NIHRDataInsights/HRCSData huggingface dataset:
Title: High resolution metabolite and peptide mass spectrometry
Funder: Wellcome Trust

Abstract: We request funding for a “High-resolution and mass accuracy Mass Spectrometer with sequential fragmentation (MSn) capability”, hereafter termed a Lumos, for researchers across the University of Edinburgh and collaborators. This will be housed at the Institute of Genetics and Molecular Medicine (IGMM) Mass Spectrometry Unit (IGMM-MS). This new instrumentation will be transformative for the types of proteomic and metabolomic analyses that IGMM-MS performs to support researchers at the University of Edinburgh and collaborators. The Lumos provides technological advances that will afford the applicants and our collaborators access to the following cutting edge analyses: 1. Accurate stable isotope tracing of biosynthesis and metabolism of lipids and large metabolites (through its ability to distinguish 15N- and 13C-containing molecules at higher masses). 2. Unequivocal identification of normal and modified/damaged lipids on a lipidome-wide scale. 3. High confidence assignment of complex post-translational modifications in proteins. 4. Quantitation of the proteome in multiple samples on a high-throughput scale, through 10-plex tandem mass tagging (TMT). The Lumos is the only mass spectrometer that will provide IGMM-MS with these transformative capabilities and we present preliminary data that support our scientific need for this instrument.

Lay abstract: The Institute of Genetics and Molecular Medicine hosts over 500 scientists who try to understand how cells work in health and disease. Many different types of disease are studied at the IGMM and different types of molecules need to be investigated. Proteins, small metabolites (e.g. sugars) and lipids (e.g. fats) make up the bulk of our bodies and of each individual cell and the current state-of-the-art technique for analysis of these molecules is mass spectrometry. We are requesting a new mass spectrometer that will allow us to analyse these molecules in ways that we previously could not. It will allow us to identify many more healthy and damaged molecules and to measure how quickly they are made and degraded, as well as enabling analysis in clinical samples from many patients. Lack of access to this machine is holding back the progress of investigators, including those funded by the Wellcome Trust.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Mechanics and execution of homologous recombination - biophysics to the organism
Funder: Wellcome Trust

Abstract: Homologous recombination (HR) is an essential mechanism for the repair of DNA double-strand breaks and damaged replication forks and is associated with genetic disorders, cancer and aging. HR repairs DNA damage by copying the correct genetic information from an intact chromosomal template, which is critically dependent on the recombinase RAD51. To ensure its timely and accurate completion, HR is positively and negatively regulated by RAD51 co-factors and anti-recombinases. How these HR regulators function at the molecular level remains poorly understood and represents a significant challenge to the field due to the lack of mechanistic resolution afforded by conventional bulk biochemical approaches. We recently pioneered several cutting-edge biophysical approaches to interrogate the HR reaction in unprecedented detail. Importantly, we demonstrated the power of integrating data from these complementary methodologies to uncover the mechanism of action of the Rad51 paralogs in modulating RAD51 to promote HR. The aim of our proposal is to extend this paradigm to study multiple different HR regulators to gain insights into how they work individually and how they act cooperatively during HR. Deciphering how HR regulators work will provide an improved understanding of the molecular mechanisms relevant to carcinogenesis and may present unique opportunities for therapeutic intervention.

Lay abstract: DNA, the molecule that encodes our genetic information, is under constant attack from both external and internal factors, such as UV light from the sun and by-products of cellular metabolism. These factors induce damage to the DNA, which must be repaired correctly to prevent mutations and more complex chromosome alterations, which drive aging, cancer and other human diseases. Arguably the most severe form of DNA damage is the double strand break (DSB), in which both strands of the DNA duplex are severed. Fortunately, cells possess several DNA repair mechanims that can rejoin or replace the genetic information at a DSB, including non-homologous end joining and homologous recombination (HR). The aim of this proposal is to apply cutting-edge experimental methods that have been pioneered in our respective laboratories, to investigate the mechanics of the HR process in unprecedented detail. Much of our collaborative work will focus on the regulation of a key HR protein Rad51, which posesses the remarkable ability to catalyze invasion of a broken DNA molecule into an intact template DNA duplex. Since many of the factors we will study are commonly mutated in breast and ovarian cancers, our work will lead to an improved understanding of human diseases.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Revealing how cells read the environment to regulate adult gut renewal
Funder: Wellcome Trust

