NIHRDataInsights/HRCSData huggingface dataset:
Title: The Molecular Mechanisms of Eukaryotic DNA Damage Tolerance Pathways
Funder: Wellcome Trust

Abstract: The key goals of my proposed research are to fully elucidate the mechanistic details of the two main eukaryotic DNA damage tolerance (DTT) pathways, translesion synthesis (TLS) and template switching (TS), and to decipher what factors influence pathway choice. To do this, I will reconstitute each DDT pathway in the presence of a eukaryotic replisome using state-of-the-art biochemical systems, before integrating both pathways together to investigate how pathway choice is governed. After successfully reconstituting each pathway, I will use cutting-edge force and fluorescence-based single-molecule techniques to provide in-depth characterisation of the response of the replisome to DNA damage and the details and regulation of each DDT mechanism. I aim to address a number of outstanding questions relating to these mechanisms including: 1. How and where do TLS and TS operate in the context of a progressing replisome? 2. How do post-translational modifications to PCNA mechanistically affect pathway choice? 3. Is TLS or TS the predominant DDT pathway? For the first time, recent advances in reconstitutional biochemistry provide the opportunity to fully elucidate the details of these pathways and how they integrate with the eukaryotic replisome. This approach will therefore significantly enhance our understanding of DDT in eukaryotes.

Lay abstract: Life can be viewed as an elaborate mechanism for the replication and propagation of DNA. Cellular division and organism reproduction rely on the accurate duplication of the genome. However, hundreds of thousands of DNA damage events occur per cell daily, this damage can block DNA replication leading to genome instability and cell death. In order to overcome this, cells possess mechanisms to tolerate such damage. I aim to elucidate the molecular details of each DNA damage tolerance mechanism, and how the choice between different pathways is regulated. To do this, I will combine state-of-the-art reconstitution biochemistry with fluorescence and force-based single-molecule techniques. Reconstituting these mechanisms outside the cell will improve our understanding of the proteins involved and their interplay with the core replication machinery. This will enhance our understanding of how these mechanisms can break down during cancer development and how this can be exploited to treat the disease.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Engineering a novel human bone marrow organoid to target myelofibrosis.
Funder: Wellcome Trust

Abstract: Megakaryocytes (MKs) are platelet-producing bone marrow cells with critical roles in the regulation of the bone marrow microenvironment. In myelofibrosis, a progressive and debilitating condition with inadequate treatment, MKs drive extensive fibrosis of the bone marrow. The mechanisms behind myelofibrosis remain unknown, largely due to limitations in existing assays which fail to replicate the complex and multicellular bone marrow milieu. There is a pressing need for better human models with which we can study MK biology and drive the development of therapies for severe conditions like myelofibrosis. In this fellowship, I will generate the first artificial bone marrow organoid which replicates the architecture of this tightly regulated and carefully organised microenvironment in 3D. I will use this to study MK migration, maturation and platelet production in healthy and myelofibrotic conditions, and test my hypothesis that the fibrosis observed in myelofibrosis is the result of impaired MK migration leading to the intramedullary release of platelets and/or profibrotic cytokines. By differentiating pluripotent stem cells in a 3D printed scaffold mimicking human bone marrow within a bioreactor, I will engineer an organoid which will be a paradigm-shifting tool not only for the study of haematopoeisis, but with broader applications in drug development.

Lay abstract: Megakaryocytes (MKs) are rare bone marrow cells which are responsible for making platelets - cells which make our blood clot on injury. In a rare bone marrow cancer called myelfibrosis, MKs drive scarring of the bone marrow which results in the loss of its ability to make healthy blood cells. Myelofibrosis remains a severe condition with limited treatment options, and like many other bone marrow cancers, is difficult to study because of a lack of models which re-create a very complex and carefully organised organ. We urgently need better systems with which we can look for and test novel therapies for conditions like myelofibrosis. During this fellowship, I will create a completely novel artificial bone marrow ‘organoid’ to study MKs in myelofibrosis. This system will not only help us develop and test new treatments for myelofibrosis, it will also be applicable to other bone marrow conditions like leukaemia.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: LncRNA scaffolds of nuclear RNP compartments, and their roles in embryogenesis.
Funder: Wellcome Trust

Abstract: Several long non-coding RNAs (lncRNAs) are capable of scaffolding ribonucleoproteins (RNPs) that condense and phase-separate nuclear membraneless compartments. Both, lncRNAs and RNPs play many key regulatory roles in development and their function in cell fate choice is further enhanced by their cross-regulation. To demonstrate the importance of such cross-regulation for fine-tuning developmental decisions I will build a framework of experimental and computational methods to study how lncRNAs scaffold nuclear RNP compartments and hence understand how interactions between lncRNAs and RNPs within these compartments create networks that coordinate molecular processes in early development. To interrogate the developmental role of these network motifs between lncRNAs and bound RNPs, I will also establish new tools that allow rapid manipulation of lncRNA condensation in advanced in-vitro embryogenesis system. Collectively, this proposal will reveal the cross-regulatory mechanism of nuclear rewiring by lncRNA condensation that plays a role not only in development, but when perturbed, could also be implicated in diseases such as ALS and cancer.

Lay abstract: Long non-coding RNAs (lncRNAs), by acting as molecular scaffolds, can condense ribonucleoproteins (RNPs) into phase-separated lncRNP compartments that concentrate and sequester nuclear molecules. It is becoming apparent that such compartments can critically influence post-transcriptional regulatory networks that guide cellular differentiation in embryogenesis, regeneration and disease. The extent and scope of this novel regulatory layer, and the molecular mechanisms controlling its assembly and operation, remain to be discovered. This proposal, by addressing these questions, has the potential to lead to novel fundamental insights into embryogenesis and regeneration, as well as in the ontogeny of diseases such as ALS and cancer.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: “Killing me softly”: role of forces in stiffness-dependent tumour-cell killing by T cells
Funder: Wellcome Trust

Abstract: My research proposal aims to study the influence of forces on immune cell activation and killing during tumour cell/target cell engagement. T cells have been reported to be stiffness dependently activated, but while this phenomenon is well known, the underlying mechanism remains unclear. With the following proposal I will link intracellular stresses in the actin network to actin assembly impairment and T-cell activation on various stiff targets. This will give unique insights into the mechanism of stiffness-dependent T-cell activation and killing and the role of actin in these processes. I propose to introduce stress reporters into T cells to visualise the distribution of stress within the actin network. I will then study the changes in force distribution upon T-cell activation on different stiff substrates using cell lines with mutations in the actin assembly machinery. I will continue the work by imaging primary cytotoxic cells and using state-of-the-art lightsheet imaging tools to visualise the 3D force distribution during T cell/tumour cell engagement. This is the first study able to uncover the role of intracellular forces in T-cell activation and killing, if force thresholds need to be met to lead to full activation, and what role actin assembly has in this process.

Lay abstract: Our immune system constantly clears our body from malignant cells through T killer cells. These scan the surface of their surrounding cells for signs of abnormality and initiate killing when activated. How T killer cells decide to kill the target cell is not yet understood, but the decision-making process is shown to be influenced by target cell stiffness and the ability to push and pull on the other cell. As such it seems, that softer tumour cells are able to escape the immune response, possibly through presenting less resistance to the T cell. In this proposal I want to uncover the mechanism of this stiffness-sensitive killing by visualising the forces inside T cells encountering targets of varying stiffnesses, and identify the decisive factor in the killing decision. This will not only give us insights into mechanisms of T-cell activation and killing, but possibly also open new avenues for therapeutic intervention.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Enhancer DNA methylation dynamics during early mammalian development
Funder: Wellcome Trust

Abstract: Early mammalian development is characterised by profound transcriptional rewiring and epigenetic remodelling, including a global change in DNA methylation (DNAme) levels. Enhancers, regulatory elements that direct spatiotemporal gene expression, are the most variably methylated regions in the genome, but the functional consequences of these alterations are not known. Enhancer DNAme has been associated with both gene repression and gene activation, but the majority of studies are correlative or focus on single loci and a systematic analysis is currently lacking. I aim to use a novel reporter-screen based approach together with cutting-edge (epi)genomics approaches (capture Hi-C, ChIP-seq, RNA-seq) to determine whether DNA methylation affects (1) enhancer activity and (2) enhancer-promoter looping interactions in mouse embryonic stem cells (mESCs). I will determine the functional effects of enhancer methylation using in vitro differentiation assays and by generating chimeric embryos. Genome wide methylation will be perturbed using DNA methyltransferase knockout cells whilst locus-specific manipulation will be achieved using epigenetic editing techniques (dCas9-TET1 fusion proteins). The proposed research will generate valuable datasets for the fields of epigenetics and development and the principles discovered will be applicable to developmental biology and other scenarios where DNAme is substantially altered, including cancer and aging.

