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Award Year:
2018
Recipients:
University of Cambridge
Currency:
GBP

Results

Cellular mechanisms of metabolic sensing by human hypothalamic neurons 06 Jun 2018

Obesity is a major public health problem, but its molecular mechanisms are poorly understood, frustrating efforts to develop broadly effective treatments. Obesity is characterised by abnormal energy homeostasis, which is regulated in large part by hypothalamic melanocortin neurons. The activity of melanocortin neurons is in turn regulated by circulating nutrients and hormones (e.g. leptin), but the mechanisms by which they sense and integrate these metabolic signals have been difficult to study using traditional model systems. Functional human melanocortin neurons derived from pluripotent stem cells provide an unprecedented opportunity to illuminate and therapeutically harness the molecular mechanisms by which human melanocortin neurons sense metabolic signals to regulate energy homeostasis. In particular, they enable a detailed characterisation of leptin signalling and direct testing of hypothesised mechanisms of leptin resistance. At a cellular level, the role of primary cilia in sensing and integrating leptin and other metabolic signals can be studied. Third, the synergistic action of different metabolic signals in vitro will be used to predict effective anti-obesity therapies in vivo. Together these studies will shed light on obesity disease mechanisms and facilitate the development of effective anti-obesity treatments.

Amount: £1,212,840
Funder: The Wellcome Trust
Recipient: University of Cambridge

Use of 68Ga-DOTATATE PET to identify the “inflammatory phenotype” in cardiovascular disease: a clinical translational programme 23 May 2018

Inflammation drives atherosclerotic plaque rupture underlying most coronary events. While vascular inflammation can be visualised using 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET), as a glucose analogue this method lacks cell specificity and is unreliable for coronary imaging owing to myocardial signal overspill. My research has shown that 68Ga-DOTATATE, a somatostatin receptor-2 PET ligand, provides a more accurate readout of macrophage-related atherosclerotic inflammation than 18F-FDG, with superior ability to distinguish high-risk versus lower-risk coronary lesions. In three parallel studies, I will test the clinical utility of 68Ga-DOTATATE PET to: i) quantify treatment response to high-intensity lipid-lowering with statins and proprotein convertase subtilisin/kexin type 9 inhibitors in patients with stable cardiovascular disease and familial hypercholesterolaemia; ii) predict disease progression post-myocardial infarction by identifying individuals with "residual" on-treatment coronary arterial inflammation; and iii) diagnose large-vessel vasculitis and monitor treatment response. 68Ga-DOTATATE imaging will also be compared to established MRI, CT, and intravascular imaging markers of atherosclerotic disease severity; and validated in vasculitis using autoradiography, histology and gene expression analyses. Moreover, transcriptomic and epigenetic profiling of monocyte-derived macrophages will be performed to better understand mechanisms underlying residual inflammatory risk defined by 68Ga-DOTATATE. This research will accelerate translation of 68Ga-DOTATATE inflammation imaging to the clinic.

Amount: £716,938
Funder: The Wellcome Trust
Recipient: University of Cambridge

Dissecting the neural circuits for visual perceptual learning 06 Jun 2018

Visual perceptual learning (VPL) is an improvement of performance of a visually guided task as a result of visual experience. However, it is not well understood whether improvements are due to changes in lower-level visual or in higher-level association brain areas, and whether improvements can transfer to untrained visual features and tasks. Here I propose to dissect the neural circuits of VPL in mice and test the theory that VPL consists of both: 1) enhanced task-independent encoding of sensory features in low-level areas in cells with selective feature-preference, generalising poorly to different features. 2) enhancement of task-relevant features, initially in higher level areas with broad feature-preference, generalising to a broader range of features. Mice can quickly learn a visual discrimination task, which I will combine with cell-type specific 2-photon calcium imaging and optogenetic manipulation of neural activity. I hypothesize that PV interneurons are necessary for task-independent learning, while SOM interneurons can gate feedback from high-level areas to boost task-relevant features. I will address the following specific aims: 1) identify the changes during VPL in visual and association areas during learning 2) determine the role of task-engagement in VPL 3) determine how VPL generalises to untrained or previously irrelevant features.

Amount: £1,317,544
Funder: The Wellcome Trust
Recipient: University of Cambridge

The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge 30 Sep 2018

Since 2013, the University of Cambridge Metabolic Research Laboratories (MRL) has developed into a world-leading centre for basic and applied research in obesity and related metabolic disease. Underpinning funding from Wellcome, which has provided new clinical research facilities and other crucial core support, has been central to this success. Importantly, this endeavour has been undertaken in partnership with the MRC, who have funded a new Unit, the Metabolic Diseases Unit (MDU), which is embedded in the MRL. The MRL, together with the MRC Epidemiology Unit (Dir. Wareham) and cognate clinical facilities, form the Wellcome Trust-MRC Institute of Metabolic Science (IMS) which operates seamlessly from basic science through to population science, translational research and delivery of ambulatory care within a single co-ordinated institute. The current bid is focused on further developing world-class metabolic research within the MRL through core support for clinical and animal model research as well as underpinning laboratory science at an internationally leading level. Given the centrality of bioinformatics to all contemporary biomedical research, we have placed a particular emphasis on development of this area for the next phase of our evolution.