Abstract: The intestinal epithelium constantly regenerates from stem cells which adjust their behaviour to the changing physiological conditions the gut is exposed to. For example, stem cell proliferation rates can transiently increase to speed up regeneration after tissue loss, or in response to the diet, before reverting to steady-state levels once correct tissue size is reached. This plasticity is essential for intestinal function, as lack of regeneration causes tissue atrophy whereas unrestricted stem cell proliferation causes cancer. I propose to use the genetically tractable Drosophila gut to identify the secreted and physical factors regulating gut plasticity. Thanks to targeted genetic screens done in contexts that trigger reversible changes in gut size and functional analyses, I will identify novel extracellular signalling molecules regulating cell proliferation. I will also examine the relationships between physical remodelling of the gut and stem cell proliferation by characterising the transcriptional output of integrin signaling and establishing how physical remodelling of the visceral mesoderm (a source of growth factors in the gut) contributes to regenerate the intestinal epithelium. As the regulation of intestinal proliferation is largely conserved between Drosophila and mammals this work has the potential to uncover important new principles relevant to intestinal maintenance and degenerative diseases.

Lay abstract: The gut is composed of thousands of specialised cells allowing it to fulfill complex roles underpinning organism health. Whilst digesting food and fighting off pathogens, the gut must also maintain itself by replacing cells after they pass their “expiry date”. This relies on stem cells that divide to maintain the necessary number and type of cells. Our goal is to understand how the stem cells ‘know’ how fast they are required to proliferate. This is important, because when the information that instructs stem cells is not conveyed properly, it can lead to degenerative diseases or cancer. We focus on 1) discovering molecules that instruct cells to multiply in the gut and 2) understanding how gut distortion influences decisions about cell proliferation. We work with the intestine of the fruit fly Drosophila due to the ease with which it can be genetically manipulated and imaged with sophisticated microscopy, its rapid lifecycle and because it is cost-effective. Importantly, this organism shares a surprising ~70% of its DNA with human disease genes, meaning that our basic research may contribute to the design of new therapies against a range of intestinal diseases affecting human health.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Development of a new generation of biostable nucleic acid antibodies (aptamers), catalysts and nanostructures for precision biomedicine using synthetic genetic systems
Funder: Wellcome Trust

Abstract: Xeno nucleic acids (XNA) are analogues of DNA with alternative sugars or backbones providing physicochemical properties beyond natural polymers, including biostability and bioorthogonality. Directed evolution of XNAs could be a powerful platform technology for generation of novel tools for molecular medicine. However, replicating artificial polymers has been technically challenging, hampering repertoire selection (SELEX) experiments using fully-artificial molecules. Recently, polymerases have been engineered that permit cycles of synthesis and reverse transcription of various XNAs; ‘synthetic genetics’. My lab will apply and develop synthetic genetics toward: 1. XNA ligands ("aptamers"/chemical antibodies) specific for biomedically-relevant targets, including live tumour cells, stem cells and pathogens (i.e “cell-SELEX” using XNAs). 2. XNA catalysts (“XNAzymes”) for precision knock-down of disease-associated RNA (e.g. oncogenic mRNA/microRNA) using chemistries capable of invading structured RNAs & retaining activity in vivo. To achieve this, we will exploit XNA systems in hand, plus seek to expand the chemical palette with modified bases through new building blocks developed by the Herdewijn lab, e.g. C5-ethynyl HNA triphosphates, which will allow side-chain attachment by ‘click’ chemistry. Long-term, we will explore using XNA aptamer conjugates and nanostructures for: 1. Targeted delivery of therapeutic payloads. 2. Modulation of immune effector responses, e.g. directed activation of T-cell cytotoxicity.

Lay abstract: Beyond their role in genetic information storage, nucleic acids (DNA and RNA) can form 3D structures capable of recognising other molecules ("aptamers" or chemical antibodies) or orchestrating chemical reactions ("ribozymes"/"DNAzymes"). In principle, such functional nucleic acids can be used to identify and combat cancerous cells or harmful bacteria and viruses. However, DNA and RNA are rapidly destroyed within the body, or can trigger problematic immune responses. Recently, engineering DNA-replicating enzymes has allowed us to use artificial equivalents, xeno nucleic acids (XNAs), which are more robust and well-tolerated. I propose to develop XNAs to target disease-associated molecules and cells ("XNA aptamers" and "XNAzymes"), and explore using XNAs to precisely target tumors, delivering medicines and turning on or off the immune system in response to disease. In principle, nucleic acids can be made, shipped and stored more easily than protein-based medicines, without using cells or animals, offering significant advantages over current therapeutics.

