- Total grants
- Total funders
- Total recipients
- Earliest award date
- 17 Oct 2005
- Latest award date
- 30 Sep 2018
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
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We recently showed that I-dsRNA specifically binds a stress-complex , which comprises various stress granule (SG) components. Furthermore, we demonstrated that I-dsRNA in cells downregulates both endogenous and reporter gene expression, in trans. We provided evidence that this was the result of both reduced mRNA levels and impaired translation initiation. We therefore proposed a model in which hyper-editing of dsRNAs by ADARs results in downregulation of gene expression via SG formation. W e now plan to carry out experiments aimed at further understanding the proposed model, and to elucidate the mechanistic details. The I-dsRNA stress-complex will be further characterized. We will investigate whether SGs assemble in response to I-dsRNA, and which SG components are important for downregulating gene expression in the presence of I-dsRNA. We will explore the pathway by which I-dsRNA results in degradation of reporter mRNAs, and will identify the nuclease(s) responsible. The mechanism by which I-dsRNA inhibits translation initiation will also be investigated. We will analyze the effect of I-dsRNA on endogenous gene expression. Finally we will look at the potential relationship between ADARs and SGs. A key part of the model predicts that editing of an unknown substrate(s) by ADARs will lead to SG assembly.
The generation of specific types of neurons in the correct place and at the appropriate time is essential for the formation of neural circuits. The neocortex, the major, mammal-specific, executive and integrative centre of the brain is a modular structure. Composed of six layers of neurons, the cortex is divided into anatomically distinct areas. Anatomical areas serve different neurological functions, with areas dedicated to vision, hearing, motor control and somatic sensation. This research pro poses to study the processes by which neocortical stem and progenitor cells generate neurons in the correct place in the mouse neocortex. In previous work, we found that the spatial identity of neocortical stem and progenitor cells is encoded by gradients of expression of transcription factors. We now wish to understand how those gradients are translated into the generation of spatially discrete populations of neurons for each area.
SUMMARY Signalling between cells and tissues is critical for their development and for their continued modulation post-embryonically. Several key signalling pathways, which include the Notch pathway, have been implicated in mediating this communication during development. In contrast, other pathways, notably the pro-inflammatory Tumour necrosis factor (TNF), have predominantly been considered in the context of the immune response and tissue homeostasis. From our investigations into targets of N otch activation we have found that dTraf1, a key signalling adaptor for the TNF receptor is up-regulated following Notch activation, suggesting a potential link between Notch and TNF pathways. This raises questions about the contribution of dTraf1 and the TNF pathway to developmental patterning and conversely about the contribution of Notch to modulation of the immune response. Here we propose to investigate these questions, using Drosophila melanogaster as a model. AIMS The aims of our prop osed research are 1. To determine the extent of cross-talk between Notch and dTraf1 and its consequences; 2 To ascertain the roles of dTraf1 in developmental signalling and its relationship to TNF/TNR signalling; 3. To identify domains in dTraf1 required for its function and localisation in different signalling contexts.
The structural basis for the interaction of carbohydrates with PfEMP1 proteins in malaria pathogenicity. 24 Oct 2007
We have developed an expression system that allows us to produce large quantities of correctly folded, functional DBL domains from carbohydrate binding PfEMP1 proteins. We have also produced crystals of a chondroitin sulphate A (CSA) binding DBL domain. We will use these tools to investigate the structural basis for binding of CSA to this domain. (i) We will determine the structure of a CSA binding DBL domain. (ii) We will develop a quantitative binding assay to investigate the interaction be tween CSA and the DBL domain. (iii) We will use this assay to determine the minimum fragment of CSA that binds to the DBL domain. We will also mutate residues of the DBL domain that are predicted to play a role in CSA binding and determine the effect of these mutations on the interaction. (iv) We will grow crystals of the DBL domain in the presence of CSA disaccharides, and short defined fragments of CSA, enabling us to determine the structure of the complex between the DBL domain and CSA. ( v) We will express and purify heparan sulphate binding DBL domains and use similar structural and biochemical studies to investigate the interaction between the DBL domains and heparan sulphates.
Structural and functional neuroimaging studies in adolescents with disruptive behaviour disorders. 10 Oct 2007
In this project, we will use event-related functional magnetic resonance imaging (fMRI) to investigate whether adolescents with early-onset CD exhibit a differential pattern of neural activation during the fear conditioning process relative to adolescents with adolescence-onset CD and healthy controls. We will also measure brain activation while participants are viewing emotional pictures from the International Affective Pictures System (IAPS) to replicate and extend findings from the only prev ious functional imaging study in children with CD. The final functional task will be a probabilistic reversal task, which will enable us to examine differences in the neural substrates of reward processing and decision-making. In addition to functional studies, we will assess prefrontal cortex, anterior cingulate cortex, and amygdala volumes and morphology in this sample using automated structural MRI methods (voxel-based morphometry). Functional connectivity between these regions will be meas ured using network modelling approaches. This will be achieved using a cross-sectional case-control design involving an existing sample (n=73) of 14-18 year old post-pubertal adolescents with CD, consisting of cases with early (n=43) and adolescence (n=30) onsets ascertained from a community population. These groups will be compared with age- and sex-matched controls (n=40) with no lifetime history of antisocial behaviour.
Molecular characterisation of NOD1 and NOD2 mediated ligand recognition and signal transduction. 23 Jun 2008
NOD1 and NOD2 are intracellular pathogen recognition receptors that detect fragments of bacterial peptidoglycan. Receptor activation induces up-regulation of NFkappaB and proinflammatory mediators. Single nucleotide polymorphisms (SNPs) have been associated with inflammatory barrier diseases; for example, the NOD2 mutations R702W, G908R and 1007fsincC predispose individuals to Crohn's disease. This Fellowship will be held at the University of Cambridge. It will investigate the molecular nature of ligand detection and signal transduction by NOD1 and NOD2 and the impact of non-synonomous SNPs on protein function. The signalling domains of NOD1/2 and their co-factor RICK will be expressed in E.Coli. Individual domains and signalling complexes will be characterised using biophysical and x-ray crystallographic techniques, complemented with cell based functional reporter assays. The composition of NOD1/2 containing complexes will be determined pre and post ligand stimulation in physiologica lly relevant cell types by combining immunoprecipitation and mass spectroscopy experiments. Moreover, experiments will be performed to finally ascertain whether NOD1/2 directly bind their respective ligands. Coupled with functional studies assessing the impact of SNPs on receptor function this work will significantly enhance our knowledge of the molecular basis of both NOD1/2 receptor function and dysfunction. This will aid development of potential therapeutics for inflammatory barrier diseases .
Transcriptional regulatory elements -- both promoters and enhancers -- contain clusters of transcription factor binding sites. I propose a two-stage strategy for finding and annotating these elements. The first stage is to build a dictionary of transcription factor binding motifs (TFBMs). I have previously demonstrated that a novel motif inference tool (NestedMICA) can recover a significant fraction of fruitfly TFBMs. By refining this strategy and applying it to whole-genome datasets, I in tend to build comprehensive motif dictionaries for several key metazoan genomes. I also plan to find and annotate individual regulatory elements on a genome-wide scale. Since enhancers can occur far from the genes that they regulate, this will require new strategies for predicting these elements and -- where available -- integrating these predictions with functional genomics datasets such as ChIP/chip. The second part of this project will be to develop such tools to the point where they can be used to make good-quality regulatory annotation. The goal is to produce regulatory annotation of quality and utility comparable to the computational annotation of protein-coding genes by Ensembl.
Insulin and IGFs are key coordinators of growth and metabolism in multicellular organisms. I have identified a set of neurons that express Ilp7, one of the Drosophila insulin-like peptides. These neurons are distinct from the known insulin-producing cells of the Drosophila brain that secrete into the circulation and regulate global growth. Instead, Ilp7 neurons contact the hindgut muscles, thus raising the possibility that they regulate local gut functions. This proposal is aimed at: 1) characte rizing the developmental and physiological functions of Ilp7 neurons 2) identifying new factors regulating the survival and differentiation of Ilp7 neurons. To investigate the functions of Ilp7 neurons, I will use RNA interference to downregulate Ilp7 expression and the Gal4-UAS system to silence/ablate these neurons. Gut development and physiology will be assessed using defined local and whole-organismal parameters. To identify new genes specifying Ilp7 identity, I will carry out a high-through put F1 screen of pre-mapped mutant lines. The readout for this screen utilises a double reporter line, allowing Ilp7 neurons to be visualised in living embryos under the microscope. Finally, in a candidate gene approach, I will investigate the mechanisms by which homologues of known vertebrate insulin and enteric regulators control insulin production in Ilp7 neurons.