Amount: £249,032
Funder: The Wellcome Trust
Recipient: University of Cambridge

Ultrastructural imaging 05 Jul 2018

This application is for funds to purchase a Transmission Electron Microscope (TEM) and necessary accessories to enable post-embedding immuno-electron microcopy to be performed. We also request funds for salary support for a full-time specialist technician to oversee tissue preparation, sectioning and microscope operation. The microscope will be located within the imaging facility within Capella (due to open in November 2018), a new research-dedicated building on the Cambridge Biomedical Campus (CBC), which will house the Wellcome Trust-MRC Cambridge Stem Cell Institute (CSCI) and the Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), and will be available for use by other research groups across the CBC. Access to ultrastructural imaging is essential for many research groups working with Capella and across the CBS. This investment will establish a core EM facility on the CBC, the importance of which is reflected by both the large number of PIs who have expressed their support for this application and the wide range of research questions for which ultrastructural imaging data will play a key role. The establishment of a core EM research facility for the CBC will provide the basis for subsequent expansion, enabling, for example, addition of equipment for 3D ultrastructural reconstruction.

Amount: £251,744
Funder: The Wellcome Trust
Recipient: University of Cambridge

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Complete humanisation of adaptive cellular immunity in the mouse: Vaccine and therapeutic TCR discovery 30 Sep 2018

Adaptive cell mediated immunity is one of the central components of immunological homeostasis. While the basic mechanisms are conserved the components that encounter antigen are subject to rapid evolutionary change driven by species specific pathogens co-evolving with the host and divergence of the host genome against which antigen receptors are negatively selected. Thus, epitopes that direct protective immunological responses differ between species. Consequently, translation of results obtained from immunisations conducted in model organisms to humans remains a pernicious issue. The long term goals of this proposal are to identify and validate vaccine candidates and discover therapeutic T cell receptors To achieve these goals we will build mice in which all components of adaptive cellular immunity have been humanised, building on the technical success, biological insights and health-care benefits accrued from the construction of a mouse with a complete human immunoglobulin repertoire. We will use this humanised mouse as platform to isolate therapeutic T cell receptors for acute myeloid leukaemia in which the nucleophosmin gene has been mutated. In an independent and parallel work stream we will systematically explore the Plasmodium falciparum genome to identify vaccine candidates protective against the liver stage of the pathogen.

Amount: £5,000,000
Funder: The Wellcome Trust
Recipient: University of Cambridge

Tissue Resident Regulatory Cells 30 Sep 2018

Not available

Amount: £234,092
Funder: The Wellcome Trust
Recipient: University of Cambridge

Resolving mesodermal diversification with single cell transcriptomics 30 Sep 2018

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

The roles of mechanotransduction in pluripotency and cell fate decisions 30 Sep 2018

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

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

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigating stiffness alterations in spinal cord injury 30 Sep 2018

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

What is the functional significance of the interaction between Eros and OS9? 31 May 2018

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Protein level knockdown of transmembrane proteins 31 May 2018

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.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Viral silencing and immune evasion pathways 10 Apr 2018

Studying how viruses manipulate their host cell machinery provides insight into both viral function and cellular regulation. Invasion by viral DNA threatens cellular integrity and therefore needs to be detected and suppressed. As the default cellular response is to silence incoming DNA, how do viruses subvert and exploit this hostile environment to their advantage? Our identification of the ‘Human Silencing Hub’ (HUSH) as a novel epigenetic transcriptional repressor complex, responsible for silencing newly integrated retroviruses, has motivated us to further investigate chromatin regulation of viral infection. We wish to: (i) Understand how HUSH silences newly integrated viral DNA. (ii) Determine how lentiviruses antagonize chromatin-mediated repression and identify shared mechanisms by which unrelated, non-integrated viruses counteract silencing. (iii) Understand how and why the lentiviral-Vif accessory protein manipulates the host cell phosphoproteome. To help address these questions we have developed and optimised powerful genetic and proteomic screening technologies. Repurposing CRISPR-Cas9 will enable RNA-guided genome manipulations, including selection of informative point mutants and isolation of DNA sequence-specific regulatory proteins. Together these approaches provide a discovery platform to determine how repressive heterochromatin is established and maintained, how viruses manipulate their chromatin environment and ultimately to develop new therapeutic tools to enable their eradication.

Amount: £3,724,493
Funder: The Wellcome Trust
Recipient: University of Cambridge