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Theses - Haematology


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  • ItemOpen Access
    Characterisation of protease activated receptors
    Fox, Mark Thomas
    The thrombin receptor is a seven transmembrane G-protein-coupled receptor that is activated by thrombin cleavage. In order to characterise the thrombin receptor three mouse monoclonal antibodies were isolated by using a recombinant fusion protein incorporating the extracellular domain of the human thrombin receptor as an antigen. An affinity matrix prepared from one monoclonal antibody was used in the successful purification of the native receptor from human platelet membranes. Two of the monoclonal antibodies were capable of blocking thrombin-induced platelet aggregation, and this may lead to their use as possible platelet thrombin receptor antagonists. To characterise further the monoclonals, their epitopes were mapped. In addition the cDNA encoding the antibodies variable regions were cloned and sequenced. By using this procedure, the amino acid sequence of the antibody complementarity determining regions (CDRs) involved in binding to the target epitope were determined. The recently discovered human protease activated-2 receptor (hPAR-2) was studied using rabbit polyclonal antibodies generated with a multiple antigenic peptide comprising a region around the protease cleavage site. Immunocytochemistry and flow cytometry using affinity purified antibodies detected expression of hPAR-2 on human umbilical vein endothelial cells, keratinocytes and granulocytes. The expression of both PAR-2 and thrombin receptor were analysed using Northern analysis in a wide variety of murine haematopoietic cell lines. This analysis revealed the widespread distribution of the thrombin receptor. In contrast expression of PAR-2 was not observed in haematopoietic cells, suggesting that it is a marker of terminal differentiation in granulocytes. To identify potential activators of PAR-2, a variety of serine proteases were tested using a chloromethylketone inhibitor based on the peptide sequence of the cleavage site. These experiments revealed three potential trypsin-like enzymes that may activate PAR-2 *in vivo* namely, pancreatic trypsin, mast-cell tryptase and acrosin. Thrombin is a serine protease that plays a central role in blood coagulation. Its specificity is determined by interactions with both the active site and a region distant from the active site called the anion-binding exosite. A surface loop from the thrombin anion-binding exosite, involved in binding to the hirudin-like domain of the thrombin receptor was grafted onto a recombinant single chain antibody using protein engineering. Molecular modelling indicated that incorporation of this loop into a framework region of the well-characterised anti-lysozyme antibody DI.3 would result in a chimeric molecule that would both bind its target antigen hen egg lysozyme, and also bind the platelet thrombin receptor through the engineered loop. The mutant antibody was unfortunately expressed as an incorrectly folded protein.
  • ItemEmbargo
    Single-cell Protein-Transcriptome Atlas of Haematopoiesis across the Human Lifespan
    Quiroga Londoño, Mariana; Quiroga Londono, Mariana [0000-0003-2352-0773]
    The haematopoietic system is responsible for the continuous production of stem and progenitor cells, which divide and mature to produce a wide variety of specialised circulating blood cells. Proper regulation of lineage decisions is key to ensure the right balance between the generation of differentiated cells and the maintenance of the haematopoietic stem cell compartment. A dysregulation in this balance can lead to blood-related diseases. Throughout an individual's life, human haematopoiesis demonstrates significant variations, likely in response to changing demands and physiological needs. Moreover, age-related alterations in the blood system are believed to impede its functionality. Both transcriptomic and proteomic regulation play crucial roles in maintaining the haematopoietic balance. Interestingly, many classical haematopoietic populations showcase heterogeneity in both expression and function. Studying genes and cell surface proteins at the single-cell level using a combination of scRNA-seq and index sorting has proven valuable for inferring individual cellular states and identifying subpopulations for further research. Nevertheless, traditional methods that measure both transcripts and proteins from single cells via index sorting/FACS often have scale limitations. Typically, they can provide data on only a limited number of genes and proteins when used in parallel. These constraints make it difficult to capture the full complexity of cellular states and do not enable tissue scale analysis. We take advantage of the method Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), which enables the simultaneous combination of highly multiplexed cell surface protein marker detection with unbiased transcriptome profiling for thousands of single cells, allowing more detailed characterisation of cellular phenotypes than transcriptome measurements alone. To investigate age-related changes, cells can be gathered from tissues representing various stages of human development. By taking advantage of a custom panel of 198 oligo conjugated antibodies, we successfully profiled over 250,000 cells collected from tissues representing prenatal development (including yolk sac, embryonic and fetal liver, and bone marrow), newborn (cord blood), paediatric (bone marrow), as well as adult (bone marrow and spleen). Our prenatal and neonatal analysis revealed unique surface protein profiles and differentiation biases already present in the prenatal tissues. In the analysis of CD34+ cells, the yolk sac had a pronounced presence of eosinophil, basophil, and mast cell progenitors compared to other tissues. Distinct differences in cell proportions for myeloid groups like megakaryocyte progenitors, myeloid-erythroid progenitors, and common lymphoid progenitors were observed between yolk sac and the other examined tissues. The paediatric exploration suggested a preference for lymphoid differentiation in the earlier stages of childhood. Furthermore, we noted a higher expression of the dendritic cell marker HLA-DR in older children, providing insight into potential maturation of certain immune cell populations as children age. In our analysis of extramedullary haematopoiesis, we observed differential surface protein expression in medullary and extramedullary haematopoietic stem cell and multipotent progenitors. We emphasised, for example, the prevalence of lineage-priming/activation markers in the spleen compared to the bone marrow, and the notable expression of lymphoid markers in the spleen. Most of these analyses rely on the transcriptomic landscape and use the protein for validation and further exploration. However, I developed a bioinformatic pipeline to enable integrated analysis of samples from numerous individuals and tissues across the different developmental stages, and our latest unreported findings for differential abundance analysis indicated an initial lymphoid-skewed tendency emerging from fetal bone marrow, intensifying during the paediatric phase, and gradually diminishing as one approaches adulthood. Preliminary data also revealed the presence of a distinct haematopoietic stem cell population exclusive to the fetal stages. This extensive atlas serves as a valuable resource and reference to understand blood-related diseases, shedding light on the stage and tissue-specific characteristics of human haematopoiesis.
  • ItemEmbargo
    Investigating the anti-leukaemic effect of tamoxifen in the myeloproliferative neoplasms
    Fang, Zijian
    Myeloproliferative neoplasms (MPNs) commonly result from the acquisition of somatic mutations affecting the Janus kinase 2 (JAK2) (such as JAK2V617F) or the multi-functional protein CALR (such as CALRIns5 and CALRDel52), causing the constitutive activation of signal transducer and activator of transcription (STAT) proteins. The selective oestrogen receptor modulator (SERM) tamoxifen (broadly used in ER+ breast cancer) was effective on eradiating JAK2V617F+ haematopoietic stem and progenitor cells (HSPCs) in preclinical mouse MPN models, which prompted a Phase-II clinical study (Effects of TAMoxifen on the Mutant Allele Burden and Disease Course in Patients with MyeloprolifeRatIve Neoplasms, TAMARIN). In the TAMARIN study, the safety and activity of tamoxifen on reducing mutant allele burden was observed in a subset of MPN patients. This thesis investigated the mechanism of action of tamoxifen in human MPN. According to the transcriptomic analysis, HSPCs from patients meeting the primary or secondary endpoints (tamoxifen responders) were highly enriched with specific molecular pathways such as integrated stress response (ISR), proinflammatory signalling, mitochondrial activity and proteotoxic stress at baseline, implying a predictable transcriptomic signature for tamoxifen susceptivity. Moreover, immunoblotting showed that tamoxifen treatment induced a proapoptotic ISR in tamoxifen–sensitive JAK2V617F+ human cell lines, which manifested as increased translation of activating transcription factor 4 (ATF4) and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The knockdown of ATF4 and inhibition of eIF2α phosphorylation partially rescued tamoxifen-induced apoptosis. Despite the oestrogenic transcriptional regulation found in HSPCs from responders or JAK2V617F+ human cell lines, full-length oestrogen receptors α (ERα), the canonical transcriptional receptor of tamoxifen in mouse haematopoietic system, was not highly expressed in these cells, and the expression of the alternative receptors ERβ and GPER was lower in HSPCs from responders compared with non-responders. Searching for alternative candidate receptors, aryl hydrocarbon receptor (AHR) was found to be highly expressed in responder HSPCs, and its transcriptional activity was activated after tamoxifen treatment. On JAK2V617F+ human cell lines, tamoxifen incubation induced the nuclear translocation and transcriptional activity of AHR, the knock-out of which protected cells from tamoxifen- induced apoptosis, indicating the activation of AHR contributes to its proapoptotic mechanism on tamoxifen-sensitive HSPCs and MPN cell lines. Interestingly, longitudinal transcriptomic analysis of HSPCs from study subjects showed a high baseline expression of OXPHOS-related genes and their specific reduction after 24-week tamoxifen treatment in tamoxifen responders, compared with non-responders, suggesting that tamoxifen exerts a metabolic inhibition on OXPHOS in responder HSPCs. The suppression of cellular respiration and energy synthesis was reproduced in tamoxifen-sensitive JAK2V617F+ human cell lines, bone marrow lineage-negative HSPCs from JAK2V617F+-driven MPN mouse model and patient peripheral blood mononuclear cells (PBMCs), where 4-hydroxytamoxifen (4OH-TAM) directly inhibited mitochondrial respiratory complex I and decreased adenosine triphosphate (ATP) synthesis. This reduction of intracellular ATP significantly suppressed the cytokine-induced pathogenic activation of JAK2 and STAT5 in JAK2V617F+ human cell lines, which was rescued by the respiratory complex II activation and ATP replenishment, explaining the high selectivity of tamoxifen on the JAK-STAT pathway of JAK2V617F+ human cell lines and HSPCs. Overall, tamoxifen exhibits multiple mechanisms in inducing apoptosis of JAK2V617F+ human haematopoietic cells with specific metabolic vulnerabilities (e.g. high OXPHOS dependency and energy requirement) through a combination of transcriptional regulation and metabolic inhibition.
  • ItemEmbargo
    Cholinergic signals promote the quiescence of normal or leukaemic stem cells through the activation of the α7-nicotinic receptor in bone marrow mesenchymal stromal cells
    Fielding, Claire; Fielding, Claire [0000-0002-5984-9127]
    The sympathetic nervous system has evolved to respond to stress and activates haematopoietic stem cells (HSCs) using noradrenergic signals. However, the pathways that maintain HSC quiescence and maintenance during proliferative stress are not well understood. This study has investigated the role of cholinergic signals during healthy and malignant haematopoiesis. During healthy haematopoiesis, sympathetic cholinergic signals maintain HSC quiescence in bone-associated (endosteal) bone marrow (BM) niches. During stress haematopoiesis, cholinergic neural signals increase and are amplified through cholinergic osteoprogenitors. If cholinergic innervation is absent, responses to chemotherapy or irradiation become unbalanced and HSC quiescence and self-renewal decrease. Cholinergic signals activate the α7 nicotinic receptor in BM mesenchymal stromal cells (BMSCs), leading to increased CXCL12 expression and HSC quiescence. In vivo, exposure to nicotine increases endosteal HSC quiescence and impairs haematopoietic regeneration after HSC transplantation in mice. In acute myeloid leukaemia (AML), cholinergic fibres are preserved despite a decrease in sympathetic noradrenergic innervation. Loss of sympathetic cholinergic innervation or α7 nicotinic acetylcholine receptor (α7nAChR) signalling in the BM microenvironment promotes HSPC proliferation and accelerates leukaemogenesis. Blockade of peripheral α7nAChR renders chemoresistant quiescent AML cells sensitive to induction chemotherapy and shows synergistic therapeutic effects in the AML model in vivo. These results suggest that the cholinergic system has a regenerative function in healthy haematopoiesis and could be a promising target to boost induction chemotherapy’s efficacy in AML.