Leprosy and society in Rouen, c. 1100 - c. 1500. 02 Apr 2008
My research project will build upon my PhD thesis, Charity in Rouen in the twelfth and thirteenth centuries (with special reference to Mont-aux-Malades) . I will expand my study of the leper house of Mont-aux-Malades to encompass the longer period c. 1100 c. 1500, and will investigate other medical and charitable activities at Rouen in this period. In the Archives d partementales de la Seine-Maritime, Rouen, I will examine charters and other documents relating to Mont-aux-Malades, the leper h ouse of Salle-aux-Puelles, the hospital of La Madeleine, and other monasteries that assisted the sick. I will also study documents in the Biblioth que Municipale, Rouen, the Archives d partementales de l Eure, vreux, the Archives Nationales, Paris, and the Biblioth que Nationale, Paris. I will write a 120,000-word monograph, Leprosy and society in Rouen, c. 1100 c. 1500 . In the context of the recent studies of Carole Rawcliffe and Fran ois-Olivier Touati, the book will aim to establish the exact identity of lepers in medieval society. It will consider the status of lepers within and outside leper house communities at Rouen, and will investigate the responses of the healthy to the leprous. It will aim to increase our understanding of medieval leprosy, medicine and society.
This research sets out to explore how the medieval experience of disease and disability was mediated through the representations of healing miracles found in saints narratives. This goal will be achieved by exploring four main areas. The first will search these texts for evidence of practical healthcare; the ailments suffered and the treatments administered by the medieval medicus . The second part will situate these practices within the contemporary context by drawing upon sources such as Old English medicinal manuals, penitentials and sermons, with the aim of discovering whether the miracle narratives show a similarly syncretic attitude towards healing. The third part of this research will concentrate on the texts themselves, examining the manuscripts in which they were found, the scriptoria where they were produced and the conditions accounting for their composition. This will feed into a discussion of the narrative strategies employed by the authors and their subversion of the te xts testimonial format. The final section will consider the occasion of their communication; how these carefully crafted stories were relayed to the public. This will involve reconstructing the conditions of performance; assessing how the architectural and liturgical arrangements of the shrine may have affected the dissemination of the stories.
Epigenetic regulation of the temporal responsiveness of human embryonic stem cells to generate motor neurons. 03 Jul 2008
I propose to ask whether there is a temporal restriction of responsiveness of human ES derived neural stem cells to patterning signals known to be sufficient to induce a MN fate with a focus on the epigenetic regulation of such restriction. Motor neurone disease (MND) is a progressive and fatal neurodegenerative condition.There is a great need for renewable sources of human motor neurons (MNs) as an experimental resource to learn more about the aetiopathogenesis of MND and to enable the deve lopment of potential therapies. The ability to generate defined neuronal cell types from hESCs offers unique experimental opportunities to study mechanism(s) underlying neural diversity. hESC derived NSCs predictably respond to morphogenetic signals permitting the generation of region specific neurons including MNs. An unresolved question is whether responsiveness to such signals is developmentally restricted. I will generate enriched populations of MNs (using established methods). Character isation will include qPCR, FACS / IHC as well as functional assays. Our hESC neuralisation system provides an ideal platform to begin characterising the epigenetic regulation of MN specification from hESC-NSCs. Analysis of the temporal responsiveness of hESC-NSCs has several important implications including defining the optimal timeframe for in vitro generation of MNs from hESC-NSCs.
The aims of this project are three-fold: to clarify how frequently ectopic lymphoid tissue (ELT) occurs within heart grafts and whether its presence correlates with development of chronic allograft vasculopathy (CAV), to examine how graft-ELT functions independently of conventional secondary lymphoid organs (SLOs), and to assess whether blocking ELT formation abrogates CAV development. Foci of ELT are frequently found in explanted failed solid organ transplants. In autoimmunity, immune respon ses originating within ELT may drive disease progression. Whether allograft-ELT responses similarly influence graft survival is, however, unknown. ELT formation will be studied in two mouse models of chronic cardiac allograft rejection. Heart allografts will be excised 50 days after transplantation and the presence of ELT correlated with the degree of CAV. To distinguish the alloimmune response in ELT from conventional SLOs, mice that lack SLOs will receive previously transplanted heart graft s and activation of na ve alloreactive T and B cells, dependent upon graft-ELT, assessed. These experiments will be extended to examine whether graft-ELT is a site for alloreactive T cell epitope spreading and alloreactive B cell somatic hypermutation. Monoclonal antibodies will be used to block key signalling pathways required for ELT formation and their effect on CAV development determined.
The principal research objective is to elucidate how anaesthetic drugs interfere with cognitive functions (such as attentiveness, memory and processing of auditory stimuli) during sedation. We will use functional MRI and continuous EEG monitoring to identify these effects in volunteers sedated with propofol, nitrous oxide or sevoflurane. We will aim to ascertain the role of the inferior frontal cortex in the resolution of semantic ambiguities during the sedated state. Painful stimuli during seda tion and anaesthesia are associated with arousal activity in the brain; we will attempt to identify the influence of such stimuli on the inferior frontal cortex. These data will help establish whether anaesthetic agents interfere with cognitive function during sedation by interfering with the brain's ability to maintain attentiveness, by interfering with individual perceptive processes, or by interfering with the ability of the brain to pass information between different regions and then bind th e whole into a unified conscious experience. This could potentially lead to an improved understanding of anaesthesia, and consciousness, and may inform the development of practical, EEG based methods of detecting awareness during anaesthesia. These studies will also provide important insights into the hierarchy of cortical language processing.
Mechanisms underlying Glucose-dependent Insulinotropic Polypeptide (GIP) secretion and function. 09 Apr 2008
Modulating glucose-dependent insulinotropic polypeptide (GIP) secretion is a potential strategy for the treatment of diabetes and obesity. However, the cellular mechanisms underlying GIP release from K-cells are poorly characterised because the cells have not, historically, been readily distinguishable from their epithelial neighbours. I recently created transgenic mouse strains expressing a fluorescent marker under the control of the GIP promoter (GIP-Venus mice), enabling, for the first time, the identification and purification of living primary K-cells. These will be characterised by electrophysiology, single cell fluorescence imaging, gene expression analysis and secretion studies, to identify agents and pathways that modulate GIP secretion and that could be targeted to control GIP release in vivo. GIP-Venus mice will be crossed with the Immortomouse to develop K-cell specific lines, which are not currently available but would be a valuable tool for research requiring a higher cell number or longer culture times. Further transgenic mouse strains will be created, expressing the diphtheria toxin receptor under the control of the GIP promoter, enabling conditional K-cell ablation in murine models of obesity. The metabolic consequences of acute and chronic GIP depletion will be investigated in these mice, to evaluate the potential effects of reducing GIP secretion in vivo.
Myosin motor proteins move membrane and vesicles along actin tracks. Myosin VI unlike almost all other myosins moves towards the minus end of actin filaments. Our intracellular localisation of myosin VI at the Golgi complex, in clathrin coated pits/vesicles and at kinetochores in mitotic cells indicates that it functions in a wide variety of intracellular processes. The diverse roles of myosin VI are mediated by interaction with a range of different binding partners. The aim of my research is to characterise the roles of myosin VI and its interacting partners, which we have recently identified: 1. In the maintenance of Golgi morphology, sorting of cargo in the trans-Golgi network and in post Golgi transport of vesicles to the plasma membrane; 2. In endocytosis at the apical domain of polarised cells. Using cell lines from the small intestine (Caco-2) and the kidney (MDCK) as well as primary kidney cells isolated from the Snell's waltzer myosin VI knockout mouse, I will determine the sequential steps of the endocytic process and the class of receptor for which myosin VI is important; 3. In mitosis to confirm their localisation at kinetochores and spindle poles and determine their function. I will study how the interaction and activity of these multi-protein complexes containing myosin VI are regulated at various cellular localisations by overexpressing myosin VI of deletion mutants followed by co-immunoprecipitation for subsequent analysis. I will visualise how myosin VI moves in cells using live cell imaging and investigate how its surprisingly wide variety of intracellular functions is so precisely regulated. To investigate which type of motor is required to fulfil this variety of intracellular functions I will study the kinetic and mechanical properties of myosin VI at the single molecule level in collaboration with the group of John Kendrick-Jones at the MRC-Laboratory of Molecular Biology in Cambridge. The information I will gain from these studies will help us to understand how the loss of active myosin VI results in a form of deafness in humans and how alternations in its binding partners cause other disorders such as glaucoma. An understanding of the role of myosin VI in these diseases may allow us to devise therapeutic strategies.
Regulation and transduction of cell polarity. 20 Feb 2008
Cell polarity is critical for many functions of animal cells and loss of cell polarity is a contributing factor in cancer. We are exploiting the one-celled C. elegans embryo to investigate conserved mechanisms of cell polarity establishment and transduction. It allows study of the polarity cue and its reception by the actomyosin cytoskeleton, of the polarisation events that occur in response to the polarity cue, and of how polarisation leads to downstream events such as asymmetric spindle posi tioning. I propose both focused projects that have come out of our current studies and new screens to identify missing molecules in the cell polarity network. We will (1) carry out directed screens to identify the initial polarity cue, (2) study newly identified regulators of non-muscle myosin, and (3) investigate roles for phosphoinositides in asymmetric spindle positioning. With a long-term goal of finding the complete set of genes functioning in these processes and their relationships, we w ill (4) apply high-throughput genetic interaction RNAi screening genome-wide together with (5) biochemical purifications to further expand the cell polarity network. (6) We will carry out functional studies of newly identified genes, taking advantage of C.elegans genetics, genomics and cell biology.
The ability to repair injuries to the DNA that arise from exposure to genotoxic agents or during nucleic acid metabolism is critical for cellular life. Equally vital is the faithful replication of the genetic information encoded in the DNA in preparation for mitosis. Cells have evolved complex molecular systems in order to repair and replicate our DNA, and failure to maintain the integrity of DNA is a major contributing factor in human disease. The goal of this proposal is to define atomic struc ture and mechanism of action of macromolecular assemblies responsible for the closely associated processes of DNA recombination and replication. Thus, we are interested in determining the structural basis of strand transfer between homologous DNA molecules, which lies at the heart of DNA recombination, as well as the regulatory interactions that alter the structural state of the nucleoprotein filament, the central molecular species of recombination. In DNA replication, we seek to determine the s tructural basis for the crucial step of initiation of nucleic acid synthesis by the pol a/primase complex, the macromolecular assembly responsible for starting DNA replication in eukaryotic cells.
Control of antigen processing by Fc receptors. 05 Jun 2008
My proposal seeks to understand how Fcgamma receptors (FcgRs) control antigen processing by dendritic cells and macrophages. Since FcgR subtypes are variably expressed, have different binding affinities for different IgG subclasses and lead to fundamentally distinct patterns of intracellular trafficking and processing, it is not understood how, for a given repertoire of expressed FcgR, a specific antigen will be processed and what programme of effector functions will be triggered. My first key g oal will be to systematically define functional outcomes (binding and internalisation, antigen degradation and presentation) for ligand-receptor combinations using a receptor expression library in U937 cells expressing 90 different patterns of receptor subtype and disease-associated polymorphisms. Findings will then be confirmed using primary cells from healthy volunteers. I will then define the critical signal transduction events responsible for determining immunogenic and tolerogenic antigen p rocessing focussing on 1) the effect of receptor membrane sub-localisation on receptor function and cellular behaviour and 2) the role of macro-autophagy in modulating or possibly mediating FcgR-triggered intracellular antigen trafficking.
Regulation of cell surface receptors is essential to maintain cell homeostasis, especially following receptor stimulation to limit the duration and intensity of signalling. Receptor ubiquitination is an important mechanism for regulating expression of critical immunoreceptors. The K3 and K5 viral E3 ligases ubiquitinate cell surface class I, promoting its endolysosomal degradation, and we showed an absolute requirement for lysine-63 linked polyubiquitination of class I for its downregulation. Th ese viral genes accelerate a constitutive pathway of class I regulation. Continued studies on K3 and K5 will further define this receptor regulation pathway. Biochemical inhibitors of class I ubiquitination, and RNAi screens will identify the cellular ligase(s) responsible for class I ubiquitination, and establish the role of lysine-63 linked polyubiquitination in regulating class I and other immunoreceptors. Investigations into the viral ligases identified an unrecognised family of cellular lig ases the MARCH proteins, whose expression downregulates surface receptors. We have developed screening and mass spectrometry-based techniques, as well as MARCH9 knockout mice, to identify the substrates and function of these proteins. Biochemical studies on the regulation and expression of MARCH9, together with an analysis of MARCH9 deficient mice will determine the physiological role of the MARCH9 E3 ligase in immunoreceptor regulation.
Regulation of neural regeneration and cell fate in the Central Nervous system of Drosophila. 28 May 2008
The central nervous system (CNS) comprises an extreme diversity of cells, whose stereotyped fates and connections enable information processing. Injuries of the adult CNS impair dramatically the behaviour, motricity and sensitivity of an organism. Repair would require neurogenesis, including the regulated division of neural progenitors and the differentiation of the daughter cells. Interestingly, cellular damage is able to generate neurogenesis, albeit limited, in the vertebrate adult CNS. Howev er, the factors that trigger mitosis, the identity of the progenitors as well as the pathways inducing appropriate differentiation are virtually unknown. Here we propose to use for the first time the Drosophila embryonic midline, similar to the vertebrate floor plate, as a genetic model for CNS regeneration. Indeed, preliminary data of the Brand lab have revealed the unique property of midline cells to regenerate their injuried siblings, through extra cell division and appropriate differentiati on. We will analyze the expression profile of individual midline cells by microarray, comparing cells that are able to regenerate with those that cannot, or comparing different cell fates. We thus aim to uncover genes controlling neural regeneration and differentiation after brain injury, that will further be targeted by misexpression and mutagenesis, and regarded in the adult fly CNS.