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Recipients:
University of Cambridge

Results

Intracellular membrane traffic in hereditary spastic paraplegias. 03 Apr 2006

In humans, the main pathway over which voluntary motor signals are carried from the brain to the spinal cord is the corticospinal tract. Insults to the "upper motor neurons" that constitute this tract can result in a clinical picture of spastic paralysis. The hereditary spastic paraplegias (HSPs) are single gene disorders in which the axons of the corticospinal tract show progressive, length-dependant, distal degeneration after initially normal development. Functional characterisation of the responsible genes is therefore likely to reveal cellular processes that are important in long tract axonal maintenance and perhaps of relevance to common neurological diseases. More than 20 HSP loci exist and 9 of the responsible genes have been identified. Until recently, there was no discernable common underlying cellular pathological mechanism for groups of HSP conditions. However, several lines of evidence now point to defects in intracellular traffic as being the underlying cause of several different genetic types of HSP. This evidence includes the known functional role of KIF5A, a gene that I have identified as being involved in HSP, and preliminary experimental data that I have generated that implicate spastin and spartin in intracellular membrane traffic events and suggest a possible functional relationship between them. This proposal focuses on spastin and spartin and its aims are to test the hypothesis that the two proteins are functionally related components of an intracellular membrane traffic pathway that is disrupted when they are mutated. I will test this hypothesis by: a) examining whether the tissue and sub-cellular expression patterns of spastin and spartin overlap, b) further investigating preliminary experimental data that suggest physical interactions between spastin, spartin, and 2 other proteins, CHMP1.5 and gp25L2, that I identified in a yeast two-hybrid screen using spastin as bait, and which are known to be involved in intracellular membrane traffic events, c) examining in cultured mammalian cells whether knocking down expression of spastin or spartin, controlled expression of wild-type spastin or spartin, or controlled expression of mutant forms of spastin or spartin, results in any morphological or functional abnormality of intracellular membrane traffic pathways or components.

Amount: £180,069
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of cardiac myocyte apoptosis in the transition from compensated hypertrophy to dilated cardiomyopathy. 03 Apr 2006

Cardiac myocytes undergo hypertrophy in response to haemodynamic overload, an adaptation that initially maintains systolic cardiac function. Over time, however, the hypertrophied ventricle often becomes thin-walled, dilated and hypocontractile. Mechanisms that mediate this transition are poorly understood but work over the past 5 years has documented increased - yet quite low - levels of cardiac myocyte apoptosis in human hearts with dilated cardiomyopathy (0.1-0.3% vs 0.001-0.002% in controls). Moreover, Kitsis et al have demonstrated in transgenic mouse models that low levels of cardiac myocyte apoptosis (0.08-0.023%) are sufficient to generate dilated cardiomyopathy. In prior work, I investigated the role of heme oxygenase-1 in angiotensin II-induced hypertrophy and apoptosis using cultured neonatal cardiac myocytes. I now wish to continue to study the relationship between cardiac myocyte hypertrophy and apoptosis in an in vivo setting. I propose to test the importance of cardiac myocyte apoptosis in the development of overload-induced compensatory hypertrophy and the transition to cardiomyopathy in vivo. I will compare the molecular, cellular, structural and phyhsiological responses of wild type mice subjected to transthoracic aortic constriction with those of two transgenic models developed in Dr. Kitsis' lab in which cardiac myocyte apoptosis has been potently suppressed. These studies will test whether cardiac myocyte apoptosis plays a causal role in overload-induced hypertrophy and the transition to cardiomyopathy, an important clinical scenario in which the role of cardiac myocyte apoptosis is yet to be defined. Third generation pan-caspase pseudosubstrate inhibitors are currently in Phase I clinical development (Idun Pharm.) and Dr. Kitsis has successfully used one of these to attenuate ventricular dysfunction and dilatation in 2 different genetic cardiomyopathy models. I plan to also perform aortic constriction on wild-type mice and test the effect of this pharmacological agent on the trasition from hypertrophy to cardiomyopathy. Mosst of this work will be carried out in Dr. Kitsis' lab (Albert Einstein College of Medicine, NY) for the first 2½ years of this 3-year fellowship. All animal procedures will be done in Dr. Kitsis' lab and I will continue experiments with animal tissue collected from the aim 3 of the project (Caspase Inhibitor) in Cambridge.

Amount: £126,107
Funder: The Wellcome Trust
Recipient: University of Cambridge

Stem cell differentiation-recapitulation of development? 30 Aug 2006

Stem cell differentiation - recapitulation of development?

