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Recipients:
University of Cambridge

Results

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.

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

Analysis of conserved pathways involved in maintaining homeostasis and survival in mammals and Drosophila. 09 Mar 2011

Our aim is to uncover the molecular basis for toxin clearance from animal body fluids. Using a simple tissue, nephrocyte cells, in Drosophila as a model for blood filtration and detoxification in vertebrates we focus on two molecular pathways essential for these functions; Ig domain proteins (Nephrin, Neph1) required for construction of the podocyte filtration slit diaphragm and the cytoplasmic protein (Rudhira), essential for the micropinocytotic activity in reticulo-endothelial cell scavenger s. Drosophila homologues of these proteins are expressed in nephrocytes. Our goals are to exploit ongoing Drosophila screens to identify candidate molecular interactors to establish the protein interactions that regulate the activity of these pathways, to analyse the roles of identified protein networks in nephrocyte filtration and toxin clearance and to extend our analysis into vertebrate tissues. We aim to test vertebrate homologues for conservation of the molecular interactions identified in Drosophila and, using podocyte cell lines and mutant mouse strains to establish the roles of Ig domain and Rudhira interactors, focussing on podocyte filtration. Overall we shall identify molecular activities that are fundamental to the stability and function of the slit diaphragm and for regulated endocytosis and vesicle trafficking.

Amount: £240,228
Funder: The Wellcome Trust
Recipient: University of Cambridge

Wolbachia as a defence against RNA viruses in insects. 07 Mar 2011

The bacterial endosymbiont Wolbachia protects insects against viral infections. The aim of this project is to understand this new form of antiviral protection, using Drosophila as a model system. Our results will be relevant for attempts to use Wolbachia to control viral diseases of humans and animals that are transmitted by insect vectors. To understand the causes of this antiviral resistance, we will first determine which stage of the viral replication cycle Wolbachia affects (entry into ce lls, replication, translation or exit). In Drosophila, we will screen for host genes and pathways that are required for the antiviral effects of Wolbachia and examine whether the antiviral protection relies upon the insect immune system or other host pathways. From the bacterium s perspective, we will identify Wolbachia genes up-regulated upon host viral infection and test these for their antiviral effects. Additionally, we will use next-generation sequencing to identify genes associated with n aturally occurring variation in the antiviral properties of Wolbachia. Finally, to see if interventions using Wolbachia will be sustainable, we will test whether viruses can evolve to overcome the antiviral effects of Wolbachia and identify the mutations that underlie these changes.

Amount: £548,004
Funder: The Wellcome Trust
Recipient: University of Cambridge

Structural studies of heterochromatin protein 1 (HP1) interaction with chromatin . 09 Mar 2011

HP1, which exists as three homodimeric isoforms (HP1alpha, beta, gamma), plays a critical role in constitutive heterochromatin, regulation of the expression of heterochromatic and euchromatic genes, and DNA repair. The monomer unit comprises a chromodomain (CD), a homologous chromoshadow domain (CSD), which dimerises, and a connecting hinge region of variable length. Most attention has focussed on binding of HP1 through its CD to the epigenetic mark H3K9Me, but this does not explain all aspec ts of HP1 binding to chromatin. We will focus mainly on two other regions in HP1 the CSD, which interacts with nucleosomal histone H3 at a second (occluded) site in a methylation-independent manner; and the hinge region, which interacts with DNA in vitro. Building on our preliminary work we will determine the structure of the HP1α CSD in complex with a peptide containing the second H3 site by NMR, and characterise the structure and the DNA-binding properties of the HP1 hinge region. We wi ll also study the interaction of full length HP1 with histones H3 and H1, and through a combination of biochemistry and novel NMR we will characterise the nature of HP1 binding to chromatin.

Amount: £233,640
Funder: The Wellcome Trust
Recipient: University of Cambridge

Does the potential for proteins to be allergenic depend on structural homologies with IgE targets on metazoan parasites? 07 Mar 2011

IgE is found only in mammals, and is presumed to have evolved to combat metazoan endo- and ecto-parasites (helminths and arthropods). Recently, a closer molecular relationship between major allergens and metazoan products has become apparent. We have tabulated the known allergens and found that 8 of the 10 most abundant allergen protein superfamilies are represented in metazoan parasite genomes, although in only 3 cases have metazoan proteins been experimentally verified as IgE targets. We prop ose to study 5 metazoan gene families containing homologues of known allergens, to establish if these are indeed targets of IgE in natural and experimental infections. To fully validate the hypothesis that all allergens contain structural homology to metazoan antigens, we will also analyze the 2 gene families for which homologues with recognizable sequence homology have yet to be found in metazoan parasites. These will be subject to surface structural feature analysis, which will enable us to se arch metazoan genome databases for structural homologues which will then be tested for IgE reactivity. The technique will then be extended to provide a searchable database of the metazoan parasite surface features which can be searched to predict allergenic epitopes in food and environmental organisms.

