NIHRDataInsights/HRCSData huggingface dataset:
Title: Defining the Mechanisms Underlying Tandem Repeat Protein Functions
Funder: Medical Research Council

Abstract: Almost 20% of the proteins estimated to be encoded in the human genome contain small (20-50-residue) structural units repeated multiple times in tandem. Disruption of the structure, function and homeostasis of these ?tandem repeat proteins? plays a central role in several highly prevalent diseases including cancer. However, our ability to design therapies is severely hampered by a poor understanding of the basics of their control. Unlike globular proteins, tandem repeat arrays stack to produce elongated, superhelical structures, thereby presenting extended surfaces for molecular recognition. However, rather than functioning as rigid scaffolds it is likely that these proteins utilise highly dynamic and elastic modes of action, as suggested by their solenoid shapes. The modular nature of repeat proteins makes them uniquely malleable (they can be subjected to gross perturbations such as insertion or deletion of repeats) and amenable to rational design, and these properties will allow us to probe the multitude of inter-converting species that make up the functional energy landscape to an unprecedented degree. We believe that the specific properties of repeat proteins that arise from their uniquely one-dimensional architectures will only be manifest by the study of larger arrays and therefore we propose to focus on HEAT and ARM proteins, which have not been subjected to any biophysical analysis to date. We will investigate proteins that play important roles in disease, in particular cancer - gankyrin, beta-catenin and the PR65 subunit of PP2A. We will ask: How is the behaviour of the repeat array different from the sum of its parts? The objectives will be to define the structural transitions sampled by repeat proteins and determine how these regulate their functions. Importantly, the conformational space accessible to repeat proteins is very heterogeneous ? a feature that distinguishes them from globular proteins and that was first hinted at by our previous studies, but has not yet been directly detected because of the ensemble averaging of bulk solution studies. To answer these questions we will assemble a new toolbox, encompassing emerging biophysical and single-molecule techniques in combination with site-directed mutagenesis, with which to resolve the full spectrum of conformational fluctuations of a repeat protein and determine how these direct its function. Using this approach we will obtain novel and fundamental insights into the distinctive facets of repeat proteins, which we will exploit to develop new chemical approaches to manipulate them for therapeutic benefit.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Measuring and Incorporating Financial Protection into Economic Evaluation in Health Care
Funder: Medical Research Council

Abstract: Despite some recent interest in the topic of financial protection, leading to the 2010 WHO World Health Report on health systems financing and universal coverage, this remains a largely unexplored area in health economics. As one policy result, the lack of adequate risk protection against the financial consequences of costly diseases, affecting hundreds of millions of people each year in developed and developing countries, has received little attention in practice for health care funding decisions. The key aim of this research programme is to investigate the theoretical and empirical implications, for health resource allocation, of incorporating the benefits of financial protection against costly illnesses into the methods of economic evaluation in health care. Three inter-related projects address specific questions forming a coherent and innovative research plan with the potential to make an important contribution to the field. Project 1 will use economic theory to develop a model of individual and government behaviour to provide answers to two main research questions: (1) How can conventional cost-effectiveness analysis be augmented to account for financial protection objectives?; and (2) What are the implications of explicitly valuing and incorporating the benefits of risk protection into the social welfare function, particularly for the profile of services in a statutory health insurance package? The theoretical analysis will inform data collection and the interpretation of the empirical results. Based on household survey data, Project 2 will provide robust estimates of the health and monetary benefits of risk protection for individuals and society, through rigorous econometric analysis of the impacts of health interventions aimed specifically at improving financial protection. Measures of financial protection currently applied in the literature will be augmented by a proposed econometric methodology aimed at obtaining broader estimates of the incidence and magnitude of catastrophic health spending, by also accounting for under-use and non-use of needed health care due to inability to pay. Finally, in Project 3, the theoretical framework developed under Project 1 to augment conventional cost-effectiveness analysis, alongside the quantitative parameters obtained in Project 2, will be used to assess the implied changes in the profile of services included in a statutory health insurance package when financial protection benefits are explicitly accounted for. Simulations will be performed for different national contexts to investigate how the conventional cost-effectiveness ratios for selected interventions-and thus funding decisions under a fixed health budget-change when risk protection is included among the treatment benefits.


Research activity codes: 8.2 HEALTH AND WELFARE ECONOMICS
Health categories: GENERIC HEALTH RELEVANCE
Title: THE ROLE OF ENDOPLASMIC RETICULUM PROTEIN MISFOLDING IN CELL DEATH AND DISEASE
Funder: Medical Research Council

Abstract: Aims: To understand how endoplasmic reticulum (ER) dysfunction affects tissue growth in disease and how its manipulation might provide novel therapies. 1. How is the PERK/GADD34 axis modulated during ER stress? 2. How are ER stress and cell growth signals integrated? 3. Can modulation of ER dysfunction promote the maintenance of secretory tissues in disease? Methods: 1. We will identify GADD34‘s phosphorylated residues by mass spectroscopy and mutate these to determine their regulatory function. Using reagents we have generated (including GFP-GADD34 inducible cell lines, transgenic drosophila) we will determine how phosphorylation affects GADD34 localisation, protein interactions, phosphatase activity and stability. 2. We will determine how PERK mediates CHK1 activation. We will induce ER stress or activate PERK directly using reagents we have described in cells (Marciniak et al., 2006 J Cell Biol. 172: 201) and flies (Malzer et al., 2010 J Cell Sci. 123: 2892) and measure DNA damage, CHK1 activity and CHK1-directed phosphatase activity. We have shown that PERK activation is insufficient for G1 cell-cycle arrest during ER stress (Malzer et al., 2010) and plan to identify the additional signals necessary. We will induce ER stress in cells deficient in UPR components and measure cell-cycle indices during ER-stress. We observe enhanced ER-stress-induced G2 arrest in p53-/- cells (unpublished) and so will determine how p53 modifies this. Using cell and fly reagents we have developed (Davies et al., 2009 JBC 284: 18202; Kroeger et al., 2009 JBC 284: 22793) we will determine the consequences of ER accumulation of ordered and disordered protein aggregates in order to dissect the pathways linking ER distension to NFkB activation. 3. In disease, ER dysfunction can manifest as ER-stress or ER-overload leading to loss of secretory tissue. We will use cell, mouse and fly models of (i) diabetes (ER-stress) and (ii) 1-antitrypsin deficiency (ER-overload) to determine how manipulation of these pathways can be employed for clinical benefit. (i) We will use genetic and pharmacological means to determine if manipulation of GADD34 toxicity can promote tissue survival in cell and mouse models of diabetes. We will determine how manipulation of CHK1 and p53 signalling affects the survival of ER-stress in these models. (ii) We will manipulate NF B signalling during ER-overload in cell and fly models of serpinopathies to determine its role in maintaining secretory tissue mass. Opportunities: To identify novel targets for therapeutic intervention when ER dysfunction impairs tissue survival.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Structural characterisation of the tetrasome and histone chaperones
Funder: Medical Research Council

Abstract: The genomes of eukaryotes exisist as chromatin. The fundamental subunit of chromatin is the nuclesome. Nucleosomes are dissassembled and reassembled to allow access to genetic infromation for example during DNA replication. An early intermediate in the assembly of nucleosomes is the tetrasome, consisting of histones H3 and H4 associated with DNA. We aim to study the structure of the tetrasome using a range of techniques including SDSL, PELDOR and site directed crosslinking. Histone binding proteins known as histone chaperones are know to facilitate the assembly of tetrasomes. In order to gain insight as to how chaperones achieve this we will investigate how histones are bound by chaperones with the aim of gaining further insight into this important initial step in chromatin assembly.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Establishing the GRN for the Germ Line Mesoderm of Axolotl Embryos
Funder: Medical Research Council

Abstract: Understanding the genetic regulatory networks (GRN)s that govern the pattern of developing tissues is critical for understanding the underlying causes of genetic diseases, and for devising methods for the production of cells for cellular therapies. Embryos from lower vertebrate animal models are particularly useful for unpicking GRNs because they are readily accessible, so material is acquired with relative ease, and they facilitate the testing of predictions from the networks experimentally. An important problem in stem cell biology is how to derive gametes from pluripotent stem cells as a means of combating infertility, and as a vehicle for genetic manipulation. We developed axolotl embryos as an experimental model to understand how germ cells are specified because they share a conserved mode of primordial germ cell (PGC) specification with mammals, and this is not conserved in embryos from other models, like frogs and teleost fish. The axolotl system has the additional advantage that this species is representative of the ancestral amphibians from which all land-dwelling vertebrates evolved, and so can be used to understand how developmental mechanisms evolved in mammals. In axolotl embryos PGCs emerge in the posterior lateral plate mesoderm, which we term germ line mesoderm. This tissue is ancestral to vertebrates, but was lost during the evolution of teleosts and frogs, so it has not been characterized before. In axolotl embryos, PGCs are closely related to haematopoietic stem cells (HSC)s, and these cells are induced with the PGCs in germ line mesoderm. The focus of this proposal is to understand how the germ line mesoderm is patterned to segregate these two closely related stem cell populations, and to establish the GRN that governs their production. We propose to use the inducible axolotl animal cap system to identify the mechanisms that pattern this novel mesodermal tissue, and we will use it to produce material for the GRN, which will be obtained under a variety of experimental conditions, and at several time points. We will validate predictions from the GRN in axolotl embryos, as well as in an in vitro system for the induction of PGCs from mouse embryonic stem cells. At the completion of this project we will deliver the conserved GRN that controls the production of PGCs from pluripotent precursors, an important step towards the efficient production of germ cells for therapeutic purposes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: In cell analysis of Myosin Va function in organelle transport.
Funder: Medical Research Council

Abstract: Importance. Cytoskeleton tracks and motors are vital for intracellular organisation and transport by regulating organelle movement and distribution. In vitro data reveal that dimeric motor MyosinVa (MyoVa) ?walks? along actin filaments, indicating a cargo carrying function in cells. Meanwhile cellular studies indicate that it tethers cargo at the cortical actin. Also MyoVa interacts with other cytoskeleton components, suggesting that it may co-ordinate cytoskeleton structure and function. Aims The overall objective of this proposal is to determine the mechanism by which MyoVa functions in organelle transport in cells. Specifically it will determine to what extent MyoVa functions in cells as a motor or a tether (Aims1+2)? And how interactions with other cytoskeleton proteins and intra-molecular regulatory interactions affect its function (Aim3)? Methods. These issues will be addressed using a recently established melanosome transport assay that reports MyoVa function in the movement of physiologically relevant and easily observable cargo (melanosomes) within primary mammalian cells (melanocytes). In this assay MyoVa, and mutants altered in structural and enzymatic properties e.g. dimerisation, are introduced into MyoVaKO melanocytes using adenovirus expression vectors. The functionality of introduced protein is then tested by assessing its ability to rescue loss of endogenous MyoVa, measured by observing redistribution of pigment from the perinuclear cluster into peripheral dendrites. Beyond this simple assay further functional tests will be performed to better understand the cellular role of the mutated part of the protein. For non-rescuing mutants the basis of loss of function will be further defined by testing cargo targeting and motor activity. For rescuing mutants more subtle alterations of function will be investigated by measuring the efficiency of rescue, indicated by the kinetics of rescue and the expression level required for rescue. Applicant. The applicant has 10 years experience working with cultured melanocytes, and molecular biology techniques required to fully exploit this assay system. He has successfully developed similar complementation assays to investigate the function of Myosin activating proteins Mlph and Myrip. Additionally he maintains a network of collaborators who will provide essential reagents (e.g. libraries, cell lines, viral vectors and mutant mice). Outcomes. This program will generate valuable information on the role of MyoVa and other motors by establishing their role as regulators of cytoskeleton activity, tethers or cargo carriers. Many motor proteins, including MyoVa, have been linked to human disease and advances in the basic understanding of their cellular function will aid the development of treatments of these diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Replication fork stability and fork restart
Funder: Medical Research Council

Abstract: The DNA in our cells is at its most vulnerable when it is being replicated. A significant proportion of the mutations, chromosomal rearrangements and epigenetic changes associated with human genetic disease and cancer are thought to be generated by stochastic and DNA damage-induced replication errors. Furthermore, many chemotherapeutic drugs are specifically toxic to replicating cells, a feature that imparts therapeutic index but which also limits their utility due to side effects on rapidly dividing normal cells. The DNA Damage Response (DDR) pathways act to suppress genetic change in the face of endogenous and exogenous replicative stress. It has become evident that the majority of cancer cells are defective in one or more DDRs. Some DDRs, such as the intra-S phase checkpoint, are proposed to be mutated in order for a pre-cancerous cell to overcome the barrier to carcinogenesis imposed by oncogene-induced senescence. Mutations in other DDRs are proposed to be selected for because they allow increased genome stability, thus promoting tumour development and evolution. My long-term aim is to provide detailed mechanistic information that will assist the development of rational ?synthetic lethality? therapies in order to target cancers with specific changes or combinations of changes in DDR proteins and pathways, as well as to help identify potential new targets for drug development. Therefore, in this program I propose to study the relationship between DNA replication and the DDR to investigate how these pathways contribute to maintaining genome stability. Specifically, I will use a model eukaryote, S. pombe, to help to elucidate and understand how the intra-S phase checkpoint regulates DNA metabolism to maintain replication forks in a stable conformation. I will also explore the mechanisms and consequences of replication fork restart, which is necessary when these stabilisation pathways fail. I hypothesise that a relatively under-explored protein, TopBP1, plays important roles in coordinating the transitions made by the replication and repair apparatus during fork arrest and restart by virtue of its ability to act as a multi-functional phospho-protein binding scaffold: TopBP1 can respond to a range of regulatory phosphorylation events and thus act as a ?node? coordinating signalling pathways by integrating disparate phosphorylation events. I will therefore continue my exploration of TopBP1 functions in yeast and higher eukaryotes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: How cells measure time during pattern formation
Funder: Medical Research Council

Abstract: The integration of growth and patterning is critical during embryogenesis to ensure the specification of complex structures. This was the focus of my previous work that revealed the link between digit number and digit identity in the chick wing. I showed that both these processes are controlled in the digit-forming field by the same signalling molecule - Sonic hedgehog (Shh) - produced by the adjacent polarizing region at the posterior margin of the limb bud. Now I will explore how the control of digit number and identity is integrated over time. Most knowledge of developmental timing comes from in vitro work and it is unclear how this relates to tissue development. To address this, I have performed a series of in vivo experiments in the chicken by grafting wing bud tissue that constitutively expresses Green Fluorescent Protein into normal buds. My preliminary data suggests that both the polarizing region and digit-forming field cell populations proliferate for a defined time. However, polarizing region cells have self-renewing properties similar to a stem cell population, whilst digit-forming field cells have a limited potential to proliferate similar to a transit-amplifying cell population. My preliminary data suggests that both cell proliferation timers are set by Shh signalling and this could integrate the extent of digit field expansion with the duration of Shh transcription. This self-terminating mechanism can be applied to other systems such as the neural tube. I have identified putative regulators of cell proliferation timers in the chick wing. I will elucidate how cell proliferation timers function using molecular, cellular and genetic approaches as well as mathematical modelling in both the chick and mouse limb. I will also identify the factors that limit or permit the self-renewal of putative polarizing region stem cells by microarray analysis. In the longer term, I will try to identify polarizing region stem cells by looking for stem cell markers and this could have major implications for tissue repair and limb regeneration. This work will also have implications for cancer biology and Congenital caused by temporally deregulated growth and patterning.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: EVALUATING THE PERFORMANCE OF RISK PREDICTION MODELS
Funder: Medical Research Council

Abstract: Making predictions on the prognosis of disease or the likelihood of developing a disease is an important role of doctors and healthcare policy makers. Risk prediction models are tools that examine the likelihood of patient outcomes in relation to multiple patients and disease characteristics. They are used to inform individuals about the future course of their illness (or their risk of developing an illness) and to assist doctors and patients in making joint decisions on future management, treatment plans and lifestyle changes. After a risk prediction model has been developed it is crucial that the generalisability of the model is appropriately evaluated minimising any optimism in the initial results. Contributing to this optimism are inadequate sample size of the validation cohort, inappropriate handling of missing data, the categorisation of continuous risk factors and the treatment of nonlinear risk factors. While these issues are quite well recognised in studies to develop risk prediction models, to date their importance has not been appreciated in relation to studies that evaluate model performance on data sets not used to derive the model (validation studies). We aim to evaluate the impact and provide guidance on the choice of sample size of the validation cohort on the performance of the risk prediction model. We will assess the impact of missing data in both the derivation and validation cohorts on the performance of the risk prediction model. Finally we will address the treatment and handling of continuous risk factors and evaluate the effect this has on the generalisability of the risk prediction model. The overall goal is to inform guidance on the design and conduct of reliable validation studies for future risk prediction models.


Research activity codes: 8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES)
Health categories: GENERIC HEALTH RELEVANCE
Title: Dynamic Wales Electronic Cohort for Children (D-WECC): a dynamic e-cohort to assess the impact of health and social factors on inequalities in educational outcomes and child health in Wales
Funder: Division for Social Care and Health Research (Wales)

Abstract: Research has shown that poor education is associated with ill-health. We propose a study to improve child health and education through “translational research”. We intend to achieve this using routinely-collected anonymised data within an electronic cohort (e-cohort) of 804,290 children; the Wales Electronic Cohort for Children (WECC). We will convert WECC into a dynamic e-cohort (D-WECC) capable of tracking individuals across datasets monitoring life events including health and education. We will include new births and flag migration. We will create novel links from D-WECC to a traditional cohort, the Millennium Cohort Study (MCS), to explore the effect that additional variables, such as socio-economic status and perceptions of the local environment, has on the well-being of children in Wales who overlap between the two cohorts. In this way we will be able to assess the robustness of using each data source alone compared with the increased power of using a hybrid design. Results will aid in assessing the ability of using routine data to identify health inequalities of children in Wales. This study offers a unique opportunity to inform policy and intervention studies to ensure limited resources are most effectively used to improve health and well-being for the most disadvantaged children.


Research activity codes: 2.3 PSYCHOLOGICAL, SOCIAL AND ECONOMIC FACTORS
Health categories: GENERIC HEALTH RELEVANCE