  • ItemOpen Access
    Single-cell genomics in rabbit and mouse elucidate eutherian embryonic development
    Ton, Mai-Linh
    Biomedical research relies heavily on the use of model organisms to gain insight into human health and development. Traditionally, the mouse has been the favoured vertebrate model, due to its experimental and genetic tractability. Non-rodent embryological studies however highlight that many aspects of early mouse development, including the egg-cylinder topology of the embryo and its method of implantation, diverge from other mammals, thus complicating inferences about human development. To get a better understanding of rabbit development, we constructed a morphological and molecular atlas of rabbit development, which like the human embryo, develops as a flat-bilaminar disc. We report transcriptional and chromatin accessibility profiles of almost 180,000 single cells and high-resolution histology sections from embryos spanning gastrulation, implantation, amniogenesis, and early organogenesis. Using a novel computational pipeline, we compare the transcriptional landscape of rabbit and mouse at the scale of the entire organism, revealing that extra-embryonic tissues, as well as gut and primordial germ cell (PGC) cell types, are highly divergent between species. Focusing on these extra-embryonic tissues, which are highly accessible in the rabbit, we characterize the gene regulatory programs underlying trophoblast differentiation and identify novel signalling interactions involving the yolk sac mesothelium during haematopoiesis. Finally, we demonstrate how the combination of both rabbit and mouse atlases can be leveraged to extract new biological insights from sparse macaque and human data. The datasets and analysis pipelines reported here set a framework for a broader cross-species approach to decipher early mammalian development, and are readily adaptable to deploy single-cell comparative genomics more broadly across biomedical research. Due to the genetic tractability of mouse, we aimed to interrogate the role key transcription factors (TFs) such as Zic2/3, Mix-like 1(Mixl1), Eomesodermin (Eomes), Stat3, and Runx1 in early development. These key TFs are involved in a variety of roles, such as metabolism, as well as the fate decision of mesoderm, and blood development. Constructing an understanding of the stepwise role that these TFs play in sequence for developing blood and mesoderm allows for a better understanding of the consequences of genetic mutations. To perform this analysis, we generate a series of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knock out cell lines. Using a chimaera model system where knock-out (KO) cells are injected into wild-type (WT) host blastocysts, we can understand the cell-autonomous role that each TF plays. In conclusion, studying single-cell transcriptomics unveils the molecular profiles behind each cell type; however combining with spatial information, chromatin accessibility, and gene perturbations allows for further understanding of the signalling niche and regulatory elements behind cell type diversification. Augmenting this analysis by a variety of model organisms allowed for a nuanced understanding of eutherian development, such as macaque and human development by understanding the divergent and convergent features of embryonic development. This also allows for the optimisation of in vitro differentiation protocols in the future. Additionally, this has implications for biomedical research due to the species- and cell-type-specific responses to drug screens. A comprehensive understanding of each target cell type of interest allows for researchers to better adapt model systems to their intended target.
  • ItemOpen Access
    The Effects of Methyltransferase Genes on Human Haematopoietic Stem Cell Function
    Vedi, Aditi
    The highly structured and hierarchical haematopoietic system is defined by the existence of a pool of haematopoietic stem cells (HSCs) at the apex, which possess both multipotent differentiation and self-renewal properties. The HSC pool is heterogenous in terms of self-renewal and differentiation capacity, and HSCs with various genetic alterations lie intermingled with wild type (WT) HSCs. Clonal expansion of HSCs is an ineluctable feature of advancing age, and age related clonal haematopoiesis (ARCH) is associated with an increased risk of haematological malignancy, atherosclerosis and cardiovascular disease. The functional effects of these clonal expansions remain to be thoroughly investigated in primary human HSCs. In this thesis, I have focused on 2 methyltransferase genes, PRMT5 and DNMT3A, which are both epigenetic modulators of HSC function. Pharmacological inhibition of PRMT5 in healthy human cord blood (hCB) haematopoietic stem and progenitor cells (HSPCs) leads to reduced HSPC proliferation and a relative differentiation block at the CD34+ progenitor level in a dose dependent manner. While PRMT5 has been identified by others as an important therapeutic target in the context of haematological malignancies, my work indicates its clinical applicability may be limited by the likely toxicity of PRMT5 inhibition on HSPCs, which could lead to significant myelosuppression. Mutations in the gene DNMT3A are the most common in ARCH and are associated with haematological and non-haematological diseases with significant morbidity and mortality. The R882 mutation is the most common within DNMT3A, especially in the context of haematological disease. I used single cell biology tools to obtain functional data from 12 primary human samples from ARCH individuals and acute myeloid leukaemia (AML) patients. A total of 2990 single HSC/multi-potent progenitor (MPP) derived colonies from DNMT3A mutant HSCs/MPPs and internal WT control HSC/MPPs from within each individual were compared. DNMT3A R882 mutation confers an intrinsic differentiation phenotype, with DNMT3A R882 mutant HSC/MPPs displaying more efficient differentiation towards neutrophils and less efficient monocyte differentiation in vitro compared to internal WT control HSC/MPPs in 6 independent individuals. No overall lineage bias is observed in DNMT3A R882 mutant HSC/MPPs, and no selective differentiation advantage. RNA sequencing of single HSC/MPP derived colonies containing mature monocytes confirmed that DNMT3A R882 mutant HSC/MPPs produce less mature monocytes, and are marked by upregulation of metabolic and inflammatory gene pathways. Overall, these functional phenotypes provide critical insight into the inherent functional effects of DNMT3A R882 mutation at the HSC/MPP level in humans, beyond the previously described transcriptional priming towards more quiescent HSCs and increased self-renewal phenotypes. Further mechanistic understanding will be required to elucidate the link between the myeloid differentiation phenotypes observed in my study and the inflammatory conditions associated with ARCH.
  • ItemOpen Access
    Pathogenicity assessment of genetic variants in von Willebrand disease using quantitative, qualitative, and functional approaches
    Sims, Matthew
    Von Willebrand disease (VWD) is the most common inherited bleeding disorder. It is defined by a deficiency or dysfunction of plasma von Willebrand factor (VWF), a glycoprotein with a multifaceted role in haemostasis. The majority of circulating VWF is synthesised and released by endothelial cells (ECs). VWD is caused by rare DNA sequence variants in the VWF gene. However, coupling genotype with phenotype is complicated by factors including incomplete penetrance and the trans-acting effect of the ABO histo-group. High throughput sequencing (HTS) is becoming the standard of care for the diagnosis of inherited bleeding disorders, including VWD. This raises several challenges. First, how should candidate VWF variants be searched for and their pathogenicity assessed? Second, if a pathogenic variant (PV) for VWD is identified how does this influence bleeding risk? Third, if the mechanism of the identified PV is unknown, how can its effect be elucidated? These questions are sequentially addressed in this thesis. I curated 1,455 unique VWF variants into a single repository called VWDbase. Variants were only included if they had been previously linked to VWD. Two thirds of VWDbase variants had previously been deemed causal of VWD and were termed Putatively Aetiological VWD Variants (PAVVs). Of these, 194 PAVVs were identified in the whole exome sequencing data of 140,327 participants in UK Biobank (UKB). These data were used to accurately determine the minor allele frequency (MAF) of these PAVVs. The pathogenicity of each PAVV was then scrutinised using published data. Seventy three of 194 PAVVs were rejected as being pathogenic for VWD. In over half of cases this was because the PAVV occurred too frequently to be compatible with VWD prevalence. The PAVVs that were accepted as being pathogenic for VWD were identified in 401 UKB participants (the ‘genetically accepted VWD’ [ga] group). Hospital inpatient data were analysed for UKB participants from 1997 to 2020. These were used to create the ICD-bleeding assessment tool (ICD-BAT) to assess the presence or absence of bleeding episodes across 16 different domains and the time over which UKB participants lived without experiencing an episode (bleeding free survival). There was no difference in the ICD-BAT score or bleeding-free survival when the gaVWD group was compared to the rest of the UKB population. However, blood group O predicted for both an increased ICD-BAT score and a reduced risk of bleeding-free survival over the observation period. VWDbase was then utilised to analyse 10 patients with VWD in whom no molecular diagnosis had previously been identified. The patient with the most severe (type 3) VWD phenotype was homozygous for a rare PAVV, c.8155+6T>A, situated in the donor splice site of the penultimate exon-intron junction. Analysis of platelet mRNA demonstrated that c.8155+6T>A results in a transcript with a frameshift and premature termination codon (PTC). Evaluation of patient-derived endothelial colony forming cells (ECFCs) revealed that c.8155+6T>A resulted in VWF that was mostly retained in a perinuclear position as opposed to being packed into Weibel-Palade bodies (WPBs). In order to overcome the finite supply of ECFCs and assess the effect of c.8155+6T>A in a different genetic context, a new cellular model of VWD was created. Human induced pluripotent stem cells (hiPSCs) were edited using CRISPR/Cas9 to contain a PTC in exon 50, positioned 10 nucleotides 5’ of c.8155+6T>A. They were then differentiated to ECs and the findings in the patient ECFCs were replicated. The effect of c.8155+6T>A is likely to be due to the truncation of VWF prior to the C-terminal cysteine knot (CK), the domain which is crucial for VWF dimerisation and exit from the endoplasmic reticulum. In summary, this thesis highlights the utility of large reference populations and hiPSC-derived ECs (iECs) in the critical appraisal of PAVVs.
  • ItemOpen Access
    The Impact of Clinically Relevant Culture on Human Haematopoietic Stem Cells: A Kinetic Analysis at Single Cell Resolution
    Johnson, Carys
    Haematopoietic stem cell (HSC) ex vivo gene therapy is now successfully used to treat an in-creasing number of monogenic disorders affecting the blood system. The ultimate efficacy of this therapy depends upon successful targeting of long-term haematopoietic stem cells (LT-HSCs), which are characterised by a predominantly quiescent status and sustained self-renewal capacity. Clinical ex vivo gene therapy protocols target the heterogenous mix of stem and pro-genitor cells encompassed in the CD34+ fraction with a limited understanding of how ex vivo manipulation impacts a purified LT-HSC subset. Importantly, long-term repopulation capacity is lost over culture, although the kinetics and molecular drivers of this process remain unclear. To address these questions, I perform single cell RNA-Seq, in vitro functional assays and in vivo transplantation in a time resolved manner following cord blood (CB) culture in differentiation facilitating conditions and mobilized peripheral blood (mPB) culture during a lentiviral ex vivo gene therapy protocol. First, I characterise the molecular impact of a 62 hr lentiviral ex vivo gene therapy protocol on mPB LT-HSC, short-term HSC (ST-HSC) and CD34+ cells. I reveal that the ex vivo protocol dramatically rewires the quiescent LT-HSC transcriptome and demonstrate that the majority of transcriptional changes are attributed to the culture conditions rather than lentiviral transduction. Secondly, I refine the kinetics associated with HSC functional attrition over the first complete cell cycle ex vivo. Long-term repopulation capacity is maintained for the first 6 hr irrespective of HSC source or tested culture conditions. I identify the 6 hr time-point as encompassing an adaptation period where LT-HSCs are rapidly responding to the instructive signals of culture and is molecularly underpinned by transient upregulation of cell stress response signalling. Follow-ing the 6 hr time-point, long-term repopulation capacity drops dramatically by 24 hr in CB culture and 62 hr in mPB culture. Loss of HSC function is correlated with reduced survival, cell cycle progression (late G1-M), sharp upregulation of MYC and a reduced ability to resolve proteostatic stress. Taken together, my results demonstrate that the 6 to 24 hr transition point is instrumental for the determination of LT-HSC cell fate ex vivo. Finally, using an ex vivo culture system of reversible early G1 arrest, I formally test whether cell cycle progression drives loss of self-renewal capacity in culture. Long-term in vivo transplantation approaches conclusively establish that cell cycle progression is not responsible for the loss of long-term repopulation capacity associated with clinically relevant HSC culture.
  • ItemOpen Access
    Translational regulation in aggressive B-cell lymphomas
    Krupka, Joanna Alicja; Krupka, Joanna [0000-0003-0369-0329]
    The Germinal Centre (GC) reaction is a dynamic process where B-cells undergo recombination and somatic hypermutation of immunoglobulin genes in response to antigen stimulation. This essential component of the adaptive immune system is associated with cycles of intensive proliferation and selection, which carries a risk of malignant transformation. Aggressive lymphomas arising from the GC stage of B-cell development are the most common haematological malignancies with heterogeneous molecular mechanisms and clinical presentation. Although the last decade witnessed considerable advances in the biology of GC reaction and related tumours, the studies focused predominantly on the network of transcription factors. The advances in Next Generation Sequencing technologies have opened new possibilities to explore mechanisms of regulation beyond the level of transcription. Ribo-Seq is a technique combining ribosome footprinting with deep sequencing of mRNA fragments that allows to map the position of translating ribosomes with single nucleotide precision. Here I investigate the mechanisms of translational regulation contributing to lymphoma development. Firstly, I introduce RiboStream, an automated bioinformatic pipeline designed to streamline processing of Ribo-Seq datasets while maintaining transparency and reproducibility of the computational workflow. Then, I provide an overview and benchmarking of current methods for identifying translationally regulated genes. Based on these I select a strategy to reveal that overexpression of two B-cell oncogenes, BCL6 or MYC, is followed by preferential translational of selected transcripts. Next, I show that loss-of-function mutations in RNA-helicase (DDX3X) promote early development of MYC-driven lymphoma by buffering the effects of MYC on translation of ribosomal proteins and the rate of global protein synthesis. Finally, I explore a genome-wide distribution of translating ribosomes to study the scope of non-canonical translation in lymphoid cells. Taking advantage of a large dataset of 79 Ribo-Seq libraries I reveal pervasive translation of ostensibly non-coding regions, and design a knock-down CRISPR screen library to identify those important for B-cell survival.
  • ItemOpen Access
    Identification of Novel Drivers of Haematopoietic Stem Cell Fate Using Low Cell Number Proteomics and Single Cell Profiling
    Bode, Daniel
    The abrogation and de-regulation of cellular decisions in adult haematopoietic stem cells (HSCs) have been widely recognised as key contributing factors in ageing and disease. In particular, acquired mutations altering HSC self-renewal contribute to the formation of pre-leukaemic disorders, such as myeloproliferative neoplasms (MPNs), and ultimately to their transformation to acute myeloid leukaemia. Therefore, we urgently require a complete characterisation of the underlying molecular pathways of HSC self-renewal in order to provide crucial insights into disease progression and to help inform novel strategies for ex vivo expansion for gene therapy applications. To date, advances in functional and molecular single cell technologies have provided unprecedented resolution of heterogeneous HSC populations and their transcriptional landscapes. In contrast, the scarcity of functional HSCs and technical limitations of unbiased proteomic screening technology have prevented comprehensive characterisation of protein networks governing HSC fate. To overcome these technical limitations, we developed an optimised mass spectrometry workflow to interrogate as few as 10,000 HSCs and multiplex up to 16 cell fractions. Using this approach, we were able to quantify in excess of 4,000 proteins, while reducing the required cell input 30-fold. To identify key molecular drivers of in vivo HSC self-renewal, we probed HSC populations with increasing self-renewal potency, including TET2-deficient HSCs exhibiting a self-renewal advantage in MPN mouse models. Here, we observed reshaping of extracellular matrix protein networks, indicating a potential physical role of the neighbouring bone marrow niche cells for regulating HSC fate. Next, we integrated proteomic and transcriptomic to characterise the molecular pathways underlying HSC self-renewal across numerous -omics technologies. Here, we identified a wide range of intrinsic regulatory pathways and described molecular mechanisms regulating intracellular calcium levels in HSCs. In the final results chapter, we explored the ex vivo expansion of HSCs, since this is of paramount importance for the delivery of gene and cellular therapies against a plethora of monogenic haematological diseases. Recently pioneered HSC expansion protocols greatly enhanced the yield of phenotypic HSCs, but such cultures exhibit significant clone-to-clone variability in long-term self-renewal potency and differentiation. By linking the transplantation outcomes with transcriptional profiles of individual single cell-derived clones, we derived a novel gene signature for ex vivo expanded HSCs with long-term self-renewal potency and characterised key molecular pathways governing HSC fate throughout expansion. Furthermore, we identified a reporter strategy for prospectively isolating expanded HSCs. Together, these findings provided an insight into the molecular machinery underlying HSC self-renewal within the native bone marrow niche and during ex vivo expansion. Comprehensive multi-omic profiling also revealed the intricate relationship of gene expression profiles with the proteomic phenotype within the HSC compartment. Finally, we propose an optimised workflow for performing comprehensive proteomics on any rare cell populations which will be of use to researchers investigating a wide-range of cellular biology questions.