Lay abstract: Enhancers are regulatory elements (specific DNA-sequences) that can drive the expression of protein-coding genes, even when located a great distance away from them. They interact with their target genes via three-dimensional loops that form in the DNA molecule. DNA methylation is an important epigenetic mark (a chemical modification of DNA) that can inhibit gene expression when located nearby (at the promoter), but it is not known how DNA methylation affects enhancers. My aim is to understand the effect of DNA methylation on enhancer activity. I will use a novel screen-based approach and cutting-edge genomics approaches to interrogate this question in cell-based models of early development (embryonic stem cells), where DNA methylation levels can be controlled dynamically. My findings will improve our basic understanding of gene regulation, and indicate how we might modulate enhancer activity in disease contexts.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Generating a global view of cell lineage trees during early mouse embryo development
Funder: Wellcome Trust

Abstract: Understanding the routes through which a single cell populates the adult organism is one of the most fundamental yet elusive areas of biology. This project aims to use recent innovations in single-cell technology to generate a blueprint of cell fate decisions during early mouse development, and to examine the mechanisms behind the regulative nature of embryogenesis. Firstly, CRISPR-Cas9 technology will be employed to induce thousands of unique barcodes in cells, enabling the interrogation of cellular clonal history. Secondly, lineage tracing analysis will be combined with single-cell RNA sequencing which will provide the molecular resolution to precisely classify cell types and build up a fate map of early development. These data will be used to answer fundamental, outstanding questions in developmental biology. Finally, these analyses will be performed in chimeric embryos which contain cells unable to contribute to certain lineages to analyse compensatory behaviour. This will shed light on the largely unexplored area of the plasticity of cell fate decisions and the molecular control behind them. Together these analyses will generate a detailed, publicly-available chart of the clonal history and expression profiles of every cell during gastrulation and thereby provide an important reference point toward understanding a wealth of cellular processes.

Lay abstract: All life starts out as one single cell which divides to generate every single cell type in the body. How this incredibly complex process is achieved still remains a mystery in biology. This project aims to use a new technology to mark cells in the developing mouse embryo with unique ‘barcodes’. By comparing cells with shared barcodes, we can infer relationships between cells and use this knowledge to build up an ancestry tree of development. The technology also allows us to investigate the mechanisms and pathways involved in cell differentiation. We aim to use our findings to build a map of cell fate decisions during development and to answer basic questions in biology such as understanding how cells from different tissues are connected, and calculating how many founder cells make up each organ. Among the applications of this analysis will be improving tissue regeneration protocols for patient benefit.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Microtubule-independent organisation of DNA by the kinesin KIF4
Funder: Wellcome Trust

Abstract: The 3-dimensional organisation of chromatin is critical for genome function and inheritance. 3-D organisation is particularly important in cell division, when chromatin must be condensed to form chromosomes that are sufficiently compact to be accurately segregated on the microtubule-based spindle. Condensin and topoisomerase II have been extensively studied for their contributions to mitotic chromosome condensation. Surprisingly, chromosome condensation also requires KIF4, a kinesin-family microtubule motor protein that acts in parallel to condensin and topoisomerase II. While KIF4 function has been investigated in microtubule-dependent processes, such as chromosome transport, cytokinesis and neuronal transport, how it acts to package chromatin remains mysterious. To elucidate new principles in genome organisation and to define a critical microtubule-independent role of a kinesin, I propose to study the role of KIF4 in chromosome condensation. My specific goals are to: 1) to understand how KIF4 function is regulated; 2) to obtain a quantitative understanding of KIF4 function in 3-D chromatin organisation in vivo, and 3) to reconstitute KIF4 interactions with DNA and chromatin in vitro. Taken together, the proposed research will help address how chromatin is condensed during mitosis and will provide the first insights into how a kinesin executes a critical cellular function independently of microtubules.

