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Recipients:
University of Cambridge

Results

The role of the immmunoglobulin superfamily proteins in the coordination of morphogenesis. 21 Feb 2006

The coordinated interaction of different cell types is required for tissue morphogenesis and organ formation. Our labs have uncovered interactions between distinctive cell populations during myogenesis and nephrogenesis. In both tissues the immunoglobulin (Ig) domain protein superfamily mediates signalling between different cell types and these interactions lead to specific morphogenetic movements. We aim to understand, at the molecular level, how signalling through this pathway acts to coordinate the activity of different cell types to produce distinctive morphogenetic responses. The Ig domain superfamily of proteins interact in specific partnerships to alter each other's activity, leading to the recruitment and activation of cytoplasmic targets. However, the outcome in terms of cell differentiation is widely divergent depending on the developmental context, ranging from cell fusion, neuronal pathfinding, synaptogenesis, cell intercalation, cell sorting and apoptosis. By focusing on two developmental contexts in a genetically manipulable organism, Drosophila melanogaster, we aim to establish interactions, partners and targets common to this pathway as well as to pinpoint those that lead to the specificity of the cell response. Adopting genetic, biochemical and cell biological approaches will allow us to confirm known interactions, to identify new partners and to establish the function of specific binding partnerships.

Amount: £173,199
Funder: The Wellcome Trust
Recipient: University of Cambridge

Strategies for the control and elimination of blinding trachoma. 01 Nov 2005

Trachoma, caused by ocular Chlamydia trachomatis infection, is the commonest infectious cause of blindness. We have shown that a single round of mass treatment with azithromycin can eliminate the infection from endemic populations for up to two years. The studies proposed here will enable this drug to be used to maximum effect. We will Determine the sensitivity and specificity of a new rapid point-of-care (POC) diagnostic test for ocular C. trachomatis infection in communities with high, medium and low prevalence of trachoma in three countries, before and after mass treatment. Use the above data, datasets from our current Wellcome Trust-Burroughs Wellcome Fund grand, to develop optimal sampling strategies to assess the need for community-based mass treatment depending on the prevalence of clinical signs of trachoma or of ocular C. trachomatis infection, determined by the POC test. Use this data to model the impact and cost-effectiveness of strategies in which a POC test is used to decide whether a community should be treated, and to compare this with the cost-effectiveness of the current strategy, based on the prevalence of clinical signs of trachoma.

Amount: £123,889
Funder: The Wellcome Trust
Recipient: University of Cambridge

The evaluation of genetic and phenotypic diversity in field isolates of Salmonella enterica serovar Enteritidis in Uruguay. 25 Oct 2005

Salmonella Enteritidis causes food-borne disease world-wide. It commonly contaminates the food chain and poultry products are a major source. Particular strains are associated with outbreaks. We have shown that in Uruguay different strains survive in the chicken population, with only a small proportion of them spreading through food to humans. We therefore hypothesize that different strains have different genetic characteristics that give rise to populations able to exploit different ecological niches. We thus propose to undertake comprehensive comparative genomic analyses of Uruguayan field isolates. Molecular typing methods will be used to allow sub-grouping of the strains. Multi-Locus Sequence Typing and microarray analysis will be applied to identify evolutionary trends, population structures, and genetic heterogeneity between strains. The genomes of three individual strains will be sequenced using new high-throughput technology at the Sanger Institute. This population information will inform selection of representative strains for use in in vitro and in vivo assays to assess their capacity to interact with different hosts.This study will provide unique information regarding the relationship between strain variation and the exploitation of different hosts by S. Enteritidis, and may inform the development of intervention measures that may be effective in developing countries.

Amount: £327,750
Funder: The Wellcome Trust
Recipient: University of Cambridge

Structural and functional analysis of RNA packaging in the lentivirus FIV. 25 Oct 2005

Lentivirus genomic RNA encapsidation involves specific recognition of the low abundance genomic RNA in the infected cell by the viral Gag protein. It is dependent on RNA structures in the viral leader. Studies on these have elucidated several novel RNA motifs and RNA/protein capture mechanisms with relevance to both viral and cellular processes. Preliminary studies on FIV suggest that the packaging mechanism is very distinctive compared to that of HIV in that in FIV the process is dependent on two discrete regions in the leader separated by a redundant sequence. The aim is to map by mutagenesis and two and three dimensional structural analysis the extent and conformation of the packaging signal region of FIV. We predict that this will lead to novel data on RNA structures and on RNA/RNA and RNA/protein interactions of direct importance to normal cell function. It will also lead to an improved understanding of the packaging process in FIV. This will improve the efficiency and safety of the promising lentiviral vector system based on this virus.

Amount: £288,791
Funder: The Wellcome Trust
Recipient: University of Cambridge

Development of an influenza virus sequencing pipeline 20 Sep 2006

Influenza viruses are a continual threat to both humans and animals. The current threat of an influenza pandemic from the H5N1 virus highlights the need for the application of new techniques to ensure that surveillance and research into influenza viruses use 'state-of-the-art' methodologies for the analysis of the sequence of the virus genome. The establishment of an influenza virus sequencing pipeline, through which to sequence large numbers of viral genomes, is proposed and encompasses scientists from five organisations. High-through-put sequencing is not available at the existing UKvirological sites where influenza viruses are handled but is available at the Wellcome Trust Sanger Institute. A work programme taking advantage of the complementary expertise available in different sites would provide access to an efficient influenza virus sequencing platform for the UK. The proposed pipeline will be set up encompassing isolates from seasonal influenza samples received during surveillance programmes, H5N1 samples from both humans and animals, isolates from reference banks of human and animal influenza, and in addition samples from on-going experiments to understand in detail within-hostselection of virus variants in extant model animal studies.

Amount: £218,565
Funder: The Wellcome Trust
Recipient: University of Cambridge

Diurnal corticoid rhythms and regulation of neurogenesis in the adult hippocampus by fluoxetine. 04 Jul 2006

We have found that a diurnal rhythm in corticosterone in required for the stimulating action of fluoxetine on the proliferation of progenitor cells and thus increased neurogenesis in the dentate gyrus of the adult hippocampus. This application seeks to explore the mechanism for this effect. The approaches are: to explore possible changes in rhythm-flattened rats in : (i) 5HT1A and 5HT7 receptors (ii) the serotonin uptake receptor (iii) the expression of gluco- and mineralo-corticoid receptors (iv) the diurnal variation in clock genes in the dentate gyrus (v) the interactions between 5HT, nitric oxide and the corticoid rhythm (vi) the action of BDNF on proliferation and maturation of progenitor cells (vii) whether a behavioural response to fluoxetine (and other SSRIs) is also dependent on the presence of a corticoid rhythm.

Amount: £436,176
Funder: The Wellcome Trust
Recipient: University of Cambridge

Specification of Circumferential Ascending Interneurons in Zebrafish Spinal Cord. 04 Jul 2006

Our ability to treat neurological disorders or spinal cord injuries is currently constrained by our limited understanding of how the central nervous system develops and functions. In particular, we know very little about how different functional types of interneurons develop. The key goal of this research is to understand how a specific functional class of vertebrate interneurons, Circumferential Ascending interneurons (CiAs), are specified in zebrafish spinal cord. We will test the spec ific hypothesis that Hedgehog, Retinoic Acid and BMP signalling pathways interact to specify the CiA domain in vivo. We will also determine the individual and combined functions of Pax2 and Engrailed1 in the specification of different CiA characteristics, including neurotransmitter expression; cell morphology; axon trajectory; synapse formation and correct activity during embryonic swimming behaviour. In many of these experiments, we will use stable transgenic-GFP lines to observe CiA deve lopment in live embryos. As CiAs share many characteristics and are probably functionally homologous with amniote V1 cells our findings should also be applicable to mammals. This research will significantly increase our understanding of vertebrate interneuron development and it will also facilitate future studies to establish causal links between particular genes and CiA activity during specific behaviours in live embryos.

Amount: £246,134
Funder: The Wellcome Trust
Recipient: University of Cambridge

Nuclear reprogramming in Xenopus using non-aqueous oocyte nuclei. NCE of 9 months approved, orig end date 30/09/09. SA 27 Jun 2006

We aim to identify some of the molecules and mechanisms by which an oocyte can reprogram the nuclei of readily accessible adult somatic cells to express embryo- or stem-cell specific genes such as Oct4 and Nanog. This is a step towards our ultimate aim of deriving rejuvenated cells for therapeutic replacement, starting with adult cells of the same individual, and thereby avoiding the need for immunosuppression. We inject mammalian somatic cell nuclei into the germinal vesicle (large specializ ed nucleus) of an amphibian oocyte and monitor nuclear reprogramming by the transcriptional activation of genes. In this project, we plan: (i) to do protein knockdown experiments, to test the necessity of identified oocyte chromosomal proteins for nuclear reprogramming; (ii) to do real-time imaging of injected nuclei in live germinal vesicles to relate the loss and gain of known proteins from and by somatic nuclei and their necessity for reprogramming; (iii) to use oocyte and egg extracts to id entify, by fractionation, so far unknown components of oocytes that are required for nuclear reprogramming. Having identified candidate reprogramming components of Xenopus oocytes, we expect to recognize mouse (or human) homologues, and to test these for function in mouse (or human) embryonic stem cells.

Amount: £663,161
Funder: The Wellcome Trust
Recipient: University of Cambridge

Engineering bacterial chondroitinase for expression in mammalian neurons and glia: a strategy for treatment of human brain and spinal cord injury. 04 Jul 2006

The main aim of this proposal is to express the bacterial gene for chondroitinase in mammalian neurons and glia, with the ultimate purpose of developing a novel strategy for treating human brain and spinal cord injury. Injection of chondroitinase at the injury site has been shown to promote nerveregeneration by degrading axon growth-inhibitory molecules. Production of the enzyme by cells at the site of injury will make local delivery of the enzyme much more efficient, and we have therefore modified the bacterial gene so thatit can be synthesized and secreted by mammalian cells. We will optimize expression and delivery of the modified cDNA in cultured astrocytes and neurons. We will also generate a construct to provide enzyme with an altered N-terminus to direct secretion to the neuronal growth cone. An in vitro model of axon regeneration will then be used to assess the ability of chondroitinase-transfected astrocytes and neurons to promote axon regeneration. We anticipate that by the end of the project we will be in a position to test the efficacy of the constructs, expressed by neurons and glial cells via an optimized lentiviral vector, in promoting structural and functional regeneration in vivo in rat models of brain and spinal cord.

Amount: £207,908
Funder: The Wellcome Trust
Recipient: University of Cambridge

What is the mechanism whereby microRNAs cause repression of translation of their target mRNAs in mammalian systems?. 27 Apr 2006

Bioinformatic studies have indicated that the expression of ~30% of all human genes may be regulated by the combinatorial action of several hundred microRNAs, yet the mechanism of action of miRNAs remains obscure and quite controversial. The aim of this proposal is to resolve these controversies: whether miRNAs repress translation of the target mRNA rather than promoting mRNA degradation, and if so, whether they inhibit the initiation rather than the elongation step of mRNA translation. Specific ally, we will use HeLa cell transfection assays and HeLa cell-free extracts to: attempt to recapitulate miRNA-mediated repression in cell-free extracts, and exploit this to analyse the mechanism of repression; identify what proteins are associated with the repressed mRNAs, and use siRNA-mediated knock-down of these and other candidate proteins to test whether they are essential for miRNA-mediated repression. examine possible explanations for the discrepancy between some authors finding inhibition of initiation, while others find the repressed mRNA in polysomes; critically examine the reasons for the reported immunity of some viral IRESs to repression whether this is because they do not require eIF4E, or because no ribosome scanning is involved, or because they were tested as Appp-capped RNAs rather than m7Gppp-capped.

Amount: £83,221
Funder: The Wellcome Trust
Recipient: University of Cambridge

Human cytomegalovirus evasion of natural killer cell functions 24 Apr 2006

Human cytomegalovirus (HCMV) provides a paradigm for how a complex viral pathogen persists and evades immune responses. 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 in a clonally dependant manor. The specific goals of the work proposed are to: (i)Define the mechanism of action of the novel viral NK evasion gene product (UL142) by identifying its ligand and how this mediates evasion of NK cell cytotoxicity.(ii)Construct deletion mutants of HCMV, for UL142 and other NK evasion genes, using Bacterial Artificial Chromosome (BAC) methodology , and use NK cell clones to determine whether CMV encodes further proteins, which mediate NK cell evasion. We will use these BAC-HCMV mutants to determine if the known evasion genes are redundant or directed at NK clone subsets, (this might explain why HCMV encodes multiple mechanisms to evade NK cells). (iii) Investigate subjects with HCMV infection to determine whether infection with HCMV shapes the expressed NK cell receptor repertoire. (iv)Determine if HCMV proteins can functionaly interact with NK receptors on HCMV specific CD8+ cells impairing effector function.

Amount: £250,076
Funder: The Wellcome Trust
Recipient: University of Cambridge

Killer cell immunoglobulin-like receptors (KIR) and reproductive success: the interaction between uterine NK cells and trophoblast HLA-C. 24 Apr 2006

Failure of trophoblast to invade and transform the uterine arteries during early pregnancy is associated with diseases such as pre-eclampsia and fetal growth restriction. However we do not know how trophoblast invasion is regulated. In a recent immunogenetic study, we showed that particular combinations of two polymorphic gene systems, Killer Immunoglobulin-like Receptors (KIR) in the mother and HLA-C2 in the fetus were associated with pre-eclampsia. The KIR are expressed by uterine Natural Kil ler (uNK) cells and their ligands are HLA-C molecules that are displayed by trophoblast. In this proposal we aim to define the biological and molecular mechanisms underlying this finding. We will use isolated primary uNK cells and placental trophoblast to define which activating and inhibitory KIR are expressed by uNK cells and what forms of HLA-C they recognise on trophoblast cells. A key goal is to determine how uNK cells respond when KIR on the uNK cells bind trophoblast expressing either HL A-C1 or HLA-C2 allotypes. By defining how NK responses to C1 and C2 allotypes differ we seek to understand the mechanism by which NK cells and trophoblast co-operate to establish a normal blood supply to the placenta and how this fails in pregnancies with particular KIR/HLA-C combinations.

Amount: £285,055
Funder: The Wellcome Trust
Recipient: University of Cambridge

The mechanism of activation of the polyketide toxin mycolactone. 21 Feb 2006

The genes for the biosynthesis of the lipophilic polyketide toxin mycolactone, which plays a decisive role in creating the lesions that are characteristic of the emerging disease known as Buruli ulcer, are known to be clustered on an unusual plasmid housed in Mycobacterium ulcerans. This strain is very difficult to work with, requires specialised P3 facilities, taking months to grow, and there are no reliable systems developed for its genetic manipulation. In this project we aim to uncover the identity of the key enzyme that catalyses the activation of the polyketide core of mycolactone by the specific attachment of a polyketide sidechain. Recombinant candidate enzymes will be cloned, expressed and purified, assayed using purified core and chemically synthesised acyl donors, and the formation of mycolactones monitored by LC-MS. Once identified, the joinase will be studied to reveal its mechanism of action and substrate specificity. In principle, purified joinase can be used to synt hesise a library of variants of mycolactone differing in the side-chain. It is already known that natural cytotoxic mycolactones differ in their side-chain, and in parallel we will also determine the structure of several novel natural mycolactones from extracts of clinical isolates of M. ulcerans.

Amount: £197,008
Funder: The Wellcome Trust
Recipient: University of Cambridge

Epigenetic control of gene activity and repression: Regional influence of large repetitive domains in mammals. 21 Feb 2006

The function of the mammalian genome is regulated by epigenetic modifications to DNA and chromatin that affect a wide range of processes including gene activity and repression and chromosome architecture. It is now well established that alterations in such genome modifications are associated with disease. Understanding and describing the epigenetic state of the normal mammalian genome is one of the big challenges in the post-genomic era and high-throughput genomic microarray-based approaches are currently being developed to determine the 'epigenome' and define the DNA sequences that direct epigenetic states. Repetitive sequences constitute around 45% of the mammalian genome and by their nature are intractable to genomic array analysis. Growing evidence suggests that in some contexts these sequences might define epigenetic processes, but little is known about the function of stretches of repeat sequence in mammals. Here we propose the deletion of a 250kb block of repetitive DNA located at the boundary of a mouse imprinted domain to assess its consequences for the establishment and maintenance of regional epigenetic modifications and for the epigenetic control of gene activity and repression. In particular, we will determine whether imprinting in the domain is affected and whether the repetitive DNA acts as a barrier preventing the spread of imprinting to adjacent regions.

Amount: £192,245
Funder: The Wellcome Trust
Recipient: University of Cambridge

Molecular genetics of planar polarity in Drosophila 21 Feb 2006

Planar cell polarity (PCP) is exemplified by the direction of ciliary beat, the orientation of hairs or the alignment of stereocilia. The problem is pervasive and long range (how does information set up over an entire developing field convey axial information to single cells?) and local (how does a single cell read this polarising information and how does it liaise with its neighbours?). We have worked on PCP for 9 years using the Drosophila abdomen. We and others have shown that PCP depends on a novel mechanism. The pervasive system relies on interacting cadherin molecules (Dachsous, Fat) and the local system depends on the Frizzled receptor and another cadherin, Flamingo. We have published evidence that each cell is polarised by comparing the levels of activity of Frizzled in its neighbours, using the intercellular bridge made by Flamingo to do so. Now we want to test this model at the molecular level, using constructs made with all three cadherins and with Fz, assaying them both in flies and in cell culture. We will also investigate the long range system, asking how it receives information from the organising morphogen (Hedgehog) upstream and passes information to the local system downstream

Amount: £389,685
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of Z alpha-1 antitrypsin in the pathogenesis of emphysema. 23 Feb 2006

Alpha-1 antitrypsin (AT) is a major serine proteinase inhibitor whose main function is to protect the lung from proteolysis by neutrophil elastase. The Z variant (E342K) is prone to form polymers which accumulate as inclusions in the hepatocyte predisposing Z-AT homozygotes to cirrhosis. The low plasma levels of Z-AT predispose early onset panacinar emphysema. Polymeric AT may in part be responsible for the previously described exaggerated inflammatory response in Z-AT homozygotes. Polymers of Z-AT are present emphysematous lungs, and are chemotactic to neutrophils and preliminary data suggests that they also induce the secretion of interleukin-8 from type II alveolar cells. The factors that cause the formation of polymers within the lung are not known. In vitro, their formation is accelerated by increasing concentration and temperature. Furthermore, Based on the structural biology of polymerisation we have identified peptide inhibitors of polymerisation. The key scientific objectiv es of this work are to (1). elucidate the factors that cause polymerisation of Z-AT within the lung (2). assess whether polymeric AT causes Interleukin-8 secretion from type II pneumocytes (3). to assess whether potential therapeutic agents are efficacious in vivo.

Amount: £126,420
Funder: The Wellcome Trust
Recipient: University of Cambridge

Role of Bone Morphogenic Protein 8b (BMP8b) in thermogenesis. 07 Nov 2005

Long-term success to treat obesity is dependent on strategies aimed at preventing the concomitant fall in energy expenditure associated with food restriction (5). Energy modifications geared toward increasing energy expenditure may provide an alternative, independent means of promoting weight loss or, even more importantly, of preventing weight regain. In this project we investigate Bone morphogenic protein 8b (BMP8b) function as a novel mechanism regulating peripheral mechanisms of mammalian thermogenesis and fuel partitioning as strategies to prevent obesity. Our hypotheses are that: a) BMP8b may be an activator of thermogenesis and fatty acid oxidation and therefore a potential therapeutic target for obesity and type 2 diabetes mellitus. b) BMP8b may redirect the differentiation of white adipocyte fat pads towards more pro-thermogenic brown adipose fat pads. and c) BMP8b may promote energy dissipation in the skeletal muscle. The specific aims of this proposal are: 1. To establish the role of BMP8b on global energy homeostasis: thermogenesis, obesity and insulin resistance. 2. To establish the role of BMP8b in white and brown adipogenesis.3. Identification of signalling mechanisms mediated by BMP8b in adipocytes and skeletal muscle. These objectives will be accomplished through a multiple integrated approach involving in vivo and in vitro strategies

Amount: £175,881
Funder: The Wellcome Trust
Recipient: University of Cambridge

Schistosome life-cycle expression profiling using a full-transcriptome DNA mocroarray. 25 Oct 2005

Schistosomes undergo a complex developmental lifecycle inhabiting two different hosts and alternating between free-living and parasitic forms. Studying when gene products are expressed will contribute to a greater biological understanding of this major pathogen useful in the identification of novel chemotherapeutic- and vaccine candidates. Although recent characterization of this helminth's transcriptome has yielded putative evidence related to the expression of approximately 13, 000 S. mansoni gene products, these estimates were based solely on comparative EST numerical profiling. As a more accurate measurement of relative RNA abundance, we recently profiled greater than 50% of this parasite's transcriptome using our well-characterized oligonucleotide DNA microarrays. While this important preliminary study provided semi-quantitative information related to the expression of greater than 7, 000 gene products, we still are lacking data related to the remaining 7, 000 or so transcribed sequences. In this grant proposal, we therefore want to extend and advance these significant preliminary findings by focusing on two specific aims: 1) develop an updated and near complete transcriptome long-oligonucleotide DNA microarray and 2) profile this DNA microarray with seventeen different schistosome life-stages/samples.

Amount: £117,270
Funder: The Wellcome Trust
Recipient: University of Cambridge

Resolving the origins of gonadotrophin-releasing homone neurons in the chick. 17 Oct 2005

This year-long pilot study aims to resolve finally the developmental origins of gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus and terminal nerve, which are critical for vertebrate fertility and reproduction. These neurons migrate into the brain along the olfactory nerves from the vicinity of the olfactory placodes, and a variety of fate-mapping and ablation studies have suggested they originate from the olfactory placodes themselves. However, a recent zebrafish fate-map has re-opened this debate by suggesting that cranial neural crest cells, which become intimately associated with the olfactory placodes, form at least some terminal nerve GnRH neurons, while the hypophyseal placode (which forms the anterior pituitary gland) forms hypothalamic GnRH neurons. The proposed series of quail-chick fate-mapping experiments, performed before neural crest cells reach the olfactory placodes, should settle the question of whether forebrain GnRH neurons in tetrapods are derived from neural crest cells, the hypophyseal placode or (as generally supposed) the olfactory placodes. This will resolve a long-standing debate and clarify our understanding of Kallmann's syndrome, in which GnRH neurons fail to reach the brain, resulting in hypogonadism. It will also provide an essential baseline for future experiments on the induction of GnRH neurons.

Amount: £32,423
Funder: The Wellcome Trust
Recipient: University of Cambridge

Development of a simple, rapid and affordable HIV RNA test for early diagnosisof HIV-infection in infants and for antiviral therapy monitoring in resource-limited settings. 27 Oct 2005

Recent initiatives such as that of the World Health Organisation to provide 3 million HIV-infected individuals with antiretroviral therapy (ART) by 20051 will increase the availability of HIV treatment in developing countries. Such large-scale treatment projects will also require diagnostics and a means to monitor treatment to be fully effective. In the developed world, measurement of HIV load is standard for diagnosis and monitoring of HIV-infected individuals. Currently available HIV load assays are unsuitable for resource-limited settings because they are complex, time-consuming and require expensive instruments and test kits as well as cold-chain shipment and storage of reagents. The availability of a simple and robust test for the determination of HIV load will be crucial for the successful implementation of HIV treatment in the developing world.The objective of this project is to develop a simple and rapid HIV RNA assay designed specifically to assist health care workers in developing countries both with the early diagnosis of HIV infection, particularly in neonates, and for HIV therapy monitoring when appropriate quantitation standards are included. The proposed assays will be based on visual detection of isothermally amplified HIV RNA by dipstick. Rather than reporting absolute quantification of viral RNA, the assay will give readout of clinically relevant thresholds.

Amount: £391,805
Funder: The Wellcome Trust
Recipient: University of Cambridge