- Total grants
- Total funders
- Total recipients
- Earliest award date
- 03 Dec 2014
- Latest award date
- 30 Sep 2018
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
Background: The risk for many common complex diseases, including type 2 diabetes, increases with age. Technological advances have recently enabled large-scale investigation of genomic markers of ageing in population-based studies. Whether genomic ageing contributes to the age-related rise of diabetes and related metabolic disorders is unknown. Aim: To systematically identify and study genomic markers of ageing, including telomere length, DNA methylation, and chromosome loss, and investigate their causal roles for morbidity and mortality from type 2 diabetes and other common complex diseases. My overall aim will be achieved by addressing the following specific objectives: Objectives: 1. To perform a systematic literature review of genomic markers of ageing to identify determinants and consequences and assess methods for their characterisation in epidemiological studies. 2. To identify and characterise genetic and modifiable behavioural and environmental risk factors of genomic ageing in large-scale population-based studies. 3. To investigate causal roles of genomic markers of ageing for morbidity and mortality from ageing-related diseases using Mendelian randomization methods, and conduct exploratory studies of the underlying pathways through detailed metabolomic characterisation.
Investigating the regulation and function of gap and Hox genes during segmentation in a short germ insect 31 Jan 2017
Three of the most abundant and diverse animal phyla - the Arthropoda, Annelida, and Chordata - are segmented along their anterior-posterior axis. Embryos of the fruit fly Drosophila form all of their segments simultaneously. In contrast, most arthropods, and all vertebrates and annelids, produce the majority of their segments sequentially. Although the molecular and genetic mechanisms regulating simultaneous segmentation have been well-characterised, our understanding of the mechanisms regulating sequential segmentation, especially in arthropods, remains poor. Gap genes are among the best-characterised components of the segmentation cascade in Drosophila. They are also expressed during segmentation of sequentially-segmenting arthropods. In Drosophila, gap genes define broad regions of the embryo; however, in sequentially-segmenting arthropods, they appear to have a different role, possibly mediated via Hox genes. Determining their function in sequential segmentation may shed light on how this developmental process is regulated, and how it was modified to give rise to simultaneous segmentation. For my PhD, I therefore propose to investigate the expression patterns, interactions and functions of gap and Hox genes in a simultaneously-segmenting arthropod, Tribolium castaneum. To accomplish these goals, I will analyse gene expression, cell behaviours and embryonic development in wild type and genetically manipulated Tribolium embryos.
Single-cell genomics is a fantastic tool for studying developmental biology: it allows unbiased and large-scale study of gene expression at the correct resolution for cell fate decision making. New fluidics systems provide the capability to study tens of thousands of cells simultaneously - as many as there are in the young embryo. For my PhD, I will analyse scRNA-seq data generated on this platform, studying mouse gastrulation between E6.5 and E8. I will be able to study this process at both an exceptional cell-level resolution (thanks to the fluidics) and at an unprecedented time resolution, at 0.1 day intervals. My focus will be on identification of lineage specification, and how cells make their fate choices. I will need to develop new methods to account for the large numbers of cells assayed, the numerous lineage decisions made, and heterogeneity of speeds of development across and between embryos. I hope to produce a map of lineage specification from epiblast (E6.5) cells through to every cell type present at E8. This work will provide a developmental atlas through gastrulation, and general inferences on cell fate decisions may provide insight for cellular reprogramming and regenerative medicine.
Understanding the Pathogenesis of Inflammatory Bowel Disease via Whole-genome Sequencing 31 Jan 2017
We will use a new whole-genome deep-coverage IBD dataset (15x+ coverage, 20 000 cases, 50 000 controls) to conduct genetic association studies. Several analyses are currently planned. The first study will use the data from >1000 IBD patients, who are part of a deep clinical phenotyping experiment, on their response to treatment with anti-TNF medication. We are hoping to determine specific genetic variants associated with successful treatment, non-response, loss of response, and unfavourable drug reactions. Once more samples are sequenced, we will attempt to discover novel low-frequency, rare, and very rare genetic variants associated with IBD. A recent low-coverage sequencing study has identified a rare missense variant in ADCY7 that doubles the risk of ulcerative colitis. In addition, a burden of very rare, damaging missense variants in genes associated with Crohn's disease was detected. The increased coverage and the size of the dataset may confirm the significance of such variants. Discovery of novel rare variants brings important insights into IBD biology, and improves the overall understanding of the genetic landscape of complex diseases.
Investigating the role of R2B receptor tyrosine phosphatases in developmental signalling pathways 27 Apr 2017
Tyrosine phosphorylation is a key post translational modification that is often dysregulated in disease. The balanced actions of kinases and phosphatases are required for cellular homeostasis. However, the substrates and functions of phosphatases are poorly understood. The receptor tyrosine phosphatase PTPRK was identified as a recurrent fusion partner of the oncogene RSPO3 – an amplifier of the Wnt pathway. Additionally, PTPRK was identified in a forward genetic screen for modulators of APC min driven intestinal tumorigenesis. Given these genetic links, and hints in the literature that PTPRK can dephosphorylate beta catenin, a Wnt transcriptional activator, we plan to investigate the signalling cross-talk between PTPRK and Wnt signaling. First, this project aims to discover whether Wnt signalling influences the regulation of PTPRK at the messenger RNA and protein level. Second, we plan to test how depletion of PTPRK affects Wnt signaling output using luciferase reporter assays and western blotting. The outcome of this project will be a comprehensive assessment of the interplay between PTPRK and Wnt signalling, with important implications for the role of PTPRK in colorectal cancer, which is one of the current focusses of the Sharpe Lab.
Determining the Significance of Pathway Bias at the Calcitonin Gene-Related Peptide Receptor Family in Human Endothelial Cells 27 Apr 2017
Family B G protein coupled receptors (GPCRs), notably the calcitonin like receptor (CLR), have been implicated in cardioprotective functions. The functional GPCR is a heterodimer of CLR and one of three possible receptor activity-modifying proteins (RAMPs). There are 3 main agonists for this GPCR: calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2 (AM2). CLR is pleotropic, activating intracellular pathways through coupling to G proteins or beta-arrestins. Indeed, we recently showed, using both a heterologous yeast expression system and mammalian (HEK-293) cell lines, that the signalling bias of the CLR is dependent upon both the agonist and the RAMP. However, the cell environment of the receptor massively affects signalling bias. Therefore, to validate these results, investigation of the pharmacology of the CLR in endogenous cell lines is essential. The aim of this research is therefore to use two different human cell lines (HUVECs and HUAECs) to pharmacologically investigate CLR/RAMP2 (the adrenomedullin receptor) and CLR/RAMP1 (the CGRP receptor) when endogenously expressed. It is hoped that this will provide greater insight into the function of CLR signalling in the vascular endothelium. This information may then be used to help characterise the pathophysiology of common cardiovascular diseases such as hypertension and myocardial infarction.
A detailed understanding of the initiation of translation in kinetoplastid pathogens is still elusive. There is an innate complexity due to the number of isoforms of eIF4E and eIF4G, two of the three components of eIF4F. Second, there has been no simple way to rapidly ablate expression of a protein to render an immediate phenotype. RNAi is available but it takes 3 to 4 cell cycles to dilute the eIF4E sufficiently to detect a phenotype and then is difficult to distinguish primary and secondary phenomena. We have recently developed a system for the inducible ablation of a specific protein in less than 60 minutes. This summer studentship will exploit this method to ablate separately the two isoforms of eIF4E that probably are responsible for mRNA cap binding during translation initiation. An analysis of the qualitative and quantitative effect on protein synthesis after ablation will inform on whether the two eIF4E isoforms have different, overlapping or identical functions. The work builds on expertise in the lab and utilizes a new technique to answer a long standing question.
Discovery of altered binding and function in mutations in human apelin receptor identified from genomic sequencing of patients with rare diseases 30 Sep 2018
Apelin and ELA peptides bind to the apelin receptor on the surface of cells in the cardiovascular system to mediate vascular effects and increase heart contractility. Apelin signalling is implicated in cardiovascular diseases such as pulmonary arterial hypertension (PAH) – a rare disorder that leads to heart failure and death, even when treated with current therapies. Apelin signalling has also been shown to regulate platelets, and may signal in red blood cells (RBCs), implicating apelin in blood disorders. In this project, apelin receptor mutations, identified in patients in the UK 10,000 Genomes BRIDGE project, will be assessed to determine the effect of these mutations on apelin receptor binding with apelin/ELA and cell signalling. In addition, human blood samples from healthy donors and apelin receptor mutant patients will be assessed for platelet and RBC function. Finally, mice that have been genetically engineered to express a selected apelin receptor mutant will be treated with SU 5416 in low oxygen conditions to evaluate the impact of the mutation on development and treatment of pulmonary hypertension. The work in this project will provide important insights into the role of apelin signalling in cardiovascular disease.