Amount: £180,679
Funder: The Wellcome Trust
Recipient: University of Cambridge

The structural dynamics of modular polyketide synthases. 09 Mar 2011

Many natural products of clinical importance are made by type I polyketide synthases (PKSs), multienzyme systems that comprise modules of covalently linked catalytic domains. The key players in each module are acyl carrier protein (ACP) domains, which serve as attachment points for the growing substrate chain. The details of ACP-mediated delivery of substrate to each active site are central to understanding how new polyketide chains are assembled. Using NMR spectroscopy, we recently discovered t hat the interaction between an ACP from the erythromycin type I PKS and its downstream thioesterase (TE) works in an unexpected way: chain transfer occurs in the absence of a protein-protein interface, with contact limited to the acyl terminus of the substrate. We now aim to build on that success by determining how ACPs communicate with every other type of enzyme domain in the erythromycin-producing DEBS PKS, the paradigm type I modular system. We will then extend our study to the PKS in Mycobac terium ulcerans that synthesizes mycolactone, the causative agent of the disfiguring tropical disease Buruli ulcer.

Amount: £287,265
Funder: The Wellcome Trust
Recipient: University of Cambridge

Transcription & post-transcriptional processing in the Plasmodium chloroplast 09 Mar 2011

It is now recognized that Plasmodium has photosynthetic ancestry, being closely related to dinoflagellate algae. It has a remnant chloroplast, including a 35kbp genome, expression of which is essential for Plasmodium viability. The chloroplast is recognised as a target for new antimalarials. Very little is known about transcription and post-transcriptional processing in the Plasmodium chloroplast, although by analogy with other chloroplasts they are expected to be important determinants of gene expression. Although the Plasmodium chloroplast genome encodes some of the subunits of a bacterial-type RNA polymerase, we have identified a nuclear gene for a phage-type RNA polymerase, which algorithms predict is chloroplast targeted (probably in addition to the mitochondrion, and similar to what may be the case for dinoflagellates). Using our experience of chloroplast molecular biology, we have also identified putative genes for other important proteins, including a sigma factor for the bacte rial-type polymerase, an RNA processing enzyme and two RNA binding proteins. All of these are predicted in silico located in the Plasmodium chloroplast, with varying degrees of confidence. We wish to confirm the chloroplast location of these five proteins, and test their function. This will provide a basis for developing a detailed understanding of Plasmodium chloroplast gene expression.

Amount: £364,457
Funder: The Wellcome Trust
Recipient: University of Cambridge

Transcriptional analysis and biomarker discovery in autoimmune disease 15 Mar 2011

We have established a translational programme of biomarker discovery in a number of autoimmune diseases, recruiting over 500 patients for whom detailed prospective clinical data has been collected, and performing microarray expression analysis on purified cell subsets. Despite having analysed <25% of the samples collected we have discovered a novel CD8 T cell-based biomarker which predicts prognosis in 4 different autoimmune/inflammatory conditions (e.g. McKinney et al. Nat Med 2010; 16:586-58 9), has been patented and will be assessed in clinical trials. We now seek funding to finish the processing and analysis of this cohort, in order to: i. refine the biomarkers already found, creating a practical clinical assay ii. extend the investigation of the current biomarkers into related conditions iii. discover novel diagnostic biomarkers, improving classification of these heterogeneous conditions iv. determine if transcriptional changes in response to therapy predict prognosis v . associate transcriptional pathways with specific patient subgroups or diseases, illuminating both pathogenic mechanisms and suggesting novel targets for treatment The unique nature of this resource, together with substantial preliminary data which demonstrate its promise, underlined the potential for completion of this important work to produce results that could directly impact patient care in the short to medium term.

Amount: £242,218
Funder: The Wellcome Trust
Recipient: University of Cambridge

Structural analysis of Wntch signalling in Drosophila. 09 Mar 2011

Analysis of signalling during development has revealed a consistent and pervasive relationship between Notch and Wnt signalling suggesting that they form a single functional module. Notch encodes a member of a family of cell surface receptors that acts as membrane tethered transcription factors while Wnt represents a family of ligands that interact with receptors to modulate cytoskeletal activity and transcription. Work in Drosophila has revealed that Notch has the ability to modulate Wnt signal ling independently of its transcriptional activity and that this effect has an important function in processes of cell fate assignation. Our studies with Drosophila have uncovered an activity of Notch which targets the activated form of -catenin, the effector of the transcriptional activity of Wnt, and modulates its amount and activity. This function relies on the ligand independent traffic of the Notch receptor, provides a buffer for Wnt signalling and suggests a framework to think about Notch as a multifunctional receptor. Here I propose to probe the mechanisms that govern the interactions between Wnt and Notch signalling using Drosophila as a model system.

