst retaining the capacity to respond upon the appropriate level. Despite their importance in controlling cell fate decisions, our understanding of the mechanisms by which poised states are established and maintained in human cells is currently lacking. Here, I discover a role for the transcription factors DPPA2 and DPPA4 in maintaining poised chromatin in human pluripotent stem cells (hPSCs). I found that DPPA2/4 bind to the majority of CpG islands, poised promoters and a large subset of poised enhancers. CRISPR-Cas9 mediated knockout of DPPA2/4 in primed hPSCs led to changes in the expression of developmentally critical genes, particularly those associated with signalling. Primed hPSCs lacking DPPA2/4 exhibit increased spontaneous differentiation even in self-renewing conditions, display altered cell fate commitment III during differentiation and show axial patterning defects upon human gastruloid formation. Epigenomic profiling following the loss of DPPA2/4 revealed a marked depletion of H3K27me3 at a subset of DPPA2/4-target regions, predominantly near to poised chromatin regions. Regions losing H3K27me3 were typically highly accessible and had high levels of H3K4me3 in both wild-type and DPPA2/4 DKO hPSCs. The depletion of H3K27me3 upon DPPA2/4 loss leaves these regions in a more active conformation, potentially driving changes in the expression of these genes. These data reveal new roles for DPPA2/4 in safeguarding the robustness of poised chromatin states and in regulating genes that are important for cell fate specification. Understanding further how these transcription factors and chromatin states jointly contribute to gene expression dynamics is critical to uncover the principles of developmental gene regulation and to improve generation of specialised cell types from human pluripotent cells.Item Open Access New roles of Rac-GEFs in Neutrophils and in Glucose HomeostasisMachin, PollyRac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems.Item Open Access The roles of DPPA2, DPPA4 and SMARCA5 in mouse zygotic genome activation, epigenetic reprogramming and developmentKubinyecz, Oana NicoletaThe epigenetic remodelling that takes place in order to transform a fertilised oocyte into an embryo and then a whole organism, is one of the most intriguing cellular transformations in biology. The whole process starts with the meiotic resumption of an oocyte when the translational activation of the dormant mRNAs takes place, followed by their gradual elimination in the zygote, and ending with the full transcriptional activation of the newly reprogrammed embryonic genome. All these complex processes constitute the maternal to zygotic transition (MZT), which is accompanied by the global epigenetic reprogramming of the embryo. In this dissertation, I focus predominantly on the epigenetic factors that potentially contribute to the initiation of transcription, a process also called zygotic genome activation (ZGA). In mice, this takes place in 1 cell embryos – minor ZGA, followed by a burst of transcription - major ZGA, at the 2 cell stage. Previous screens in the lab, using mouse embryonic stem cell (mESC) identified the small DNA binding proteins DPPA2, DPPA4 and the chromatin remodeler SMARCA5 as potential inducers of major ZGA. Here I focus on validating these three factors and I assess their roles in vivo, using conditional knock-out mouse models and a targeted protein depletion system, together with the characterisation of the transcriptomic and epigenetic changes. In Chapter 1, I give an overview of the biological context for my study together with the latest findings, and depict some of the technologies used to describe the changes that take place during epigenetic reprogramming in the early embryo. Chapter 2 outlines the materials and methods used to address my questions, and Chapters 3, 4 and 5 contain a detailed description of my results and their significance in the wider context. Chapter 3 is focused on the definition of comprehensive ZGA gene lists and of control genes, building a base for chapters 4 and 5. Using six published transcriptomics datasets from independent studies using different library generation methods, I define minor and major ZGA signatures that give a complex picture of the transcriptional landscape in zygotes and 2 cell stage embryos, by waning the biases introduced with each individual study. In Chapter 4, I assess the role that maternal DPPA2 and DPPA4 play in mouse embryonic development and major ZGA. For this, I used single and double conditional knock-out models to deplete maternal deposits of these proteins, and showed that major ZGA still takes place in their absence. This work was recently published. In Chapter 5, I assess the role that the maternal ISWI ATPase SMARCA5 plays in embryonic development, ZGA and chromatin remodeling in early embryos. For this I use both a conditional knock-out mouse model lacking SMARCA5 in oocytes, but also a targeted protein depletion system in early wild-type zygotes, followed by transcriptomics, methylation and chromatin accessibility characterisation of the embryos. Both systems confirmed that SMARCA5 plays an important role not only in the transcriptional activation but also in global chromatin changes that take place at the 2 cell stage. Chapter 6 contains a summary of my findings, conclusions and their relevance in the wider context of the field, and ends with proposed future directions. My dissertation provides new insights into the mechanisms and factors that regulate mouse ZGA, and highlights the importance of validating in vitro findings in a relevant in vivo system.Item Open Access RNA binding proteins ZFP36 and ZFP36L1 limit CD8+ T cell differentiation and effector functionMitchell, TwmPost-transcriptional regulation of gene expression is mediated in part by RNA binding proteins (RBPs). The ZFP36 family of RBPs are key regulators of gene expression in the immune system. The founding member ZFP36 is a well-known regulator of cytokine mRNA stability. Paralogs ZFP36L1 and ZFP36L2 act redundantly during thymopoiesis and in developing B lymphocytes, where they limit DNA damage response and cell cycle progression. Furthermore, ZFP36L2 has been shown to repress translation of preformed Ifng mRNA and maintain quiescence in CD8+ memory T-cells. However, roles for the ZFP36 proteins in the differentiation and effector functions of CD8+ T cells remain to be explored. My project utilized mice featuring the OT-1 transgenic T cell receptor to produce cytotoxic T lymphocytes (CTLs) and memory-like CD8+ T cells in vitro. I made use of knockout mouse models with CD4cre-mediated conditional deletion of Zfp36 and Zfp36l1, to determine the role of these RBPs in limiting CD8+ T cell differentiation and effector functions. Using this approach, I demonstrated that ZFP36L1 acts to limit the cytotoxicity of CTLs and, by employing a CRISPR/Cas9 mediated gene knockout system, I demonstrated that this occurs early after initial T cell stimulation. I also demonstrated that upon adoptive transfer, memory-like CD8+ T cells form part of the central memory niche and respond to Listeria monocytogenes-OVA infection by differentiating and proliferating and that cells that lack ZFP36 and ZFP36L1 show more rapid terminal differentiation and improved effector functions. To begin to assay the molecular mechanisms by which ZFP36L1 limits CD8+ T cell differentiation, I made use of a recently published improved individual nucleotide crosslink immunoprecipitation methodology to identify directly bound target mRNAs. I demonstrated that transcripts encoding for factors in the MAPK, PI3K-AKT, and JAK/STAT signaling pathways are bound by ZFP36L1 and transcription factors IRF8 and Notch-1, and multiple cytokines and chemokines, including IFN-γ, TNF-α, IL-2 and CCL3/4. In addition, I describe a novel reporter mouse model of ZFP36L1 with an N-terminal fusion of a red-fluorescent protein expressed from the endogenous Zfp36l1 locus. Using this model, I analyzed the kinetics of ZFP36L1 expression in response to TCR stimulation. These findings demonstrate a role for ZFP36 and ZFP36L1 in coordinating both the differentiation and effector functions of CD8+ T lymphocytes. In the future, I hope to determine the mechanisms of action for ZFP36 RBPs in limiting CD8+ T cell differentiation.