During the course of development, cells divide, migrate, and specialize to form major organ systems. Furthemore, among most mammals and birds, mouse cells differentiation follows a unique morphology. Understanding the molecular mechanisms underlying such process is a core issue in Biology and a curiosity in mouse, which despite differences still share fundamental properties during the process. The challenge has been addressed by leveraging current high-throughput technologies such as single cell transcriptomics. The amount and complexity of this data requires innovative mathematical frameworks that take advantage of current computational capacities. I am intersted on resolving mesodermal diversification during mouse gastrulation. Based on the premise that single cell profiles represent snapshot measurements of expression as cells traverse a differentiation process, I will use probabilistic modeling among other statistical and mathematical methodologies to reconstruct a measure of a cell’s progression through some biological process, and to model how cells undergo some fate decision and branch into two or more distinct cell types. In particular, Bayesian Inference has shown to be a useful approach to take advantage of computational resources, and to include prior knowledge into models, by providing a formal probabilistic framework that allows learning from the data in order to make predictions.
The influence of ageing on the ability of microglia to stimulate the regeneration process of remyelination 30 Sep 2018
Remyelination is the regenerative process in which new myelin sheaths are replaced on demyelinated axons, restoring function and preserving axon integrity1. Its declining efficiency with ageing results in the accumulation of clinical signs in chronic demyelinating disease such as multiple sclerosis (MS)2. There is a requirement to understand how ageing affects both microglia clearance of cellular debris and the ability of microglia to respond to demyelination and initiate the process of remyelination. After demyelination, oligodendrocyte progenitor cells (OPCs), become activated, divide, migrate and differentiate into myelin-forming oligodendrocytes3-4. These events are involve a complex signalling environment, at which microglia are at the heart. The role of microglia in remyelination involve 1) the initial detection of injury and triggering the remyelination cascade and 2) the clearance of myelin debris, which inhibits OPC differentiation5. To understand the mechanisms to be tested in a translationally meaningful way, human microglia will be collected from patients of various ages to identify common phenotypical patterns. A quantitative trait locus (QTL) analysis will be performed to assess the association of such phenotypes with established GWAS data on demyelinating disease. Subsequently, transgenic mice will be used to demonstrate the functional implications of ageing on the process of remyelination.
Epigenetic transgenerational inheritance of metabolic, reproductive, and endocrine phenotypes through the male germline: effects of developmental bisphenol A and dexamethasone exposure 30 Sep 2018
The majority of heredity is accounted for by transmission of genetic material from one generation to another. However, in recent years evidence has accrued that some environmental factors can cause variations in phenotype that are inherited through the germline without changes in DNA sequence – so-called environmental epigenetic transgenerational inheritance. We are interested in how metabolic/reproductive/endocrine effects of developmental exposure to two exogenous endocrine insults – bisphenol A, an endocrine disrupting chemical that leaches from plastics and thermal paper, and dexamethasone, a synthetic glucocorticoid administered to pregnant women at risk of preterm delivery – may be transmitted inter/transgenerationally through the male germline. We will expose mice to human-equivalent doses of these chemicals and breed for three generations to obtain both phenotypic data and spermatozoa for epigenetic analyses (using RNA-seq, RRBS, and ATAC-seq). We will investigate the functional significance of any spermatozoal epigenetic changes detected; for example, using zygote pronuclear microinjection to determine the role of spermatozoal non-coding RNAs. The ubiquity of human exposure to these chemicals means that even small inter/transgenerational epigenetic effects would have significant implications at the level of public health; we therefore expect this work to be of interest to the wider scientific and medical community.
Embryonic stem cells have the ability to differentiate into all somatic lineages of the embryo, a property known as pluripotency. The behaviour of pluripotent stem cells is regulated by multiple signals from the microenvironment. While the soluble biochemical cues involved in maintaining pluripotency have been studied extensively, the roles of mechanical forces are not well understood. I aim to use a novel polyacrylamide hydrogel (StemBond) with tuneable stiffness and adhesive properties to study how mechanical properties of the cells' environment, particularly substrate stiffness, affect pluripotency and cell fate decisions. This will allow tighter control over pluripotent stem cell maintenance in vitro, and importantly, differentiation into mature cell types that can be used for stem cell therapies and disease modelling. Specifically, I aim to identify the factors and pathways involved in the mechanical regulation of pluripotency maintenance and exit, and study how soluble and mechanical factors interact in response to changes in substrate stiffness. Additionally, I will probe the effects of substrate stiffness, and its synergy with soluble factors, in controlling lineage decisions. These insights will be used to achieve improved derivation of differentiated cell types, such as those of the pancreas, from human embryonic stem cells.
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.
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.
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
Spinal cord injury is a devastating condition that may lead to loss of limb movement, sensation and bladder control. Despite intense research, treatment is still very limited. Most research to date has focused on biochemical signalling. However, some more recent studies have hinted that mechanics might play an important role in spinal cord regeneration. Using atomic force microscopy (AFM), a cutting-edge technique which allows us to very precisely measure stiffness maps of biological tissues, we will investigate the stiffness of spinal cord tissue at various time points after injury and compare this to the stiffness of healthy spinal cord. We will test whether artificially modifying the stiffness of the damaged spinal cord or modifying mechanosensing in spinal cord cells improves regeneration of neurons after spinal cord injury. Our studies will be carried out in a cervical contusion model in rats which closely mimics the pathology seen in the human spinal cord after injury, even though the behavioural impairments the animals show are markedly less grave.
Regulation of Neural Stem Cells 30 Sep 2018
Of all the tissues and organs in the human body the nervous system is the most intricate and complex, consisting of more than 100 billion neurons. These neurons make precise connections with each other to form functional networks that can transmit information at amazing speed over considerable distances. Neurons are produced by neural stem cells, which renew themselves at each cell division while also giving rise to all of the diverse types of neurons in the brain. The Brand lab is interested in how the environment influences stem cell behaviour, in particular how nutrition regulates neural stem cell proliferation. Uncovering the molecular mechanisms that control whether a stem cell chooses to proliferate or remain dormant is crucial for understanding tissue regeneration under normal and pathological conditions and in response to ageing. It is critical to learn not only how stem cell proliferation is induced but also how stem cells can return to a dormant (‘quiescent’) state, as uncontrolled stem cell division can lead to cancer, including brain tumours like glioma. A thorough appreciation of the signals, both extrinsic and intrinsic, that control stem cell behaviour is necessary to understand how homeostasis is achieved and maintained in the brain.
Eros is a recently described endoplasmic-reticulum transmembrane protein that controls the phagocyte respiratory burst. Eros is essential for the generation of reactive oxygen species because it is necessary for protein (but not mRNA) expression of the gp91phox-p22phox heterodimer, which is almost absent in Eros-deficient mice. Consequently, Eros-/- animals succumb quickly following infection with Salmonella or Listeria. Eros is highly evolutionarily conserved and has a human orthologue C17ORF62 , which exhibits approximately 90% sequence similarity. Dr Thomas's group have preliminary data that the role of Eros is fully conserved in humans. However, the exact mechanism by which Eros controls gp91phox-p22phox abundance remains unclear. Using a yeast 2 hybrid screen Dr Thomas has shown that Eros's most significant interaction partner was OS9, an ER-resident lectin that regulates the degradation of misfolded transmembrane glycoproteins. Given that gp91phox is a transmembrane glcyoprotein, it is possible that Eros regulates gp91phox through a mechanism that involves OS9. While OS9 is highly expressed in the immune system, its role in the respiratory burst has not been studied. Using OS9 knockout mice and lentiviral over-expression systems, I will determine whether it regulates expression of gp91phox-p22phox or indeed, Eros itself.
TrimAway is a newly described protein-level depletion method for degrading specific endogenous proteins. The technique relies on the experimental introduction of antibodies to the cell, which elicits rapid degradation of target antigens via the cytoplasmic Fc receptor and E3 ubiquitin ligase TRIM21. This enables acute depletion of proteins, enabling the functional characterisation of previously intractable proteins. TrimAway has been shown to act against diverse cytosolic substrates including membrane-anchored GFP. However, it is currently unknown whether TrimAway is capable of targeting transmembrane proteins. To address this, we will attempt to degrade three representative transmembrane proteins using TrimAway. With one, seven and twelve transmembrane passes respectively, the impact of topology on degradation rates will be determined. Targets, and control GFP, will be expressed as C-terminal myc-tagged constructs and expressed in human cells competent for TrimAway. Anti-myc antibody will be electroporated into the cells according to established protocols and the fate of target proteins will be monitored by western blot. Our targets have been selected as well-studied, disease-relevant proteins whose function may be illuminated by acute depletion. The results will help define the limits of the TrimAway technique and shed light on the cell's ability to degrade membrane proteins via the ubiquitin-proteasome system.
Signalling pathways are required iteratively during development, coordinating many different developmental processes. This diversity relies on them eliciting different qualitative and quantitative transcriptional responses depending on the context. The Notch pathway is one of the small set of core pathways and can promote distinct outcomes through different sets of target genes. How Notch responsive enhancers achieve this level of regulation is not understood. In this project, well characterized Notch responsive enhancers will be used to study how activity and specificity in response to Notch are encoded within the enhancer sequence and how different factors play a role in the control of these properties. 1. Activity: Characterize the transcriptional output of a Notch responsive enhancer and analyze how it is governed. 2. Specificity: Compare two enhancers that exhibit different response to Notch to determine how their response specificity is achieved. 3. Sufficiency: Assess if a defined enhancer is sufficient to confer a specific Notch response when introduced into a heterologous locus.