Amount: £344,942
Funder: The Wellcome Trust
Recipient: University of Cambridge

The influence of intracellular bacteria on MHC class II in relation to autoimmunity . 07 Mar 2011

MHC class II molecules, which present peptide antigens to T-cells, are associated with autoimmune disease, by mechanisms that are incompletely understood. A set of autoreactive T-cells that escape thymic tolerance and induce autoimmune disease has been identified. These Type B T-cells respond to peptide antigen but not to antigen derived by processing in the conventional class II pathway, which supplies antigen to Type A T cells. We have demonstrated that intracellular Salmonella interfere w ith MHC class II presentation, leading to partial loss of mature class II from the cell surface. We propose to investigate whether the disturbance in MHC class II by Salmonella leads to skewing in presentation of self-epitopes to Type B T-cells. We will ask whether Salmonella influences the loading of antigen in early and late compartments. This approach will be supported by investigation of the precise molecular mechanism of MHC Class II down-regulation. In addition, we intend to use a powerful mutant screen to identify genes required for presentation to Type A or Type B T-cells. These experiments would have consequences both for the way in which Salmonella manipulates the immune system as well as understanding of autoimmunity.

Amount: £244,819
Funder: The Wellcome Trust
Recipient: University of Cambridge

Recognition and evasion of Human cytomegalovirus by natural killer cells. 07 Mar 2011

Human cytomegalovirus (HCMV) provides a paradigm for how a complex viral pathogen persists and evades immune responses in humans. HCMV evades cytotoxic T cells by downregulating class I MHC, but then has to evade natural killer (NK) cells. We have recently described a novel MHC-like gene unique to clinical isolates that inhibits NK cell lysis by preventing the expression of NK cell activating ligands MICA and ULBP3. Recently we have discovered another viral gene (UL147) that prevent ULBP3 expres sion from very early post-infection. The specific goals of the work proposed are to:(i) Define the mechanism of action of the novel viral NK evasion gene product (UL147) (ii) Analyse the mechanisms that control NK cell recognition of HCMV in latently infected cells and during reactivation as all analysis to date has been on lytically infected cells. (iii) Address whether if particular NK cell subsets are refractory to HCMV mediated immune evasion and efficiently recognise HCMV infected cells?

Amount: £293,275
Funder: The Wellcome Trust
Recipient: University of Cambridge

Reward prediction in health and disease. 21 Feb 2011

Our goal is to characterise and extend our understanding of behavioural and brain signatures of prediction-dependent responding in schizophrenia. The behavioural symptoms of schizophrenia, as well as the implications of dopaminergic alterations, suggest that abnormal prediction error signals and predictions may be key to understanding the condition. Several functional neuroimaging studies have provided evidence in favour of this view. However, the functions of prediction errors in learning and b ehaviour are complex and cannot be adequately characterised solely in terms of their presence and magnitude. We hypothesise that a detailed understanding of the adaptivity of prediction error coding and the variability of reward prediction in relation to background environmental characteristics will bring us closer to understanding the positive symptoms of schizophrenia. Our aims are therefore to explore these two features of error-dependent learning using complementary studies in control partic ipants and in patients with schizophrenia. Our specific goal is to identify changes in prediction error sensitivity to adaptation and contingency in patients and to characterise the extent to which these signals in healthy controls are susceptible to perturbations induced by dopaminergic drugs (L-Dopa and sulpiride). In doing so, we will provide critical insights to the neurobiology of positive symptoms of schizophrenia.

Amount: £381,826
Funder: The Wellcome Trust
Recipient: University of Cambridge

Imaging mechanisms of energy failure following head injury. 07 Oct 2010

Traumatic brain injury (TBI) caused over 170,000 NHS hospital admissions in 2008, requiring over 0.5 million acute hospital bed-days, and over 50,000 intensive care unit bed-days annually. TBI is amongst the most important causes of death in young adults. Survivors experience an enormous burden of physical disability, neurocognitive deficits, and neuropsychiatric sequelae. Despite this, outcome following injury has improved through the provision of protocol driven therapy aimed at preventing an d treating secondary ischaemic insults. However, energy failure may be due to mechanisms other than classical ischaemia, and this may explain the failure of pharmacological neuroprotective therapies. The research detailed in this application aims to improve our understanding of the mechanisms of energy failure following TBI using magnetic resonance imaging, proton spectroscopy and positron emission tomography to map the extent and spatial distribution of metabolic derangements. These will differ entiate classical ischaemia, regional hypoxia secondary to microvascular injury and mitochondrial dysfunction. Sequential measurements of n-acetylaspartate, a mitochondrial metabolite, will address whether metabolic derangements are reversible and represent mitochondrial dysfunction, or irreversible and represent on going neuronal loss. Such data may provide important advances in the development and implementation of successful neuroprotective therapies.