Lay abstract: The DNA in the cell’s nucleus carries all the critical information required for life. Hence, its accurate duplication and subsequent distribution during cell division is vital. Sophisticated intracellular machinery is responsible for precise packaging of the DNA into chromosomes before cell division occurs. KIF4 is a molecular motor protein that is involved in this process. However, it functions in the nucleus, which is devoid of the structural polymers called microtubules that are normally required for the activities of motor proteins like KIF4. How KIF4 contributes to DNA organisation in this environment is unclear. My proposed research combines biochemistry, in vivo microscopy of living cells and in vitro DNA single-molecule assays to address this question. The proposed research will help elucidate how KIF4 organises DNA to form chromosomes and will provide the first insights into how a kinesin executes a critical cellular function in the absence of microtubules.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Care as a Complex System: Understanding the Network Dynamics of Healthcare Delivery
Funder: Wellcome Trust

Abstract: Health systems are a product of the interactions between healthcare professionals and their patients. These interactions may be understood as networks connecting all actors within the health system to one another through their clinical interactions. In doing so, the provision of healthcare becomes a complex system which may be interrogated and understood using methods of network analysis not previously used in population health research. In the fellowship I will address critical questions facing health systems today, using the latest techniques in machine learning, econometrics and network analysis. I will derive patient-sharing networks to understand how clinical data may be safely, and efficiently shared between patients and clinicians. Through the application of random forest and alternative-specific conditional logit models I will develop new ways to understand how clinical demand is redistributed following sudden or planned changes to hospital services. Using Markov Multiscale Community Detection, I will identify how current primary and secondary care services may be integrated to enhance continuity of care between clinicians while ensuring accountability for the entire population served by the National Health Service. Collectively, the techniques developed during this fellowship will empower patients, clinicians and policymakers to make informed decisions to improve how healthcare is delivered.

Lay abstract: Recent years have seen the emergence of computational techniques that are able to understand complex systems. In medicine, these techniques have largely been applied to derive new drug therapies or improve diagnosis. They have not been applied to understand how the health system itself may be more fair or efficient. In this fellowship, I will explore three pressing questions facing health systems across the world: How should clinical information be shared across the healthcare system? How will changes to clinical services affect patients and clinicians? How can we organise primary and secondary care services to make patient care safer, fairer and more efficient? I will apply the latest methodological techniques in data science and network analysis to explore healthcare in the National Health Service and internationally as a complex system, and in doing so empower patients, clinicians and policymakers to make informed decisions to improve how healthcare is delivered.

Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES
Health categories: GENERIC HEALTH RELEVANCE
Title: Making social change happen: What inspires collective action for health and gender equality?
Funder: Wellcome Trust

Abstract: Motivation: Interventions mobilising communities in collective action for their own health are some of the most effective known global health interventions. However, they do not always succeed and we cannot presently predict or explain why this happens. Aim: Taking as an example a community mobilisation programme to prevent violence against women in urban slums in Mumbai, India, I will test theory concerning the drivers of collective action from behavioural economics and social psychology. Method: (1) To unpack how a community mobilisation programme may stimulate collective action in urban India, I will collect and analyse qualitative interview, media and observational data. (2) To uncover the determinants of participation in collective action, I will conduct cross-sectional and difference-in-differences analysis of secondary data from a randomised controlled trial of the same community mobilisation programme. (3) To produce causal evidence for the drivers of collective action, I will conduct a behavioural experiment to test the behavioural effects of changing key messages on programme participants. I will use the answers to (1) and (2) to guide message design. Impact: The study results will be able to guide policy-makers and practitioners on when, how and why complex participatory interventions work to promote health and gender equality.

Lay abstract: Whether to address violence against women, prevent newborns from dying, or hold clinic staff to account for low performance, research shows interventions engaging local people in developing countries in collective action for the common good can have enormous impact. However, results have varied widely. Sometimes, whole communities have rallied in protest and solidarity. At other times, interventions have been abandoned because nobody turned up. We cannot currently predict or explain what happens, because we do not have enough evidence. I aim to find out what inspires people to participate in collective action for their health. Taking as an example a community-based programme to mobilise residents to take action against violence against women in urban slums in Mumbai, India, I will test hypotheses informed by social science using qualitative, survey and experimental methods. My findings will help governments, NGOs and researchers design better programmes to improve people’s health through community participation.

Research activity codes: 3.1 PRIMARY PREVENTION INTERVENTIONS TO MODIFY BEHAVIOURS OR PROMOTE WELL-BEING
Health categories: GENERIC HEALTH RELEVANCE