  • ItemOpen Access
    The role of Nbeal2 in the homeostasis and retention of granules in haematopoietic cells
    Mayer, Louisa
    Gray platelet syndrome (GPS) is a rare disorder primarily characterised by the absence of α-granules in platelets. Typical clinical features of GPS include macrothrombocytopenia, abnormal or excessive bleeding, splenomegaly, and bone marrow fibrosis. GPS is caused by biallelic mutations in the NBEAL2 gene, where the deficiency of platelet α-granules is attributed to a loss of function in the Nbeal2 protein. Thus, the study of Nbeal2 can provide insight into both the clinical manifestations of GPS and the essential processes that contribute to the homeostasis of platelet α-granules. To this end, this work investigates the function of Nbeal2 through the study of a large collection of GPS patients through the NIHR BioResource-Rare Diseases (NBR-RD) programme, the Nbeal2−/− mouse model, and gene-edited Nbeal2−/− cell lines to understand its role in essential granule-related processes in haematopoietic cells. Through the NBR-RD GPS study, detailed clinical phenotypes were compared and patient blood samples were examined by Sysmex, RNA-Seq, and mass spectrometry analysis. The study revealed novel clinical phenotypes, including an array of autoimmune diseases and a prevalence of autoantibodies, differences in the transcriptomes and proteomes of GPS platelets, neutrophils, monocytes, and CD4+ lymphocytes, compared to healthy blood donors, as well as a pro-inflammatory signature in GPS plasma that may be mediated by changes in the liver. Loss of Nbeal2 function in GPS patients also affected the granularity and presence of granule-related proteins in leukocytes, which provides evidence that Nbeal2’s function is not exclusive to the regulation of platelet α-granules. This work also aimed to evaluate the mechanism by which Nbeal2 controls granule- related processes in haematopoietic cells. Subcellular fractionation of human and murine platelets revealed that Dock7, a known Nbeal2 binding partner, is mislocalised and has reduced expression in the platelets of GPS patients and Nbeal2−/− mice. The interaction of Nbeal2 with Dock7, a protein that regulates cytoskeletal rearrangements, prompted an investigation of actin-related processes in megakaryocytes (MKs) and platelets, which showed that MKs from Nbeal2−/− mice exhibit appropriate proplatelet formation, but thrombin- activated Nbeal2−/− platelets do not appropriately express F-actin. The reduced abundance and mislocalisation of Dock7, and the lack of α-granule release, may contribute to the defective platelet shape change observed in GPS patients. Lastly, Nbeal2−/− cell models were generated in the haematopoietic CHRF and K562 cell lines. These gene-edited cells were compared with induced pluripotent stem cell-derived MKs and Nbeal2−/− murine platelets to assess differences in their morphology, granularity, and size. Additionally, RNA-Seq and mass spectrometry analysis of these cell lines revealed differences in the respective transcriptomes and proteomes, which were used to define cellular processes associated with Nbeal2. These results were compared to the transcriptome and proteome results of additional data sets derived from GPS patient, Nbeal2−/− mouse, and cell line samples, to pinpoint novel genes and proteins that may be essential to the Nbeal2 mechanism.
  • ItemOpen Access
    Investigating the role of rare genetic variants in the aetiology of haemostasis disorders
    Stefanucci, Luca; Stefanucci, Luca [0000-0002-4352-1151]
    High throughput sequencing and publicly accessible genomic resources increased the diagnostic yields for inherited conditions, and, nowadays, the genetic bases for thousands of Mendelian disorders have been identified. However, providing a molecular diagnosis for these conditions remains challenging, and a considerable portion of patients with inherited conditions still lack a genetic diagnosis. In clinical genomics, identifying the aetiological variants remains a significant hurdle because they are hidden amongst thousands of rare variants present in the genome of each person. Furthermore, historically only the coding portion of the genome, or so-called exome, has been explored to identify causal variants. It is reasonable to assume that, for a fraction of patients with unexplained inherited diseases, the answer lies in the non-coding portion of the genome. To date, the ability to interpret the functional consequences of variants in the non-coding space remains limited. My thesis uses large-scale genomics studies and functional genomic techniques to investigate the role of genetic variants in the aetiology of haemostatic diseases. To explore the contribution of rare coding variants to the different phenotypes, I selected all the pathogenic and likely pathogenic variants from a few well-curated resources. Then, I identified carriers of these variants in the UK Biobank cohort and explored their phenotypes. This approach allowed me to estimate the effect sizes of this class of rare variants on the haemostatic phenotypes and investigate their interplay with common ones. I then expanded my investigation to non-coding regions. I performed experiments to define the most detailed cell type-specific maps of interactions between promoters and regulatory elements for the 93 diagnostic-grade genes for haemostatic diseases. To obtain these interaction landscapes, I differentiated human induced pluripotent stem cells from the principal cell types functionally implicated in haemostasis. I also generated chromatin conformation maps for the relevant genes using a capture Hi-C approach. Finally, I characterised the captured sequences by annotating them with cell type-specific epigenomic features, and I experimentally examined the regulatory potential of some of the defined regions. These validation experiments were based on two independent approaches: (I) reporter assays (II) perturbation of the epigenetic state for a few identified regions. Furthermore, I assessed the impact of rare genetic variants found in the NIHR BioResource Rare Disease participants, 10% of whom have haemostatic diseases. The aim of my PhD project was: (i) to investigate the contribution of rare coding variants to different phenotypes, (ii) to improve the annotation of the non-coding space of a set of well-characterised rare diseases genes and, (iii) to improve our ability to provide an accurate molecular diagnosis for individuals with unexplained inherited haemostatic diseases.
  • ItemControlled Access
    The role of the Dnmt3aR882H mutation in the evolution of clonal haematopoiesis.
    (2021-10-14) Marando, Ludovica
    DNMT3A mutations are very frequent events in clonal haematopoiesis of indeter- minate potential (CHIP), and considered one of the earliest genetic events during the development of haematopoietic malignancies. Individuals with CHIP, in spite of having a normal full blood count are at increased risk of cardiovascular and pulmonary diseases as well as haematological neoplasms. Somatic mutations in DNMT3A are drivers of CHIP, but decades may elapse between the acquisition of a mutation and CHIP. Besides, only a minority of these individuals will go onto developing a haematological cancer, this wide interindividual heterogeneity suggests that environmental factors influence clonal expansion. It is in particular emerging, that individuals with CHIP have a proinflamma- tory phenotype, additionally, chronic inflammation is typically considered deleterious to normal haematopoiesis, as it leads to haematopoietic stem cells (HSCs) exhaustion. R882H Using a conditional knock-in mouse model heterozygous for Dnmt3a mon DNMT3A mutation, we have assessed the responses of Long-Term Haematopoietic Stem Cells (LT-HSCs) to inflammatory "stress" in vitro and in vivo. We have also em- ployed bulk LT-HSCs genomics studies to interrogate differences in the transcriptional landscape in response to inflammation. We have demonstrated that Dnmt3a-mutant LT- HSCs have a globally abnormal response to inflammation, characterised by faster exit from quiescence and subsequent hyper-proliferation. Interestingly, as well as a larger differentiated output, Dnmt3aR882H HSCs were also better preserving their progenitor compartment and their self-renewal ability compared to wild-type HSCs exposed to the same stimulus, giving some insight into potential mechanisms that lead to clonal expan- sion within an inflammatory microenvironment. We linked this phenotype to the reduced expression of two TP53 target genes: Niam/Tbrg1, involved in the TP53-mediated activation of the p21 pathway that leads to G1 growth arrest upon exposure to "stress", and Necdin/Ndn a regulator of LT-HSCs quiescence. We then went on to demonstrate that TP53 and DNMT3A might function redundantly within the same functional pathway, and that Dnmt3a loss-of-function can lead to re- duced stem cell quiescence in vivo, and increased genomic instability when Dnmt3aR882H progenitors are challenged with genotoxic “stressors”. Interestingly, in spite of accumu- lating more DNA damage, Dnmt3aR882H cells did not lose clonogenic ability and showed similar viability to wild-type cells, suggesting that these cells are more tolerant to the accumulation of DNA damage. Subtle changes of this nature are likely to play an im- portant role in a phenomenon that develops over the lifetime of an individual, and could create a permissive state for the retention and accumulation of subsequent mutations, eventually leading to transformation.
  • ItemOpen Access
    Computational reconstruction of mouse development using single cell transcriptomics
    Imaz Rosshandler, Ivan; Imaz Rosshandler, Ivan [0000-0001-5975-0138]
    Single cell transcriptomics has significantly contributed to our understanding of cell types across species, organs and developmental processes. The rapid development of technologies, protocols and computational methods reflects a highly dynamic field over constant improvement. Particularly for Developmental Biology, being able to study at single-cell level and over time is required for a more detailed understanding of the underlying molecular mechanisms associated with cell differentiation and fate choice. Longitudinal cohort studies, ideal for capturing the dynamic nature of developmental mechanisms delineate the temporal relationship between cell type diversity and developmental dynamics. The efforts to build cell atlases from different model organisms already include extensive transcriptomic profiling of embryonic development, where detailed non-human models are essential to compensate the limitations arising from ethical reasons. In this context, the transcriptomic landscape of mouse development is perhaps the most complete among mammals. However, as cells are destroyed when measuring their transcriptomic profiles, only snapshots of the dynamical system are effectively captured. Thus, computational reconstructions of cell differentiation trajectories are essential even when lineage tracing experiments can be performed. The work presented in this dissertation uses a time course large-scale single-cell transcriptomic experiment of mouse embryos to present an effective mathematical and computational strategy that adequately describes the dynamics of gastrulation and early organogenesis on a variety of cell types during mouse embryonic development. The experimental data generated for this project, incorporates new time points into the mouse gastrulation and early organogenesis atlas already publicly available (Pijuan-Sala et al., 2018). That is, an extended version of the existing atlas. Results show a pipeline of sophisticated computational strategies to integrate the new time points. Then, to overcome the challenge of identifying and reconstructing the dynamics of all cell lineages, previously generated knowledge was combined with a variety of state-of-the-art computational methodologies. The analysis of differentiation trajectories is then taken to a deeper level in regards to the emergence of haemato-endothelial lineages, with emphasis on resolving the so-called Primitive and Definitive waves of blood production. Furthermore, perturbations to the system using mouse embryonic chimaera KO models are included. The analysis of haemato-endothelial lineages, presents a detailed reconstruction of the in vivo developmental process not reached by previous studies with single cell transcripomics. Lastly, predicted cell fates are compared to experimental observations by leveraging lineage trace experiments using cell grafting. In summary, this work highlights the complexity associated to generating developmental atlases, how to overcome the corresponding computational challenges and leverage this resource in a variety of contexts.
  • ItemOpen Access
    Oldest-old partner’s experiences of providing end-of-life care: a narrative study
    (2021-11-05) Morgan, Tessa; Morgan, Tessa [0000-0003-4917-6149]
    Background Population ageing has rapidly increased the number of people requiring end-of-life care across the globe. Governments have responded by promoting end-of-life in the community. Partly as a consequence, older partners are frequently providing for their partner’s end-of-life care at home, despite potentially facing their own health issues. Little is known about people aged 75 and over who are providing end-of-life care. In order to prepare our health and social care systems for rapidly ageing populations, we need to understand more about this group’s experiences of end-of-life care. Aim To explore the experiences of oldest-old partners looking after their partner approaching end-of-life care. Method First, I conducted a systematic review of the extant literature published since 1985 on the topic. Second, I conducted a longitudinal narrative interview study with 17 couples (19 participants in total). Findings A systematic review of the literature identified a small and only medium quality evidence-base with important empirical and theoretical gaps that require further research. Drawing on interview data, the first key finding was that older partners navigated the carer identity in relation to external and internal factors with not all subsequently embracing the carer identity for themselves. A second key finding is that older partners are actively engaged in integrating care in their capacity as home- keepers, networkers and vigilant visitors. A third key finding highlights the creative ways in which older partners engaged with a pill organizer called a dosette box to make their daily end-of-life caring and medical management bearable. Conclusions The overarching contributions of this thesis challenge notions of the fourth age as merely comprising “decline, passivity and frailty” by emphasizing the activity and creativity of older partners providing end-of-life care. Second, by thinking about oldest-old partners needs and experiences as interconnected, I suggest that policy-makers and health and social care providers will be able to more effectively design services that meet the needs of both oldest-old partners.
  • ItemOpen Access
    Understanding the role of extrinsic regulators in normal and malignant haematopoiesis
    Belmonte, Miriam
    Numerous studies have highlighted the central role that extrinsic regulators play in both normal and malignant haematopoiesis. Recent evidence suggests that variations in key molecules can directly modulate haematopoietic stem cell (HSC) fate, implicating noncell intrinsic mechanisms for both maintaining homeostasis and for driving disease development. This thesis spans both normal and malignant haematopoiesis, assessing the impact of extrinsic regulators on HSC fate and explores their role in driving the evolution of clonal haematological malignancies. First, I explored the impact of extrinsic regulators on highly purified single HSCs and found that minimising cytokine signalling could sustain a hibernation state in HSCs with the retention of functional properties both in vitro and in vivo. This work established the principle that core functions of HSCs could be maintained in the absence of the bone marrow microenvironment and allowed the identification of key factors dispensable for HSC function (Chapter 3). Next, to understand the role of extrinsic regulators in disease evolution I shifted focus to characterising the cytokine microenvironment in patients with preleukaemic HSCderived disorders known as the myeloproliferative neoplasms (MPNs). This work identified key regulators of the preleukaemic state, including two potential biomarkers of disease evolution, and additional molecules that associated with advanced disease (Chapter 4). This correlation of cytokine levels with specific disease subtypes and disease severity led to the further exploration of one of these molecules in the context of MPNs. The final chapter of my thesis therefore focused on the functional characterisation of IP-10, whose levels correlated with a more severe disease subtype and associated with JAK2 and TET2 mutational status in both patients and mouse models (Chapter 5). Together, these findings highlight the importance of the extrinsic regulators and the haematopoietic microenvironment in influencing cellular outcomes in both the normal and malignant setting and underscores the experimental and clinical potential of modulating extrinsic regulators of HSCs.
  • ItemOpen Access
    Niche disturbances in Chronic Lymphocytic Leukaemia affect HSC residency and drug resistance
    Santoro, Antonella
    Chronic lymphocytic leukaemia (CLL) is an indolent B cell malignancy infiltrating the lymphatic system and invariably the bone marrow (BM). Although treatment options for patients with advanced disease have significantly increased in the past years and improved life-expectancies, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary BM failure. Survival of CLL cells depends on protein kinase C- (PKC) expressed in activated BM mesenchymal stromal cells (BM-MSCs) which display activation of inflammatory pathways, as well as BM endothelial cells (BMEC) (Lutzny et al., 2013). This activation provides a nurturing environment, which not only contributes to disease progression, but likely also disrupts normal haematopoiesis, leading to insufficient blood production. I hypothesised that disease-induced alterations in the tumour microenvironment of CLL cells contribute to BM failure by skewing hematopoietic stem and progenitor cell (HSPC) fate choice commitments and tissue residency. In my study, the analysis of the peripheral blood (PB) of 67 patients with predominantly early stage CLL revealed that short term (ST) HSCs and Common Myelo-Erythroid Progenitors (CMP/MEPs) are found in higher frequencies and are associated with a skewed differentiation into myeloid cells in vitro and progenitor cells are less likely to produce erythrocytes. These findings provide evidence for the mechanisms underlying BM failure in patients with CLL. Our current knowledge of how malignant B cells interact with stromal cells within the BM microenvironment is limited. I hypothesised that BMEC play a fundamental role in nurturing malignant B cells during disease progression. In a murine model for CLL, I identified that malignant B cells home preferentially to the BM in close proximity to EC, and during disease progression, sinusoidal, but not arterial vessels, upregulate the stem cell marker, SCA1. RNAseq analyses of the remodelled sinusoids revealed activation of inflammatory and cytokine regulation pathways. In lymphoid malignancies, minimal residual disease (MRD) in the BM has prognostic value to predict disease relapse after patients received chemotherapy, suggesting the BM microenvironment is not only important for disease progression, but also plays an essential role for chemoresistance. Our lab recently demonstrated that BM-MSCs contribute to drug resistance via PKC mediated activation of lysosomes, which is required for tumour-stroma cell interactions (Park et al., 2020). However, it still is unclear which type of stromal cells are involved in this environment mediated drug resistance (EMDR) mechanism. I hypothesised that BMEC are the main drivers of EMDR in CLL and dependent on the activation of PKC. My results show indeed that BMECs activate lysosome production as a drug resistance mechanism and these effects are mitigated by pharmacological inhibition of PKC. Malignant B cells are found in close proximity to EC after treatment, suggesting the endothelial niche provides a sheltering from the action of cytotoxic drugs. In vitro experiments demonstrated that pharmacological inhibition of PKC sensitises CLL to the action of cytotoxic therapies. Overall the two projects carried out during my PhD have provided novel insights; first revealing that the disease-induced inflammatory environment could affect normal blood production in patients, and second that BMECs become remodelled. This reprogramming of BMECs is likely to play a fundamental role in the process of EMDR. Results from my work are important to improve current therapies for patients with CLL as well as the prevention of BM failure.