Research activity codes: 1.3 CHEMICAL AND PHYSICAL SCIENCES
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: The mechano-biology of cell division
Funder: Wellcome Trust

Abstract: Cell division in epithelial sheets is crucial for tissue renewal, yet unchecked division leads to tissue dysplasia and ultimately cancer. The aim of this project is to discover how cell division is regulated in normal epithelial tissue and how it is perturbed by oncogenic mutations to disrupt tissue integrity. In previous work, I showed how activation of an oncogene, Ras, directly impacts cell division by controlling the shape, contractility and mechanics of single mitotic cells. I now ask how this finding plays out in a complex tissue environment. To do this, I will employ a multi-disciplinary approach, combining imaging and quantitative cell biology with biophysical techniques to study cell division mechanics in normal epithelial monolayers and Ras-mutant pancreatic cancer tumoroids. I will ask how the actin cytoskeleton is regulated during epithelial cell division to permit dynamic cell shape changes while maintaining cell-cell adhesion and preserving barrier function. I will determine how k-Ras activation perturbs this, identifying key mechanistic links between Ras/ERK-dependent transcription and morphological phenotypes. Finally, I will address how these Ras-induced changes to cell division alter the growth and organization of 3D tissue structures in pancreatic cancer.

Lay abstract: In the human body, cells constantly divide to maintain and repair tissues. However uncontrolled cell division leads to tissue overgrowth and ultimately, cancer. I want to understand how cell division is controlled in normal tissue and how it is altered by genetic mutations in cancer. I will focus on the role of an oncogene, Ras, as I have previously discovered that it directly controls how cells divide. Ras activation occurs in many different types of cancer, but particularly pancreatic cancer. Therefore, I will use cutting-edge microscopy techniques to image cells dividing within normal tissue and pancreatic cancer tumoroids. I will ask how Ras subverts the normal cell division process within these tissues and how abnormal cell divisions disrupt normal tissue organization to form tumours. By discovering fundamental principles of how cancer cells divide, this work will identify new molecular targets for pancreatic cancer treatment.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Understanding signal integration in eukaryotic cell growth
Funder: Wellcome Trust

Abstract: The aim of the project would be to understand signal integration in the TOR (Target of Rapamycin) growth signaling pathway. TOR growth signaling is mediated by the kinase activity of TOR complex 1. This is contingent on concurrent activation of two sets of small GTPases, the Rags and Rheb, which recruit TORC1 to lysosomes and activate it there. Both GTPases are regulated by GTPase activating proteins, the GATOR complex(es) in the case of the Rag GTPases, and the TSC (tuberous sclerosis complex) in the case of Rheb. We would attempt to recombinantly express, purify and reconstitute these complexes, in order to interrogate their activity in response to known signals using biochemical and biophysical assays. We would also attempt to obtain their structures, using x-ray crystallography and electron cryo-microscopy, leading to a molecular understanding of their regulation. The results would be beneficial both academically, given the key position of TOR signaling in eukaryotic cell growth, and medically, as TOR is a known to be a key target in immuno-repression, cancer and tumour syndromes. Any new information on the regulation of the tuberous sclerosis complex would almost certainly be directly applicable to this, and related, hereditary condition(s).

Lay abstract: When cells decide whether or not to grow, they take into account many considerations about their environment. Are there enough nutrients? Is there enough oxygen? Are conditions outside clement? Does the body need to grow? Are we in the right place? Incorrect interpretations can result in tumours and allergies. We know that most of these signals activate a protein called TOR, but it is still unclear which are considered more important, or how they are combined. The aim of the project would be to understand how signals are interpreted just upstream of TOR in molecular detail. We would produce the proteins responsible for carrying these signals, and get them to work outside of the cell. Known incoming signals could then be applied in a controlled environment to pick apart their effects. We would also try to look at the signaling process directly using powerful electron microscopes or light sources to visualise the molecules and understand how they work. Success in this project would improve our understanding of how the decision for a cell to grow is made, and would be applicable to immunology, oncology, metabolic conditions such as diabetes, and possibly also to our understanding of neurology and aging.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Molecular mechanisms controlling germline stem cell biology
Funder: Wellcome Trust

