NIHRDataInsights/HRCSData huggingface dataset:
Title: Integrative study of Unc5 signalling
Funder: Medical Research Council

Abstract: This study focuses on Unc5, a cell surface receptor that plays essential roles in neural/vascular tissues and in cancer. I will reveal the molecular properties of Unc5-ligand interactions by solving crystal structures of the Unc5 ectodomain in complex with its ligands. To achieve this, I will express the proteins recombinantly in mammalian cell cultures and use automated methods to produce crystals. Using synchrotron beamlines to collect X-ray diffraction data from these crystals, I will calculate their structural models. I will use Surface Plasmon Resonance (SPR) and Multi-Angle Light Scattering (MALS) experiments to characterise the binding properties of Unc5 and its ligands. Based on the structural data, I will design mutants with specific ligand-binding properties. I will study the properties of these mutants, alongside the wild type proteins, using single molecule localisation microscopy and Stimulated Emission Depletion (STED) microscopy techniques. I will thereby reveal their localisation and diffusion patterns at the cell surface. Using cell biology assays, I will reveal the effects of Unc5-ligand interactions on cell behaviour (adhesion, repulsion, migration and proliferation). I will use a range of assays to achieve this. For example, I will perform "stripe" assays where cells choose between growing on alternating surface stripes: one coated with the protein of interest, the other coated with a neutral control protein. I will also perform assays in which cells (e.g. neurons) expressing Unc5 receptor choose the direction in which they grow within in a gradient of ligand protein. In collaboration, I will also reveal the effects of Unc5-ligand interactions in model tissues. As a result, the proposed work will constitute a necessary step towards understanding this medically important signalling system on the molecular, cellular and tissue levels and generate knowledge for the specific treatment of cancers with defects in the Unc5 signalling system.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.3 CHEMICAL AND PHYSICAL SCIENCES
Health categories: GENERIC HEALTH RELEVANCE
Title: The cellular response to complex DNA damage induced by ionising radiation
Funder: Medical Research Council

Abstract: The critical cellular target for ionising radiation (IR) is DNA and DNA damage accumulation is implicated in the development of ageing, neurodegenerative diseases and cancer. However, IR is also the most effective cancer treatment and it is believed that its therapeutic effect is partially attributable to complex DNA damage (CDD), where several DNA lesions are induced in close proximity. However, very little is known about how CDD is initially recognised and/or processed in human cells. Since genomic DNA is tightly packed with histones to form chromatin there is emerging evidence, particularly during double strand break repair, that histone modifications (eg. ubiquitylation) are required to unwind or remodel chromatin to increase accessibility of the DNA damage to repair enzymes. However, the role of these modifications in the signalling and/or repair of CDD induced by IR is unknown. Therefore, I propose to understand whether histone modifications (with an initial focus on histone ubiquitylation/deubiquitylation) play an important role in the cellular response to IR-induced CDD. Using IR of different ionisation densities (ie. gamma-irradiation and alpha-particle/proton irradiation), which increases the complexity of DNA damage, together with various biochemical and molecular biology techniques, I propose to identify whether site specific histone ubiquitylation (eg. H2A-K119, H2B-K120 and H2AX-K119) is modulated in human cells at CDD sites. Furthermore, I will identify the enzymes involved using a candidate approach of known histone ubiquitylation/deubiquitylation enzymes, a screening approach by overexpressing/siRNA knockdowns of deubiquitylation enzymes, and a biochemical approach incorporating mononucleosomes containing DNA damage sites and protein fractionation. The goal is to improve our understanding of radiobiology, and ultimately identify new cellular targets for drugs and inhibitors that may enhance the efficacy of radiotherapy in cancer treatment.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Determining the macromolecular structure and cellular function of an alternative MCM complex
Funder: Medical Research Council

Abstract: Defective DNA replication can have a wide variety of effects on genome structure and information content, such as sequence deletion, insertion and duplication, point mutation and chromosome fusion. In mammals, defects in chromosome replication can lead to developmental defects, growth impairment and tumour formation. The MCM complex plays an essential role in eukaryotic chromosome replication as the catalytic core of the CMG complex, the enzyme that unwinds double-stranded DNA at the replication fork. A number of studies have drawn direct links between MCM dysfunction, chromosome instability and disease, including cancer. Recent evidence points to the existence of an alternative form of the MCM complex, in which one subunit (Mcm2) is replaced by the MCM-BP protein. The work proposed in this application aims to determine the structure of this alternative MCM complex, to investigate its function, and to establish how this function impacts on that of the archetypal MCM complex. Using fission yeast as model, the MCM-BP complex will be purified and its size and subunit composition determined by biochemical and biophysical methods. EM will be used to visualise 3D structure, and state-of-the-art protein crosslinking techniques used to map subunit-subunit interactions. The ATPase and DNA helicase activities of the complex will be examined using a variety of artificial fork-like substrates. To complement these biochemical approaches, the cellular function of the complex will be probed using a variety of molecular and cell biological methods. DNA combing will be used to measure replication origin firing, fork migration and recovery from fork stalling in MCM-BP mutant cells at the single molecule level, effects on chromosome cohesion will be monitored and the efficiency of fork recovery after stalling assayed. Overall, this work will lead to an understanding of the role played by the MCM-BP complex in the maintenance of genome stability and disease avoidance.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Enhanced integration of primary and secondary health systems and patient empowerment through improved continuity of patient care and clinical handover
Funder: Medical Research Council

Abstract: Design: Observational prospective study using mixed-methodology; Setting: One rural, urban and periurban hospitals in 2 states of India Objectives 1.situation analysis of policies, training, activities, and culture for clinical handover between primary and secondary care during referrals and discharge 2.identification of barriers and facilitators (health care system and patient related) for improving clinical handover 3.exploration of options for complex health systems and patient related interventions to improve clinical handover 4.pilot data collection tools for handover intervention evaluation 5. build health systems research capacity in order to enable further research and progress in integration of health systems for the development and implementation of a future cluster-RCT testing out potential interventions Methods: These will be achieved through mixed-methodology research to include: a)semi-structured interviews, focus groups, patient journey observation, and expert groups with health care academics, policy makers, managers, providers in primary and secondary care and patients: Views will be elicited on barriers, facilitators, potential interventions and identifying training needs b)document content review c)health care provider surveys d)patient and case note surveys on admission, discharge, 3 weeks and 3 months post-discharge for high-burden tracer health conditions requiring follow-up: stroke, diabetes, hypertension, and elderly (>65 years) with multiple co-morbidities. A range of analytical frameworks will triangulate the findings together with evidence of best practice, to identifying opportunities for improvement and options for intervention definition. The findings will be used by local policy makers, managers and practitioners to start addressing the identified low-cost barriers and solutions, while a systematic application of the findings will develop a complex-health systems intervention programme for a cluster trial to follow.


Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES;8.3 POLICY, ETHICS AND RESEARCH GOVERNANCE
Health categories: GENERIC HEALTH RELEVANCE
Title: The role of variant (v)U1 snRNAs in development and disease.
Funder: Medical Research Council

Abstract: Our recent discovery of a large family of non-coding variant U1 small nuclear (sn) RNAs, (vU1s) which, like U1 snRNA (U1), can regulate recognition of splicing and polyadenylation signals in pre-mRNA, has uncovered new mechanisms controlling gene expression in human cells. vU1 genes are most highly-expressed in human embryonic stem cells (hESCs), in dedifferentiated cancer cell lines and the cells of patients with neurodegenerative disease, including Spinal Muscular Atrophy (SMA) and down-regulated upon differentiation. Thus, vU1s may play key roles in hESCs and early development and mis-regulation of these RNAs could cause disease. We have shown that, like U1, one of the vU1s-vU1.8 is complexed with proteins in an snRNP. While some proteins are common to U1 and vU1.8 snRNPs, others are associated only with vU1.8, including DIS3L2, mutations in which cause Perlman Syndrome. The composition of snRNPs containing other vU1s has not been investigated. In addition, the direct pre-mRNA targets of any vU1 snRNP have not been identified. The proposed research aims to further characterize the composition of the vU1.8 snRNP, which is found in HeLa cells and hESCs and the hESC-specific vU1.20 snRNP and to identify the direct targets of these RNPs using immunoprecipitation and RNA-seq. In addition, the role of DIS3L2 in the vU1 snRNPs will be investigated using immunoprecipitation, siRNA-mediated knockdown and analysis of DIS3L2 enzymatic activity. The role of vU1s in ESC maintenance and pluripotency will be investigated by knock down and over-expression of vU1s in hESCs. In addition, the expression of the full range of vU1s will be analysed by qRT-PCR in cells from patients with SMA and Parkinson's disease to determine whether mis-regulation of these RNAs is a common occurrence in neurological disease. The proposed research will help to elucidate the roles vU1s play in human gene regulation and may yield insights into the molecular basis of human disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Structural Identifiability and Indistinguishability Analysis as Tools for Quantitative and Systems Pharmacology to Support the 3Rs
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: This proposal aims to overcome recognised limitations in the modelling of toxicological processes by introducing and applying established and novel systems modelling tools and techniques to exemplar problems in toxicology. The project will provide a more robust generic analytical framework to improve parameter estimation, sensitivity, robustness, reliability and validity of models in toxicology. In turn this step change in modelling will enhance experiment design, improve risk and safety assessment and thus help support the replacement, reduction and refinement (3Rs) of animal use. This research will be supported by industry (AstraZeneca) and will be applied to models for the improvement of both human and animal safety. This work will provide new tools and seed the emergence of a new field, namely Quantitative and Systems Toxicology.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: GENERIC HEALTH RELEVANCE
Title: Imprecision and importance: Probabilistic graphical models in toxicology
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: To evaluate the safety of a new chemical, existing toxicological protocols typically require 4 years, £3-4m, and over 4000 animals. This is inefficient, ethically questionable, and fails to exploit recent biological, mathematical and computational advances. We plan to use advanced computational and statistical methods to investigate how the evaluation process can be improved. More explicitly, we will use Bayesian networks to exploit existing data both to identify the key studies necessary for efficient toxicological assessment, and to quantify what levels of imprecision should be tolerated. The outputs will provide a rigorous quantification of the value of each element of existing protocols. In doing so, we will seek to answer the question of how far the detailed data from a panel of 50+ well-studied toxicants can justify applying the 3Rs in the toxicological testing necessary for future chemicals. Explicitly, reduction of the number of animals used for testing will be achieved where our models indicate that sufficient precision can be derived from a smaller battery of tests; refinement of animal testing protocols will become possible where our models identify efficiencies through the holistic assimilation of broadspectrum data; and where appropriate replacement of animal tests where they can be rationally and quantifiably justified early in a given chemical’s testing strategy.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: Uncertainty and confidence in applying mathematical models and in vitro data in toxicological safety assessments
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Over the past decade, there has been substantial research effort into the development and utilisation of systems biology models for a range of applications At the same time, there has been increasing pressure to end the overreliance on animal experiments within toxicology and to consider the information from non-animal alternatives when making decisions about human safety. A key challenge is to understand how the different proposed elements of a pathways-based toxicological risk assessment (e.g. chemical characterisation, human exposure information, in vitro toxicity testing data on compounds and metabolites from potentially diverse test systems, dose response modelling, population-based modelling and risk contexts) can be integrated to enable robust safety decisions. Our objectives are to answer how mathematical models could fit into a risk assessment alongside relevant in vitro data sources and to give toxicological risk assessors more confidence in using mathematical models. To achieve this, we must understand what the key input parameters of the models are and how existing in vivo and in vitro assays can be used to validate and parameterise the models. Using data sets (e.g. ToxCast, EPA) and models, which are publicly available or provided by Unilever, and working closely with expert toxicologists and risk assessors at Unilever, we will demonstrate the potential of existing uncertainty analysis techniques to evaluate the confidence in a toxicological risk assessment decision using non-animal data. This will take the form of prototype risk assessments based on in vitro data interpreted through a systems biology model and compared to human exposure concentrations estimated using a physiologically-based pharmacokinetic model. The approach therefore requires the linking of models at different scales and the determination of the contributions of experimental and model uncertainty to assess the confidence in the resultant risk assessment.


Research activity codes: 8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES)
Health categories: GENERIC HEALTH RELEVANCE
Title: Novel approaches to immortalise salmon fish cells.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Primary cells will be dissociated from gill, heart, kidney and spleen dissected from juvenile Atlantic salmon using procedure optimised to maintain cell type diversity. Cells suspension will be transfected using the Neon electroporation system suitable for transient or stable transfection of fish cells. GFP-expressing plasmid will be used to monitor and optimise transfection parameters for each tissue. Plasmids producing salmon proto-oncogenes, stem cell inducers and Insulin-Like Growth factor (IGF)-1 are already available in-house and can be used immediately. Coding sequences for anti apoptotic and Theileria genes are available and will be synthesised commercially and sub-cloned into expression plasmid vectors carrying suitable eukaryotic selection markers. Coding sequences for EGF and TGF-a are available for Zebrafish. The salmon genes will be isolated by a combination of PCR homology and in silico interrogation of the Salmon genome. Where antibody is available, protein expression will be verified by western blot on transiently transfected fish cells. Otherwise qPCR analysis will be used to ensure successful transcription. If success is limited double, triple or quadruple transgenic fish cell lines will be attempted sequentially. Four eukaryotic selection markers have been used successfully in fish cells and dual-expression vectors have been engineered giving a potential of 8 transgenes being over-expressed in a quadruple transgenic cell line. Double transgenic fish cell lines have been successfully generated (1). In every approach, strong proliferation will be the expected phenotype. Inhibition of apoptosis will be measured by reduction in DNA fragmentation and decrease in TUNEL staining. Immortalised cell lines will be characterised by specific staining, morphology and qPCR profiling (2). 1. Lester K, Urquhart K, Hall M, Gahlawat S, Collet B. 2012. J Virol Meth 182:1-8. 2. Collet B, Collins C. 2009. Vet. Immunol. Immunopathol. 130:92-5


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Development of a Non-Mammalian, Pre-Clinical Screening Tool (FETOX) for Predictive Analysis of drug Safety.
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Currently protocols to assess the safety of new drugs use mammalian models to predict the potential toxicity of novel compounds. Incorporating mammals as models in these early toxicity assessment protocols is undesirable due to cost effectiveness and the need to reduce mammalian testing in drug development. We propose to create a pre-clinical drug development protocol to predict the potential toxicity of new compounds integrating computer modeling, in vitro methodologies and in vivo assessment using larval forms of the amphibian, Xenopus laevis. Xenopus has long been used as a teratogenic screening model in the Frog Embryo Teratogenesis Assay Xenopus (FETAX) toxicity screening test. We plan to modify the FETAX assay to look at toxicity as well as teratogenicity. The new assay we term FETOX (Frog Embryo TOXicology assay). This assay will be used for predicting toxicity of drugs and will be combined with multi-parametric analysis on selected cell lines (related to the organs with predicted toxicity) and in silico modeling that will allow a hazard assessment decisions to be made prior to further research in mammalian models. This exciting prospect aims to fill a current gap between cytotoxicity assessment and systemic toxicity in a whole organism without the need for mammalian models in the early stages of drug development.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: GENERIC HEALTH RELEVANCE
Title: DM-MAP: drug and Metabolite Microsampling Analytical Platform for Preclinical Medicines Development
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: Preclinical testing is used to demonstrate efficacy and to characterise risks during drug development. One problem to be overcome is the provision of quality data to support decision making when progressing a compound through development phases. Discovery stage pharmacology and pharmacokinetic (PK) studies are used to assess compound developability. In this studentship we propose developing a drug and Metabolite Microsampling Analytical Platform (DM-MAP) to improve the quality of data obtained from discovery-stage study protocols. The project aims to provide a platform to the pharmaceutical research community to combine pre-clinical pharmacology and PK discovery studies by reducing the blood sample volumes required to be removed, for studies typically performed in rodents.The solution we propose is to use solid phase microextraction (SPME), an emerging analytical technique employing biocompatible, coated microfibers to adsorb compounds from complex biological sample matrices. Due to their size, microfibers appear suitable for insertion directly into organs or tissues (e.g. blood, dermis, liver) to detect the presence of drug or metabolites. Once validated, SPME microsampling will reduce sample volumes removed when determining drug concentration. Thereafter it will be feasible to perform a PK study for drug concentration in the same animal that is being studied for pharmacological effect. This will remove the need for „satellite‟ treatment groups, i.e. animals administered compounds solely for concentration determination. In combination with analytical techniques (e.g. mass spectrometry), we will validate SPME to perform sensitive qualitative and quantitative analysis of drug and metabolite concentration directly within organ tissues and body fluids.The student undertaking this project will address the shortfall in information on tissue distribution of compounds, which can currently only be determined in animals at a single timepoint using terminal procedures.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: GENERIC HEALTH RELEVANCE