Amount: £184,299
Funder: The Wellcome Trust
Recipient: University of Cambridge

Molecular and cellular mechanisms of mouse fetal lung maturation. 14 Oct 2010

Multipotent epithelial progenitors in the embryonic lung produce bronchiolar and then alveolar daughter cells. We will investigate the cellular and molecular mechanisms responsible for this bronchiolar to alveolar switch. The long-term aim of this research is to facilitate the development of therapeutic alveolargenesis. Key goals: 1. Cellular mechanism: determine if the bronchiolar to alveolar switch is intrinsic to the epithelium. We will transplant lineage-labelled multipotent epithelial progenitor cells into older, or younger, cultured lungs and quantitatively assess the fates of their daughters. 2. Molecular mechanism: define the minimal genes which are sufficient for the embryonic bronchiolar to alveolar switch. We will test which of the transcription factors expressed in the multipotent progenitors while they are producing alveolar descendents are sufficient to determine alveolar fate. The transcription factors will be ectopically expressed during the bronchiolar (pseud oglandular) phase of lung development in vitro. The molecular mechanisms of the phenotypes, in particular the transcription factor targets, will be determined in vivo. 3. Adult application: test the ability of the embryonic alveolar progenitor-specific transcription factors to convert postnatal progenitors from bronchiolar to alveolar fate. Transcription factors will be ectopically expressed in small numbers of postnatal bronchiolar progenitors in growing, steady-state, or injured animals.

Amount: £302,491
Funder: The Wellcome Trust
Recipient: University of Cambridge

Key interactions in a tripartite multidrug efflux pump of pathogenic bacteria. 05 Oct 2010

Studying mechanisms of antibiotic resistance is essential to understand and counteract bacterial diseases, and also informs us about fundamental biological processes. We propose to continue our studies on the TolC-dependent machineries, or pumps, which effect efflux of antibacterial drugs by all Gram-negative bacteria. In these tripartite systems an outer membrane-anchored TolC protein presents a cell exit duct to diverse molecules bound by inner membrane transporters. The two apposed membrane c omponents are structurally and functionally linked by a periplasmic adaptor protein that is key to the recruitment and opening of the TolC exit channel. The assembled active pumps span the entire bacterial cell envelope of two membranes and intervening periplasmic space. We will continue to establish key details of the assembly and operation of the AcrA/AcrB/TolC RND type pump widespread among bacteria such as E.coli, Pseudomonas and Neisseria. Our work will advance understanding of this medic ally important system and will allow us to assess potential targets for future resistance inhibitors. It will also provide insights into the biogenesis and activity of complex membrane machineries.

Amount: £510,682
Funder: The Wellcome Trust
Recipient: University of Cambridge

Human Monoclonal Antibody Therapy for Rhodesiense Sleeping Sickness. 05 Oct 2010

Human African Trypanosomiasis remains a serious health problem in Central Africa and treatment has been largely untouched by the advent of modern medicine. The chemotherapy used is based on drugs introduced between 40 and 90 years ago that have severe side effects including a few percent mortality. There is a desperate need for new medicines. Most trypanosomes cannot infect humans due to a primate-specific innate immunity mechanism based on apolipoprotein L-1 (apoL-1), a component of a subse t of high density lipoprotein (HDL) particles. Receptor mediated endocytosis of HDL and trafficking of the apoL-1 to the lysosome results in cell death. Trypanosoma brucei rhodesiense, one of the two human infective subspecies, counteracts the action of apoL-1 through the production of the SRA protein which binds to apoL-1 and prevents its action. The objective of this application is to produce and test a new therapeutic based on human monoclonal antibodies that bind SRA and block the bindi ng of apoL-1, thus rendering the trypanosome susceptible to killing by apoL-1. The use of human antibodies for the treatment of disease is an emerging technology that is characterized by dramatic on target efficacy, and little or no off-target effects.