Abstract: The ability of stem cells to replenish and differentiate is critical for tissue growth, repair, and homeostasis. Yet, our understanding of the molecular mechanisms governing stem cell fate transitions remains limited. Through a systematic RNAi screen using the Drosophila germline as a model for stem cell biology, I uncovered a central role for ribosome biogenesis and protein synthesis regulation in controlling fate transitions. My current goal is to dissect the molecular mechanisms by which protein synthesis control - a new frontier in genomic regulation - governs stem cell maintenance and differentiation in vivo. In particular, I propose to generate a systematic and detailed translation-based roadmap of the differentiation process. This will be achieved by coupling genetic tools with high-throughput techniques, and aims to determine the set of actively translated mRNAs at consecutive differentiation stages. This unbiased approach will allow me to reveal the set of regulated targets and to uncover the cascades dictating fate transitions during germline differentiation. In parallel, I will combine developmental and molecular analysis to explore the mechanisms by which specific translation regulators support key developmental aspects. Dissecting such regulatory mechanisms will provide new insights into the principles dictating stem cell fate transitions in vivo.

Lay abstract: Maintenance of stem cell populations, as well as the control of self-renewal and differentiation is essential for proper development and tissue homeostasis in animals. Indeed, ablation of the stem cell population can lead to organ malformation and tissue replacement defects, affecting longevity or fertility. The same is true for unbalanced shifts between stem cell self-renewal and differentiation, which can directly affect tissue architecture and regeneration capacity, as well as lead to tumorigenesis. Even though the proper control stem cell identity and commitment to differentiate must be very well controlled, our understanding of these processes remains limited. By using the germline as a model system and employing a variety of genetic, molecular, developmental analyses, my goal is to investigate the mechanisms that regulate stem cell biology in vivo. In particular, I am interest in understanding how changes in gene expression control the balance between stem cell self-renewal and differentiation. Given the importance of stem cells for development and survival, this research is not only of broad interest to biologists and stem cell researchers, but has direct implications on regenerative medicine, aging, and cancer.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: The molecular mechanisms underlying epithelial cell plasticity
Funder: Wellcome Trust

Abstract: Epithelial cells display a remarkable plasticity and while this property is vital for morphogenesis during normal development, it plays a critical role in driving the abnormal cell behaviours underlying tumour metastasis. Cell plasticity is the ability of cells to reversibly change phenotype and relies on cells undergoing an epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET). While several transcription factors regulating EMTs have been isolated, how these impinge on the cellular responses underlying cell plasticity remains poorly understood. Even less is ­known about MET: the signals required for its induction and the downstream cellular and molecular consequences remain obscure. I propose to identify the molecular mechanisms controlling and orchestrating epithelial cell plasticity. The embryonic Drosophila midgut offers an unparalleled opportunity to visualise these processes in vivo, delineate the transcriptional networks and downstream effectors involved, and, in particular, study how the processes of EMT and MET relate to each other. I will take a multidisciplinary approach, combining cutting edge live cell imaging with sophisticated genetic, transcriptomic and bioinformatics approaches. Dissecting the molecular mechanisms underlying epithelial cell plasticity and providing new targets will ultimately aid the diagnosis, prognosis and treatment of metastatic cancer.

Lay abstract: In many cancers cells acquire abnormal motile behaviours leading to metastasis, the main cause of cancer-related deaths. It is now clear that processes normally driving the tightly-controlled movement of cells during development, are reactivated in metastatic cancers in a dysregulated manner. These processes are called the epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-epithelial-transition (MET). They enable cells to reversibly switch between stationary and migratory cell states and the molecular mechanisms orchestrating both processes remain poorly understood. I will use a model organism, the fruit fly Drosophila melanogaster to study the basic biology of these processes during normal development. Long recognised as a valuable model for basic research, the fruit fly is now emerging as a powerful tool to investigate malignancy and identify new therapeutics. Not only will I use sophisticated genetic tools and systems biology approaches to identify the molecular mechanisms underlying EMT and MET, I will use cutting-edge microscopy to follow the behaviour of cells undergoing these processes in real time. My goal is to determine the molecular machinery driving the processes of EMT and MET and consequently identify novel targets that will ultimately aid the diagnosis, prognosis and treatment of metastatic cancer.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS