- Total grants
- Total funders
- Total recipients
- Earliest award date
- 20 Nov 1998
- Latest award date
- 05 May 2020
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
Clinical Characterisation of a Broad Spectrum of Genetic Variation in the General Population 30 Sep 2018
Inborn errors of metabolism (IEM) are severe and extreme changes in metabolism caused by mutations in a single gene. Recent large-scale human studies have shown that genes causal for IEM are associated with nutrients, or ‘metabolites’, in the blood. However, whether these associations cause disease or adverse health outcomes is unknown. In this project, I will use IEM genes identified in these studies to link genetic variation to clinical features in a large human population. To do this, I will assemble a list of IEM genes of interest that were identified in the literature and in large population datasets. I will then test for associations between the variants I find in these genes and a wide range of clinical features found in open-access population datasets. As the IEM genes used in this study have been associated with blood metabolites previously, linking variants in these genes to clinical features will shed light on the molecular mechanisms underlying genes and disease in the general population. Understanding how genetic variation affects disease will help identify novel therapeutic targets and enable health professionals to better manage disease risk.
Human Fcgamma receptors (FcgammaRs) are proteins found on the surface of immune cells. They bind to antibodies, which are produced by the body, in response to infection. Some antibodies produced recognise their own tissues and are found in many diseases, including rheumatoid arthritis and lupus. It has been shown that genetic changes in the FcgammaRs are found more frequently in rheumatoid arthritis sufferers compared to healthy individuals. This project will focus on FcgammaRIIa, which is present on cells which are responsible for the destruction of many antibody-bound objects. Through a combination of cutting edge techniques, spanning physics, biology, immunology and medicine, we will uncover fundamental information within this field. This information would aim to inform the production of effective therapies to treat diseases such as arthritis, which put a huge strain on the NHS every year.
A structural investigation into the action of and resistance to ribosome-targeting antibiotics 30 Sep 2018
Antibiotics are crucial to modern medicine, allowing treatment of life-threatening bacterial infections and making many surgeries like transplantations possible. However, pathogenic bacteria are rapidly evolving to resist their effects. Protein synthesis is one of the main antibiotic targets in bacterial cells. I will use structural biology techniques, principally cryoEM and single particle image processing, to understand how both novel natural products and clinical antibiotics bind to the ribosome to bring about their inhibitory effects on protein synthesis. Furthermore, I will investigate the cause of toxicity of certain ribosome-binding antibiotics by examining how they bind to the mammalian mitochondrial ribosome. Finally, I will use a combination of cryoEM and protein X-ray crystallography to elucidate how certain ribosomal-protecting proteins form complexes with the ribosome in order to bring about antibiotic resistance. On an individual level, these studies will allow an assessment of the viability of novel natural products as suitable clinical antibiotics. More generally, they will contribute to our knowledge of how different classes of antibiotics target the ribosomes of pathogenic bacteria, and how these bacteria evolve resistance. This knowledge will help the development of methods to rationally design new ribosome-targeting antibiotics that are able to overcome or circumvent resistance.
Cellular Dynamics and Regulatory Networks Controlling Endometrial Remodelling during the Window of Implantation 17 Jul 2018
The endometrium undergoes iterative cycles of menstrual shedding, regeneration, rapid growth, and differentiation in response to ovarian hormones. During the mid-luteal phase, the endometrium becomes transiently receptive to implantation, heralding the start of a process of intense tissue remodelling, characterized by secretory transformation of glandular epithelium, angiogenesis, differentiation of stromal cells into secretory decidual cells, and activation of specialized immune cells. Several reproductive disorders, including recurrent pregnancy loss, are linked to defects in tissue remodelling at implantation. However, the cellular complexity and dynamic nature of the endometrium have so far precluded precise characterization of the underlying pathological mechanisms and drivers. We will employ high-throughput single-nucleus sequencing to map the dynamic changes in gene expression and chromatin accessibility (cis-regulatory regions) in all endometrial cell types across the luteal phase in defined patient groups. The data will be back-mapped to a future successful pregnancy or miscarriage. This analysis will yield unparalleled insight into the sequence of endometrial events (i.e. changes in cell populations, cellular states, gene expression and transcriptional regulation) leading to a successful or failed pregnancy. Further, 3D organoid cultures, consisting of glands and stroma, will be used to investigate putative drivers of endometrial dysfunction and to evaluate new treatment targets.
Investigating non-canonical programmed ribosomal frameshifting in porcine reproductive and respiratory syndrome virus 30 Sep 2018
RNA viruses are under selection pressure to maintain a small genome, however they still need to produce a variety of proteins. To overcome these conflicting pressures, many viruses use non-canonical methods of translation control. One example of this is programmed ribosomal frameshifting (PRF), in which a percentage of ribosomes, while translating a ‘slippery sequence’ slip one or two nucleotides out of frame, consequently translating the remainder of the mRNA in a different reading frame and allowing expression of more than one protein from a single gene. This is normally stimulated by a downstream secondary RNA structure, however there are two known examples of a trans-acting viral protein being used as the stimulatory element. One example is found in the genome of porcine reproductive and respiratory syndrome virus (PRRSV): an Arterivirus that infects pigs, causing an estimated annual cost to the US swine industry of $664m. I will use structural techniques such as X-ray crystallography to derive information about the RNA/protein complex, and will investigate the efficiency and mechanism of this non-canonical PRF using ribosomal profiling in parallel with RNASeq. The latter will also allow me to analyse host and viral gene expression, to examine host-virus interactions in this important pathogen.
Evidence from epidemiological studies and experiments in animal models suggests that effects of environment and lifestyle can be transmitted across generations via non-genetic mechanisms. Such mechanisms are challenging to unravel in mouse and man. In mammals, non-genetic inheritance is best exemplified by the Agouti viable yellow (Avy) mouse where phenotypic differences in genetically identical animals are caused by insertion of a retrotransposon - an endogenous retrovirus (ERV) that provides a cryptic promoter driving ectopic expression of agouti. This ERV is variably DNA methylated in different individuals causing inter-individual variation in coat colour – a non-genetic influence on phenotype. Remarkably, a memory of parental coat colour is transmitted to subsequent generations. Variable expressivity can be modulated by in utero environmental exposures. ERVs represent ~12% of the mouse genome. Inspired by Avy, we propose a research programme, supported by preliminary data, to address the following questions: Aim 1 – To what extent do mammalian ERVs exhibit variable epigenetic silencing and what is the mechanism? Aim 2 – Is this transmitted as non-genetic memory across generations? Aim 3 – Are they sensors of environmental compromise? Aim 4 – Are there implications for phenotype? Aim 5 – Does a related phenomenon occur in humans?
Gene Expression Heterogeneity in the Maintenance and Coordinated Differentiation of Neuromesodermal Progenitors in vivo 08 Aug 2018
Modern imaging data in biology is essentially multi-scalar in that raw image data undergoes a series of processing steps until it is at a manageable size to perform quantification and analysis. While this processing pipeline might be beneficial to one set of scientific questions, it may be inappropriate to others. Computational biologists may be interested in improving the pipeline itself, while other researchers may be interested in accessing the already processed data. Thus, a central road-block in the open sharing of large-scale imaging data is the fact that there is no one size fits all solution. This project aims to generate a web-based database that stores experimental and data descriptors together with links to the raw and processed data files. This will greatly enhance our ability to upload this data to repositories that are based placed to share the datasets in question, from the raw unprocessed data files down to feature extracted and processed data. Upon submission, the website will link to the deposited data and thereby act as an integrated platform for other researchers to access and explore the data that is available to them. Thus, researchers will be able to access our data at all levels. In built in the project is a second evaluation phase, whereby will we reach out to collaborating laboratories to assess the effectiveness of our open research platform. These will include computational biologists interested in accessing raw data files and processing pipelines, and other developmental biologists who will interact with processed datasets.
The proposed research uses standard molecular biology, protein purification and biophysical structural analysis methods in a focused series of experiments that comprise a complete 6-week project. This builds on existing molecular genetics studies that have identified novel missense mutations in KMT2D (also known as MLL2) as the cause of a unique phenotype (renal tubular dysgenesis, choanal atresia and athelia). Previous studies have identified KMT2D mutations as a major cause of Kabuki syndrome, a comparatively common autosomal dominant congenital mental retardation syndrome. The missense mutations occur in a central region of the KMT2D protein (2841-3876) that does not have variants associated with Kabuki syndrome. This central region contains a series of coiled-coil domains that are likely to mediate protein-protein interactions. However, the effect of the missense mutations on KMT2D structure and interactions is completely unknown. This project will determine the structure-function relationships between KMT2D and a unique phenotype that are likely to be caused by altered protein-protein interactions, as well as describing the broader genotype-phenotype correlations in this important gene. The approach described in the proposal is the only tractable way to understand possible structure-function relationships, given the large size of the gene and encoded protein.
Every cell in the human body descends from the fertilised egg cell, and during the journey from zygote to adult cell, many mutations are acquired. The mutations that happen early enough during embryonic development will be present in some parts of the body, but absent in others. One of the difficulties of this project is the discovery of these early embryonic mutations, as they are hard to distinguish from mutations that happened later in life, noise from experimental procedures, and mutations that were already present at fertilisation. Here, we cut out small pieces of various tissues from multiple individuals using a laser and sequence its DNA. Mutations shared between different samples from the same individual are candidate embryonic mutations. Each mosaic mutation gives us information on the development of the human body and with enough mutations, we can construct and visualise the human body as a large evolutionary tree, with the fertilised egg cell as the single root, and all the adult cells as its leaves. This can give us many new insights on the developmental relationship between different organs, how many different early embryonic cells give rise to one tissue or organ and developmental lineages of different cell types.
The effect of nutrients on maximal fat oxidation rates in adult humans measured using indirect calorimetry. 31 May 2018
I aim to study the effects of nutrient availability and mitochondrial transport capacity on the variability of maximal fat oxidation (MFO) during exercise in healthy adults. Less than half of MFO can be predicted by variables such as gender, VO2max and body composition. There are two possible reasons for this. First, nutrient availability may have a large effect on MFO and current protocols may not adequately control for it. Second, VO2max - which combines two variables with opposite effects on MFO (oxygen uptake and fat mass) - may not be the optimal predictor. Here, I will test whether heart rates at a given power are a better predictor of MFO and whether short-term fluctuations in nutrient availability can explain some of the variability of MFO seen within the general population. Nutrient availability will be altered using a glucose meal and by glycogen depletion. I will also use nitrate supplementation to test whether MFO can be increased by induced-expression of fat oxidation enzymes. The key goals are to determine to what extent short-term changes in nutrients and the expression of fat oxidation enzymes can alter MFO and whether the resultant fat oxidation rates can be predicted using simple heart rate data.
To divide and multiply, bacteria must remodel their cell envelope to facilitate physical separation of daughter cells. FtsEX is a key player in coordinating cell division events on either side of the bacterial inner membrane. FtsEX belongs to the same protein superfamily as the MacB efflux pump and the LolCDE lipoprotein trafficking complex, collectively termed Type VII ABC transporters. Current models for FtsEX activity suggest long range conformational changes in FtsEX regulate periplasmic enzymes responsible for peptidoglycan hydrolysis while maintaining cytoplasmic interaction with the septal Z-ring. Structural and functional data are essential to understand how FtsEX works and to assess viability of inhibition using chemical compounds. This project seeks to characterise the interaction of FtsEX with its binding partners, the role of ATP binding and hydrolysis, and to obtain structural data using X-ray crystallography. The project builds on published work on Type VII ABC transporters and is supported by preliminary data showing FtsEX has been crystallised. The Seed Award will presage future applications to the Wellcome Trust, MRC or Leverhulme Trust to further explore the structure and function of bacterial cell division proteins as targets for future antibiotic development.
Understanding the Initiation of Viral Replication & its Role in Influenza Virus Pathogenicity 31 May 2018
The development of novel strategies against influenza viruses depends on our understanding of influenza virus replication and pathogenicity. Both are directly linked to the activity of the viral RNA polymerase, which copies and transcribes the viral genome, and generate aberrant RNA products that are non-contiguous in the viral genome and strong inducers of the interferon response. Despite recent crystal structures of the RNA polymerase, we only poorly understand how it interacts with and copies the viral genome, or how it generates aberrant RNA products. This project aims to use i) structure-guided mutagenesis, ii) in vitro and in vivo activity assays, iii) cell culture-based interferon production assays, to ask if RNA polymerase residues that bind and guide the viral genome are important for the initiation of viral replication and the formation of aberrant RNA products and thereby the pathology of influenza virus infections. The project will contribute to our understanding of the mechanics of influenza virus replication and the identification of putative targets for the development of new anti-influenza virus drugs.
Alternative pre-mRNA splicing (AS) is a widespread regulatory mechanism enabling individual genes to generate multiple protein isoforms. We have investigated the mechanisms controlling AS events that are regulated during the transition of smooth muscle cells (SMCs) between contractile and proliferative phenotypes. We have shown how the widely-expressed RNA binding proteins (RBPs) PTBP1 and MBNL1 regulate SMC splicing events. Recently, we identified RBPMS as a potential "master" regulator of SMC AS. RBPMS is sufficient to switch AS events to the SMC pattern and its activity is strongly modulated by its own AS and by phosphorylation. Critically, RBPMS is sufficient to switch AS to the SMC pattern in vitro. This offers a unique opportunity to determine the molecular anatomy of regulated splicing complexes. We will carry out detailed mechanistic analyses of RBPMS-regulated splicing using a combination of biochemical, proteomic, single-molecule, and structural approaches including Cryo-EM. We will identify critical regulatory interactions between regulatory RBPs and core splicing factors, and test their importance by genome editing and mRNA-Seq. In a complementary aim, we will investigate how peptide-ligand interactions equip PTBP1 to regulate AS and a range of other post-transcriptional processes, and whether a family of such peptide-mediated interactions extends to related RBPs.
Investigation into the role of RBM8A/Y14 in the development and function of megakaryocytes and platelets using a human pluripotent stem cell model of haematopoiesis 30 Sep 2018
Platelets are small blood cells, which cause blood to clot, preventing bleeding after injury. They are produced by megakaryocytes, large cells in the bone marrow. In people with low platelet counts (thrombocytopenia), life-threatening bleeding occurs spontaneously or after injury. Studying platelet and megakaryocyte development and function is important in understanding a) diseases causing thrombocytopenia, such as genetic disorders and other conditions, particularly cancer (and chemotherapy) and b) strokes and heart attacks, where platelets are excessively activated, forming clots that block vessels. Using stem cells (special cells capable of becoming any cell type) derived from adult skin or blood samples we grow & study megakaryocytes and platelets in the laboratory. We study a rare genetic disease, Thrombocytopenia with Absent Radii (TAR) syndrome, in which babies are born with very few platelets and abnormal bone formation (particularly the radius in the forearm). Our group discovered the cause of TAR, due to abnormalities in a gene called RBM8A, which helps cells control what proteins are produced; however precisely why this causes TAR is unclear. We believe our research will uncover the mechanism of this condition, helping to treat patients with TAR and improve wider understanding of how megakaryocytes & platelets develop and function.
We aim to understand in detail the dynamics of how white blood cells (specifically T helper cells) react to infections by multiplying rapidly and at the same time adapting their cell state to fight the infection. In particular, we focus on a mouse model of malaria where T helper cells differentiate into two subtypes: Th1 and Tfh. By quantitatively profiling the T helper cell population at different time points during a malaria infection, we expect to improve our understanding of the mechanisms which are responsible for the cell proliferation and specialisation. We study the cell population by detecting RNA expression, surface markers and cell divisions at the single cell level. The RNA expression will provide clues as to genes which are driving this process, and we will test a subset of genes using CRISPR knock outs. In addition to a better knowledge of the immune system, we hope to develop new mathematical and computational methods that will be widely applicable to modeling cell proliferation and differentiation data in diverse biological contexts.
Specification of human primordial germ cells (hPGCs) occurs around gastrulation, a critical juncture when the specification of the primary somatic lineages also occurs. In combination with human preimplantation embryos, in vitro models and hPGCs from aborted fetuses, our objective is to elucidate the origin and properties of the early human germline. For the mechanism of the hPGC fate, we will use experimental models that simulate early human development. We aim to investigate how cells gain competence for germ cell fate, and then respond to combinatorial effects of the critical transcription factors, which induce hPGC specification. Altogether, this study will reveal the organisation of the very early human embryo, and mechanisms of hPGC and somatic outcomes, which is essential for advances in regenerative medicine. Following hPGC specification, epigenetic resetting of the early human germline leads to extensive erasure of DNA methylation and epimutations in response to the critical regulators of chromatin organisation and nuclear architecture towards the epigenetic ground state. Some conserved resistant loci ('escapees') evade reprogramming. We will explore if some escapees have been exapted to function as regulatory elements. If so, this may have a crucial influence on human development, including brain development and neuronal diseases.
The diffusion of chemokines in the extracellular matrix is a requirement for the formation of chemokine gradients that guide immune cell migration to sites of inflammation, and controlled by matrix glycans of the glycosaminoglycan family. The focus of this research is to use well-defined models of the extracellular matrix to probe the interaction between the chemokine CXCL12 and the glycosaminoglycan heparan sulphate, and how this defines the mobility of CXCL12. The first key goal of the project is to design and produce a fluorescently-tagged CXCL12 mutant with modulated glycosaminoglycan binding which can be compared against the wild-type chemokine and other mutants already available. The second key goal is to use the biophysical method of fluorescence recovery after photobleaching to characterise the differential diffusion of mutant and wild-type CXCL12 in glycosaminoglycan-rich matrices. This project thus combines biochemistry and biophysics to gain a better understanding of the molecular mechanisms underpinning the formation of chemokine gradients in extracellular matrix.
During my fellowship, I proved the feasibility of measuring cardiac energetics in volunteers and patients using ultra-high field (7T) MRI scanners. The sensitivity and the separation of signals from different metabolites both improved significantly compared to standard research scanners. I recently secured £340k funding to fit a new phosphorus coil on the Oxford 7T scanner, which I am now testing in volunteers. Theory predicts that this coil will have several complementary technical advantages. These will enable mapping of cardiac energy metabolism across the whole heart, with sufficient spatial resolution to distinguish signals from healthy from diseased tissue. It will also enable quantification of cardiac energy metabolism with high precision to study single subjects rather than groups. I request funding to validate these new whole-heart methods, proving their value in three carefully-targeted groups of patients, via an extension of my fellowship. My goals are (A) to study patients in which the metabolic pattern is known by other means; (B) others where the metabolic pattern will reveal previously-inaccessible aspects of disease mechanism; and (C) to prove I can resolve metabolic changes in single patients. Success in each of these studies will give me the pilot data needed for competitive Senior Fellowship applications.
When viruses infect a cell, they need to hijack host machinery to produce their own proteins from mRNA, in a process called translation. The host cell requires several factors for translation, including proteins called eukaryotic initiation factors (eIFs). EIF4F plays a central role in this process and is a complex made up of the proteins eIF4A, eIF4G, and eIF4E. Together, these proteins act along with other factors to recruit the cellular machinery required for translation to begin. Influenza can promote the translation of its own proteins whilst host protein synthesis is impaired. As viral and host mRNA are highly similar, influenza virus was thought to only use the same mechanism of translation as the host. However several findings, such as the fact that influenza can translate its proteins without eIF4E, suggest that this is not the case. My hypothesis is that influenza can employ a different mechanism of translation from the host. I will use several RNA/protein analysis approaches to identify the key components required for influenza translation, and attempt to dissect the mechanism(s) of translation used by influenza. Identifying key differences between host and viral processes is important for identifying novel therapeutic targets.
INVESTIGATING THE ROLE OF DDX17 IN ANTIVIRAL INNATE IMMUNE SIGNALLING DURING VIRAL INFECTION AND ITS IMPACT ON VIRUS SPREAD AND REPLICATION 31 May 2018
The host DEAD box RNA helicases are master regulators of pathogen RNA and DNA sensing dependent IRF3 signalling and are crucial for host survival and infection outcome in response to a multitude of both viral and bacterial pathogens. Here we report the DEAD box RNA helicase DDX17 as a novel pathogen recognition receptor essential for IRF3 driven IFNbeta expression in response to immunostimulatory DNA and dsRNA. Our current data maps DDX17 to act independent to the canonical IRF3 signalling cascade at the level of gene transcription, independent of IRF3 phosphorylation. We hypothesise that DDX17 may regulate beta-catenin nuclear shuttling, an essential IRF3 transcriptional cofactor. We aim to investigate the impact of DDX17 on beta-catenin activation and phosphorylation status as well as subcellular localisation following stimulation of WT or KO MEFs. Furthermore, we aim to investigate the biological relevance in viral infection following vaccinia virus and herpes simplex virus type 1 infection of WT and KO MEFs through quantification of viral replication and spread as well as IRF3 pathway activation. This project will contribute to understanding the role of DDX17 in innate immunity and host-pathogen interaction with implications in the immunological understanding of viral infection. Key words: DDX17, IRF3, IFNbeta, helicase