Amount: £143,077
Funder: The Wellcome Trust
Recipient: University of Cambridge

Intracellular dynamics of the influenza A virus RNA polymerase and their role in influencing virus host range. 26 Apr 2006

WT Studentship for Ms Agnes Foglein : 4-year PhD studentship in Infection and Immunity. Influenza A virus has a segmented, negative stranded RNA genome. In order to replicate in an infected cell it has to transcribe this genomic vRNA into mRNA (for viral protein expression) and into cRNA; an exact copy that serves as a template for amplification of the genome for new viral particles. To achieve this, influenza virus encodes a viral RNA dependent RNA polymerase. This complex, consisting of 3 proteins - PA, PB1 and PB2 -transcribes all the three RNA species required by the virus: mRNA, cRNA and vRNA. However, to achieve this it needs to interact with the cellular transcription machinery to "steal" the cap of cellular mRNA and use it for viral mRNA. There is much evidence that the viral polymerase genes are a major determinant of host range and pathogenicity. Although all three viral polymerase subunits play an important role and the combination of these also has an influence, PB2 is especially important for host specificity. The amino acid at position 627 seems to be a "signature" for the virus, with avian strains preferentially displaying glutamic acid (Glu) and human strains lysine (Lys). Change from Glu627 to Lys induces severe pathogenicity and increased replication of avian virus in mice. As Shinya et al. show, the single amino acid substitution converts a nonlethal H5N1 virus isolated from man to a lethal virus in mice. However, the molecular mechanisms behind this host range determination are unclear. While cell tropism did not seem to be influenced by the variant of PB2 in a mouse model, the human variant showed accelerated viral spread in infected animals and higher virus titers were measure in cell culture models (Shinya et al. 2004). The aim of the project is to investigate what molecular mechanism lies behind the species specificity conferred by PB2.

Amount: £141,232
Funder: The Wellcome Trust
Recipient: University of Cambridge

The structural biology of cell signalling and regulation: multiprotein systems and the achievement of high signal-to-noise ratios. 27 Apr 2006

The control of cell differentiation, growth and proliferation is mediated by the assembly and disassembly of multiprotein complexes. My group has studied the 3-D structures of many such complexes in order to understand the nature ofthe interactions, organisation and cooperativity; examples are FGFR, human recombinase Rad51, the breast cancer associated gene product BRCA2, and Cdk6 with cell cycle inhibitor p19INK4d. We have shown that pair-wise interactions are often weak and ill-defined but lead to cooperative assembly of multiprotein complexes with well defined structures, so providing better signal-to-noise ratios in signalling. In this proposal I focus on other features that are shared between multiprotein systems at the cell surface, in the cytoplasm and in the nucleus. I plan to investigate 3-D structural features of complexes where: 1. One or more components are disordered prior tobinding. 2. Higher order clustering modulates signal transduction.3. Glycosylation controls receptor interactions. 4. Different assemblies regulatedifferent signalling pathways. In order to achieve these objectives I plan notonly to extend studies on systems where we have worked before, for example FGFR, Met receptor and Xrcc4; but also to initiate work on others where we have only preliminary studies, such as the Notch receptor. The research involves protein expression, purification, characterisation of complexes, crystallisation, X-ray analysis and bioinformatics.

Amount: £1,454,930
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

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

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

Effect of neutrophil priming and de-priming on neutrophil retention in the lung. 06 Dec 2005

Neutrophils become 'primed' after exposure to inflammatory mediators and this greatly enhances their subsequent secretory activity. Priming also alters neutrophil shape/deformability, integrin expression and longevity and hence has a profound affect on their rheological, adhesive and survival properties. Priming is a prerequisite for neutrophil-mediated tissue injury and plays a fundamental role in the pathogenesis of ARDS. Since priming is reversible, I will test whether the pulmonary capillary bed and/or spleen can trap and de-prime systemically-primed neutrophils before releasing them back into the circulation, and whether failure of this function results in neutrophil-mediated lung injury.Specifically, I will address:(i) What is the bio-distribution, preferential disposal site and circulating half-life of primed versus un-primed neutrophils? This will be addressed using 99mTc and 111In-labelled primed/un-primed neutrophils in healthy volunteers.(ii) Can pulmonary capillaries or the spleen retain circulating primed neutrophils, de-prime them and return them to the circulation? I will determine the capacity for neutrophils primed in vivo to de-prime ex-vivo and use flow-based techniques to track the intravascular fate of permanently or transiently primed 111In-labelled cells.(iii) What is the effect of lung vascular (ARDS) and airspace (pneumonia) inflammation on transpulmonary primed neutrophil gradients and does neutrophil transit time and pulmonary retention fraction predict the evolution of ARDS.

Amount: £235,646
Funder: The Wellcome Trust
Recipient: University of Cambridge

Therapeutic approaches for codon reiteration diseases 05 Jun 2006

I aim to develop therapeutic strategies for diseases associated with protein misfolding and intracellular aggregation, focussing on Huntington's disease (HD) and oculopharyngeal muscular dystrophy (OPMD). First, I will develop our findings that levels of mutant huntingtin fragments can be reduced by inducing autophagy. This strategy attenuates toxicity of the HD mutation in transgenic cell, fly and mouse models. Recent work in cell and fly models suggests that autophagy induction is beneficial for a much broader range of targets, including polyglutamine expansions underlying many spinocerebellar ataxias, and tau. Currently, the only autophagy-inducing drug that is known to reduce mutant huntingtin levels effectively in mammalian brains is rapamycin. While it is designed for long-term use, it has significant side-effects. My aims in the context of this component of the proposal are:1. Further the understanding of the machinery and regulation of mammalian autophagy to aid discovery of safer and more specific targets.2. Test autophagy upregulation as a therapeutic strategy for SCA3 and tauopathies in mouse models. Second, I will exploit our OPMD mouse model for testing 4 known safe drugs/compounds in vivo, on the basis of pilot data showing that they reduce aggregation and toxicity in OPMD cell models.

Amount: £1,645,643
Funder: The Wellcome Trust
Recipient: University of Cambridge

Fundamental associative learning processes in psychosis: combined behavioural, functional neuroimaging and psychopharmacological studies. 05 Jun 2006

Delusions are core symptoms of psychotic illnesses such as schizophrenia. Central to our understanding of delusions is the idea that they emerge from a background of abnormal formation of associations. Over the last decade, there has been an increasing understanding of the neuroscience of association formation. This work has emphasised prediction error signal as a drive to associative learning and a guide to allocation of attention and to selection of action. The goal of my proposed work is to test the hypothesis that delusional beliefs arise from changes in association formation as a consequence of abnormal prediction error signalling. In order to do so, I wish to combine behavioural, psychopharmacological and functional neuroimaging studies to:(i) Refine understanding of prediction error-driven associative learning processes in humans(ii) Establish the extent and nature of disruption of these processes, (both in terms of behaviour and brain response) a) in deluded individuals and b) under ketamine administration.(iii) Test directly the possibility of a link between symptoms of psychosis and disturbances in associative learning by evaluating the extent to which these behavioural and brain responses predict the existence or emergence of delusional beliefs (under ketamine and in psychosis).

Amount: £1,028,769
Funder: The Wellcome Trust
Recipient: University of Cambridge

VALUE IN PEOPLE AWARD. 30 Aug 2006

Not available

Amount: £300,000
Funder: The Wellcome Trust
Recipient: University of Cambridge

WELLCOME TRUST CENTRE FOR STEM CELL RESEARCH 20 Sep 2006

The new Institute for Stem Cell Biology in Cambridge will be an international centre of excellence in fundamental stem cell research. The Institute will focus on definition of the genetic and biochemical mechanisms that control stem cell fate, providing foundations for applications in disease modelling, drug discovery and regenerative medicine. This proposal is for provision of core resources for embryonic stem cell manipulation and transgenesis. A central resource of skilled personnel will maximise research productivity and continuity, promote cooperation and synergy, and accelerate technological innovation. Timely and efficient production of customised gene-modified stem cells and mice is essential underpinning. Specialised expertise will support advanced genetic engineering of mouse and human stem cells, and operation of robotic platforms to develop screening methodologies for isolating genetic, protein and chemical regulators. A dedicated PdD programme in stem cell biology will capitalise on the opportunity for high level research training provided by the intellectual environment and core facilities in the Institute. A Strategic Award will immediately establish the Institute for Stem Cell Biology amongst the best-resourced and most attractive environments for stem cell research world-wide, providing a magnet for recruitment, and a much-needed focus for UK and European stem cell biology.

Amount: £6,956,531
Funder: The Wellcome Trust
Recipient: University of Cambridge

Core support for Wellcome Trust/Cancer Research UK Gurdon Instiute. Award for years 4 and 5. 20 Sep 2006

This application requests the continuation of core support to the Wellcome Trust/Cancer Research UK Gurdon Institute. The aim of the Institute, an integrated part of Cambridge University, is to contribute to an understanding of normal animal development, including the processes of cell differentiation, morphogenesis and cell proliferation, and to explain how, should these processes fail, cancers may arise. The Institute attempts to achieve these objectives by recruiting the best scientists and by ensuring that they have the best possible environment for their work. Recruitment is carried out by a committee of Group Leaders, with the main criterion for appointment being scientific excellence. This application is concerned with providing the best scientific environment for our researchers, and indeed the Institute's core support is one of its most important assets, allowing our scientists to spend as much time as possible on their work. This support provides practical assistance, such as media preparation, and secretarial and administrative assistance, including help with grant applications and equipment procurement. It also provides computer staff and a bioinformatician, as well as expertise in confocal microscopy. Core support is essential if the Institute is to maintain its position as a leading centre of cell, molecular and developmental biology.

Amount: £3,005,149
Funder: The Wellcome Trust
Recipient: University of Cambridge
Amount: £155,658
Funder: The Wellcome Trust
Recipient: University of Cambridge