NIHRDataInsights/HRCSData huggingface dataset:
Title: Imaging cellular structure and function beyond the diffraction limit
Funder: Medical Research Council

Abstract: This partnership aims to combine strengths in basic science underlying human disease and imaging technology to develop capabilities in super-resolution microscopy (SRM) in order to study fundamental biological mechanisms inside cell nuclei, inside live cells and at the synapses of interacting cells. Initially we would establish a commercial SIM microscope to provide rapid SRM of cells with existing labels. This would enable users to rapidly gain expertise in SRM, e.g. through studies of chromatin organisation and reprogramming of lymphocytes by ES cells. We would also implement rapid FLIM FRET, e.g. for directly correlation of SRM of changes in subcellular structure with cell signalling. In parallel the Photonics team would extend their 3-D STED microscopy to live cells with GFP and YFP and also implement RESOLFT using, e.g. rsGFP. This will utilise novel fibre-laser based excitation/depletion/switching lasers developed in-house, as well as SLM technology for beam shaping and adaptive optical correction of aberrations. A key application would be to image synapses between live cells - noting that these can lie in arbitrary planes not readily accessible to other SRM techniques. Progressing to intravital imaging, we would also develop a STED/RESOLFT microscope for to study the formation and function of neurological synapses in live in mice. We would also upgrade our in-house developed TIRF-based PALM/STORM systems to live cell imaging using emerging algorithms to separate signals from closely packed fluorophores and implement 3-D PALM/STORM using, e.g. axial localisation and oblique light sheet illumination. We would then replicate this system in the CSC for studies of nuclear reorganisation during meiosis in C. Elegans and for other subnuclear imaging applications. The in-house development of SRM technology makes it cost effective and inherently upgradeable, enabling it to be sustained at the cutting edge for multiuser access beyond the project's end.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Dynamics of Fundamental Cellular Processes by Live Cell and Tissue Imaging
Funder: Medical Research Council

Abstract: This application seeks support for a strategic upgrade of two advanced imaging capabilities in the College of Life Sciences Light Microscopy Facility (CLS-LMF) at the University of Dundee. Based on previous awards from SULSA, we have established 3D Structured Illumination Microscopy (3DSIM) on a commercial "OMX" platform as a production-grade resource for scientists located in Dundee and across Scotland and the UK. We have also built and a custom, new Light Sheet Fluorescence Microscope (LSFM), with a modified design that enables horizontally mounted specimens (e.g., higher vertebrate embryos, chick, etc.) to be imaged with unprecedented temporal and spatial resolution. Under the requested award, we will extend our 3DSIM capability to include living samples, and continue our commitment to deliver this super-resolution capability as a resource for all scientists in the UK. In addition, we aim to build a new LSFM based on Bessel beam illumination that will extend our analysis of fundamental process from the cellular to the subcellular level. As always, we will use the technologies within CLS-LMF in a wide variety of applications, but to enhance the impact, we have selected specific application domains-chromatin structure and dynamics in 3DSIM and cytoskeletal dynamics in BB-LSFM-- that will directly benefit from those technologies. Finally, we will drive the use of existing and newly developed image processing tools, linked in through our ongoing, separately funded work with the Open Microscopy Environment Consortium, to maximise our ability to analyse, understand, and publish the data generated from these resources.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: MICA: High speed, high resolution imaging of excitable cell networks
Funder: Medical Research Council

Abstract: Advances in biomedicine have always been paced by developments in the physical and engineering sciences, which enable us to peer ever deeper into the nature's intricate mechanisms We are now at another watershed, with cutting-edge techniques in optical microscopy offering the opportunity to resolve the dynamic behaviour of excitable cell networks, with a combination of high speed 3D scanning and subcellular spatial resolution. The aim of this proposal is to capitalise on these UK innovations in optical microscopy, and establish the technologies in a life sciences department in which they can be applied rapidly to biomedical questions. The imaging facility will implement turn-key solutions for three synergistic technologies: a) Remote focusing two-photon microscopy, which enables scanning at kHz rates along arbitrary 3D trajectories in thick biological specimens, and thus the imaging of activity propagation within and between excitable cells. b) High speed (1000 fps) confocal microscopy, using structured light and a temporal pixel multiplexing camera, which enables simultaneous high speed imaging of cellular activity and high spatial resolution imaging of anatomical structure. c) Multi-focal structured illumination microscopy (mSIM), which enables high speed imaging at spatial resolution below the diffraction limit. These high speed microscopy techniques offer various trade-offs in temporal and spatial resolution, and will be implemented on the same microscope to offer the most flexible imaging system. The hardware and software implementations will be honed iteratively through application to cardiac and neuronal preparations, both in vitro and in vivo, yielding a sustainable imaging system for studying excitable cell physiology and pathology.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Imaging of cellular dynamics from single molecules to tissues
Funder: Medical Research Council