Amount: £293,985
Funder: The Wellcome Trust
Recipient: University of Cambridge

Dissection of mechanisms underlying the development of neuronal lineages. 14 Oct 2010

We will use the developing nerve cord of the Drosophila embryo and larva to investigate elementary organizational principles and their mechanisms that underlie the development of neuronal progenitor cell lineages. We will use genetic methods to label (and manipulate) neuroblast lineages in first instar larval stages, when these have fully differentiated, and show how key features of neuronal differentiation, namely transmitter phenotypes and projection patterns, are assigned within lineages. We will determine which (combinations of) transmitter(s) and projection phenotypes are generated within a lineage and in what temporal pattern. We will establish experimentally how these fundamental aspects are regulated by daughter cell interactions and birth order progression. In summary our aims are: 1) To determine within neural lineages the pattern and underlying developmental mechanisms for transmitter phenotype differentiation. 2) To establish how neural lineages generate diverse cell m orphologies.

Amount: £254,576
Funder: The Wellcome Trust
Recipient: University of Cambridge

Molecular mechanisms of filopodia formation. 22 Jun 2011

I have developed a reconstitution system using supported lipid bilayers and Xenopus egg extracts that recapitulates several aspects of filopodia formation in a format highly amenable to microscopy and intervention. The filopodia-like structures are comprised of long, parallel bundled actin filaments that polymerise from the membrane surface. At the membrane there is assembly of actin regulators that mimics the filopodial tip complex. To understand how the tip complex functions I propose to: (1 ) determine the assembly mechanism of the tip complex. I will do this by (a) measuring the recruitment of proteins to the tip complex (b) studying the roles of tip complex members toca, IRSp53, srGAP2 (of the BAR domain superfamily) and Cdc42 (the Rho-type GTPase) in initiation using mutant proteins, immunodepletion and kinetics using fluorescent probes and (c) testing the contribution of Ena/VASP/Evl and diaphanous-related formins using similar techniques; (2) explore the molecular architecture of the tip complex using single molecule microscopy to determine the relationship between the structure and function of the tip complex; (3) compare the kinetics of protein recruitment to filopodia formed during early development to determine whether the molecular mechanism is conserved between different cell types.

Amount: £1,168,078
Funder: The Wellcome Trust
Recipient: University of Cambridge

Genome-wide analysis of the interactions that mediate communication between central carbon metabolism and the cellular regulome. 06 Dec 2010

Cells exposed to oxidants re-route their main carbon flow from glycolysis to the pentose phosphate pathway (PPP), by two different mechanisms. One bases on a biochemical block and operates purely on a metabolic level, the other on transcriptional regulation. We found that the two processes compartmentalize over time: After contact with the stressor, the cell induces the metabolic transition in seconds-timescale but it takes minutes until transcripts raise. Remarkably, metabolic changes seem cau sally implicated in regulating this process: induction of the transition activates transcripts of the antioxidant-system. It is the aim of this proposal to identify and analyze regulatory mechanisms which surround the involved metabolic pathways on a genome-scale level. For this, we will develop multiple reaction monitoring (MRM) assays that allow fast quantification of metabolic intermediates, and screen an entire yeast knock-out library composed of 5,200 systematically generated yeast mutants to identify all gene deletions which cause specific changes in the concentration of their intermediates. In a second step, by using a targeted proteomics strategy, these mutants will be screened to identify alterations in the abundance of implicated enzymes. The identified genetic/metabolome interactions are studied for their general importance on metabolism, and analyzed in respect of yeast aging phenotypes.

Amount: £883,906
Funder: The Wellcome Trust
Recipient: University of Cambridge

Non-transcriptional mechanisms of the human circadian clock. 06 Dec 2010

Circadian (or daily) rhythms permeate biology. They are manifest at all levels of biological scale from social activity to cognitive ability - from physiology to self-sustained rhythms in cellular gene expression. It is well established that disruption of our biological clock correlates with impaired performance and increased likelihood of diseased states e.g. metabolic syndrome and obesity. Understanding the intrinsic cellular clock mechanism therefore constitutes an important goal for treatin g human disease as well as increasing workforce productivity. The consensus model for cellular time-keeping posits several inter-linked transcriptional-translational feedback loops at their mechanistic heart. However we have recently observed that human erythrocytes, which lack the capacity for gene expression, exhibit robust rhythms in post-translational modifications associated with cellular redox metabolism. We have also shown this is conserved in murine tissues and algae, and therefore li kely represents an evolutionarily ancient eukaryotic clock mechanism. If funded, this work will likely produce a paradigm shift for many aspects of cell biology with important consequences for understanding the temporal regulation of physiology at a deeper level..

Amount: £854,253
Funder: The Wellcome Trust
Recipient: University of Cambridge