Abstract: The aim of the project is to enhance synergistic interactions between microscopy technology developers and users in Manchester. Following the recruitment of the PI, M. White & D. Spiller to Manchester, we established a new Systems Microscopy Centre (SMC) to focus on development and exploitation of live cell imaging. We believe that microscopy developments depend on 1) biological developments and probes; 2) new microscopy instrumentation and 3) software development and mathematical analysis. Here, we plan a close collaboration with the leading microscopy company Carl Zeiss to develop new technologies over a 4-year period. The aim is to ensure that relevent skills and resources are fully ultilised to tackle biomedically important problems We propose development of 3 novel technologies. 1. Automated confocal fluorescence and bioluminescence microscopy. These imaging modalities have not previously been combined automatically in timelapse microscopy. (We previously used a manual strategy that led to a major publication but was labour intensive). The new microsope would be developed in collaboration with Zeiss and we will purchase the optimised system at the end of year 1. 2. Super-resolution fuctuation imaging. The great advantage of this technique is that it involves low illumination leading to less photo-toxicty than some other super-resultion approaches. With fast imaging and sophisticated data analysis, SOFI offers promise for timelapse super-resolution imaging in living cells. 3. We will access new Selective plane illumination microscopy (SPIM) technology from Zeiss (working with C Power, a Zeiss staff-member who recently relocated to UK, after managing the Zeiss SPIM development team for 6 years. Zeiss will loan a prototype system and we will purchase an optimised system in year 3. We will also investigate the potential for luminescence tomography and SPIM. There is an active community of multidisciplinary users for these technologies in Manchester


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Liverpool Imaging Partnership: Molecular physiology and drug response
Funder: Medical Research Council

Abstract: The aim of Liverpool Imaging Partnership is to support excellent science with state-of-the-art live cell imaging. We will build on our expertise in long term live cell imaging and single molecule imaging using nanoparticles, to measure intracellular molecule dynamics in a wide range of living models including cells, tissues, small embryos and plants. To improve the sensitivity of detection and accurately measure protein-protein interactions, we will install a LSM 780 (Carl Zeiss) with Fluorescence Life time imaging (FLIM) component. The considerable increase in sensitivity of the LSM 780 allows it to be used in photon counting mode (FCS/FCCS). This will provide capabilities to measure intracellular sensors (e.g. oxygen nanosensors), protein-protein interactions, protein diffusion and complex formation in living samples. It will have direct application in several projects, such as elucidation of mechanisms of pancreatic damage, testing drug specificity. To increase our resolution capabilities and correlate intracellular dynamics with ultrastructural changes, we will develop two new techniques: 1) super-resolution optical fluctuation microscopy (SOFI), which requires a limited investment in comparison with other super resolution techniques and logically builds on our expertise in quantum dot development; 2) photothermal-correlative light microscopy (p-CLEM) where the gold standard of EM probes becomes also an excellent optical probe. This will constitute a platform with high sensitivity state-of-the-art confocal microscopes coupled with photothermal imaging of nanoparticles that is dedicated for long term imaging and imaging in physiologic settings including oxygen controlled environment. Altogether, this will provide unique measurements in living cells supporting research in areas such as inflammation, ageing, cancer, drug safety, nanomedicine, plant biology, stem cell research etc. in a platform that will support users from experimental design to image analysis.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Regulation of ER-nuclear communication by CREB3 transmembrane transcription factors
Funder: Medical Research Council

Abstract: The CREB3 transcription factors play key roles in physiological pathways whose dysfunction underpin human disease. They have been shown to be required for the control of triglyceride homeostasis in the liver and are essential for normal bone and cartilage formation. They operate by intricate intercompartmental signalling pathways about which we have little understanding. Our objectives concern the elucidation of the mechanisms controlling CREB3 protein function and their regulation of cellular adaptive responses. There are two themes, each involving molecular biology, biochemical and imaging techniques to dissect of trafficking and signalling mechanisms This work involves molecular techniques for construction of plasmid vectors expressing wild-type genes and mutant variants. We will examine post-translational modification of these proteins using well established methods, examining phosphorylation in the control of transport between cellular compartments, identify the sites within the protein that are modified and the important kinases involved by in vivo and in vitro techniques. We wil also examine insulin mediated control of transport and cleavage, linking this to insulin mediated phosphorylation. In a second theme we will establish cell lines for regulated expression of wild-type and variant proteins and develop chromatographic purification schemes to identify interacting proteins by mass spectrometry. We will then develop antibodies and siRNA approaches to confirm interacting proteins and their role in transport of CREB3 proteins.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The Golgi apparatus as an initiator of ciliogenesis
Funder: Medical Research Council

Abstract: The formation and function of primary cilia is critical to the normal development of tissues, organs and ultimately organisms Defects in these processes cause severe developmental abnormalities. These diseases, collectively known as the ciliopathies, are causes by mutations in genes encoding the machinery that forms and maintains the cilium. These include centrosomal proteins, those that mediate assembly of the ciliary membrane and deliver critical signalling molecules to this highly specialised structure. Key questions remain unanswered about the origin of the membrane that docks adjacent to the mother centriole as it transitions to become a basal body. Subsequent extension of the microtubule axoneme from this basal body is accompanied by significant expansion of the specialised ciliary membrane and delivery of critical signalling molecules that define its subsequent function. Assembly of the cilium and its subsequent function requires the activity of molecular motors that drive translocation of particles along the axoneme. Dynein-2 is centrally involved in this process and considerable genetic data from experimental systems and patients implicate dynein-2 in the initial formation of the cilium itself as well. Our preliminary data have implicated the Golgi membrane protein giantin in this process. Acting through the dynein-2 motor, our model is that giantin acts to deliver critical components, possibly even the initial membrane itself to the newly forming cilium. Here, we will use a combination of advanced light and electron microscopy approaches, supplemented with biochemical assays, to define whether giantin acts as a hub to recruit dynein-2 during ciliary assembly, the role of giantin and dynein-2 in the targeted delivery of ciliary components, and the relationship to other pathways known to be required for cilium formation. Our work will provide key mechanistic insight into the processes that govern both the formation and function of primary cilia.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Tissue-specific programming of pericyte-derived mesenchymal stem cells
Funder: Medical Research Council

Abstract: Adult mesenchymal stem cells (MSCs) promise great potential for use in tissue engineering and other cell-based clinical therapies. The fact that these cells can be isolated from individual patients makes them amenable to use as an autologous cell source. Research is emerging from both the MSC and iPS field suggesting that adult cells retain a functional epigenetic memory of their developmental histology. The clinical importance of MSCs makes it of great importance to understand the basis of this memory (programme) and how it affects their differentiation and response to tissue injury. In this study we aim to investigate the molecular basis of epigenetic programming of cells (pericytes) that are now generally accepted as a source of MSC in most tissues. The general concept we have formulated is that pericytes are programmed, as a result of their developmental origins, in order to restrict their differentiation potential in vivo. If this is true it has important consequences for their clinical use. We will use the latest approaches in epigenetic analysis and gene expression profiling to identify differences between pericytes of different origins. We will use heterotypic transplantation of pericytes following tissue damage to correlate repair efficiency with epigenetic (gene expression) status and finally carry out reprogramming experiments to determine the extent to which this improves repair efficiency.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mechanisms underlying the transmission of programming effects
Funder: Medical Research Council

Abstract: Epidemiological studies demonstrate an association between early life events and cardio-metabolic disorders in adulthood, a phenomenon termed early life 'programming'. Human and animal studies suggest programming effects are not limited to the first generation but may be transmissible to subsequent generations through both male and female lines. The mechanisms accounting for the intergenerational transmission of programmed effects are unclear. We have developed a rat model of fetal glucocorticoid overexposure in which the offspring have reduced birthweight and as adults have hypertension and hyperglycaemia, a phenotype which is transmitted to a second generation. Alterations in epigenetic modifications have been described in animal models of programming and in humans and the transmission of epigenetic modifications such as DNA methylation (5mC), histone modifications and small RNAs through the germline may be one mechanism accounting for the transmission of programmed effects. We propose there may also be a key role for a newly described DNA modification 5-hydroxymethylation (5hmC). In this project we will test the hypotheses that programming effects are transmitted to a second generation as a consequence of (a) altered epigenetic reprogramming during gametogenesis and/or (b) altered epigenetic modifications in mature germ cells. This research will increase knowledge and understanding of the role which epigenetic modifications may play in the early life origins of disease and will explore the mechanisms which might account for the transmission of such effects to a second generation, providing a basis for the inherited association between low birthweight and cardiovascular risk factors. Ultimately, this study may suggest methods that would allow the early identification of individuals at risk of later disease, potential interventions to decrease this risk and interventions to prevent the transmission of disease risk across generations.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: GENERIC HEALTH RELEVANCE
Title: Regulation of the Rho GEF Pebble in Fibroblast Growth Factor dependent cell migration
Funder: Medical Research Council

Abstract: The induction of cell movements is an important cellular response elicited by Fibroblast growth factor (FGF) receptor signalling. Despite its relevance in human disease pathways, the molecular basis of FGF-dependent cell movements is only poorly understood. Our previous work has established that the fly homolog of the human proto-oncogene Ect2, called Pebble (Pbl) is required for FGF-dependent cell migration. These molecules are conserved in humans and are involved in controlling cell migration in normal and cancer cells. We discovered that the conserved carboxy-terminal tail of Pbl is important for the control of its substrate specificity and its localisation to the cell cortex in FGF-dependent cell migration, but it is unknown how FGF-signalling controls Pbl activity. To address these questions we propose to investigate the molecular requirement for the Pbl protein in cell migration and to identify and characterise regulators of Pbl using biochemical, molecular and genetic approaches. We will determine the specific molecular requirements of Pbl in FGF-dependent cell migration using a mutagenesis screen. We will identify posttranslational modifications of Pbl in response to FGF signalling and analyse their biological significance in transgenic animals. We will use proteomics and analyse novel mutants to identify proteins and genes that are involved in the regulation of Pbl in cell migration. Together these approaches will allow us to define the protein domains and amino acid motifs that are important for the regulation of Pbl in FGF-dependent cell migration and regulatory interaction partners of Pbl. Identifying those domains and their regulators will help to design assays that can be used to inhibit deregulated FGF-signalling and hyper-activated Ect2 in abnormal cell migration in diseases including cancer.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: GENERIC HEALTH RELEVANCE
Title: Identification and investigation of novel candidate genes for primary ciliary dyskinesia
Funder: Medical Research Council

Abstract: There is a need to identify the gene defects underlying Primary Ciliary Dyskinesia, a disease in which cilia have impaired motility. One approach is to sequence candidate motile cilia genes in PCD patients. Our recent discovery of the Drosophila transcription factor, Fd3F, and appreciation of its role in regulating the motile cilia gene expression programme provides an avenue for motile cilia gene discovery and validation using the power of Drosophila genetics. In Drosophila motile cilia are confined to sensory neurons (particularly auditory neurons) and sperm, so mutations causing loss of ciliary motility yield flies that are uncoordinated, deaf and male infertile. Their sensory neurons and sperm are readily accessible to structural and functional analysis. We found that two novel Fd3F target genes (Zmynd10 and Heatr2) are required for motile cilia in Drosophila. Or collaborators then found PCD-associated mutations in their human orthologues. Our first aim is to analyse these two genes in Drosophila to provide functional validation to support the studies of the PCD-associated mutations by our collaborators. We shall examine cilium ultrastructure and look at the localisation of known motor proteins within the mutant sensory cilia and sperm. We shall investigate protein localisation through tagging and immunofluorescence. Our second aim is to screen for further ciliary motility genes in Drosophila by gene expression profiling for Fd3F target genes and in vivo RNAi screening of these for motile ciliary defects. This will identify new candidate genes for subsequent analysis in PCD families by our collaborators. If homologous genes are indeed mutated in PCD families, then we shall again use Drosophila to provide supporting functional validation.


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: The Oxford Protein Production Facility-UK
Funder: Medical Research Council

Abstract: The Oxford Protein Production Facility-UK (OPPF-UK) is a National resource for protein science. The overall goal of the OPPF-UK is to provide the UK biological, chemical and biomedical communities with a world-class facility for the production of proteins for structural and functional research. The OPPF-UK has established a high throughput approach to the production of recombinant proteins using three main expression hosts (E. coli, mammalian cells and baculovirus/insect cells) which is provided as a service to the UK scientific community through a peer-review process. Research and development in the OPPF-UK will be focussed on streamlining the cloning, expression and purification of multi-protein complexes and membrane proteins using insect and mammalian cells. Methods will be rolled out as part of the user programme of the OPPF-UK. In parallel, data management systems will be enhanced to provide users with secure on-line access to all project data.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE