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Award Year:
2010
Currency:
GBP
Recipients:
University of Cambridge

Results

Ultra-Performance Liquid Chromatography Mass Spectrometry for Comprehensive Metabolomics 12 Oct 2010

This application is a request to part fund the upgrade of one of the mass spectrometers used by researchers in Cambridge to conduct open profiling of metabolites, including intact lipids. The requested instrument is a Waters UPLC QToF Xevo which will combine highly reproducible ultra-high pressure chromatography and near exact mass analysis to provide an instrument that will be able to both separate a large number of components and identify these components by a combination of MS and MS^E fragme ntation. The major aims are: 1. To increase sensitivity of the current open profiling LC-MS assays within the Griffin group. This in itself will increase the number of metabolites that can be detected by the instrument, particularly in negative mode where the current Ultima QToF is poor. 2. Allow the use of MS^E and improved software tools for metabolite ID. This will be particularly useful for lipid species where fragmentation produces spectra characteristic of different lipid species. 3. A llow increased use of the instrument by providing a machine that can be used in open access mode for routine LC-MS analysis. Currently one of the biggest bottle necks for collaborators is the availability of experienced users in the Griffin group.

Amount: £177,998
Funder: The Wellcome Trust
Recipient: University of Cambridge

Dissecting compulsivity. 25 Nov 2010

Compulsivity, or the repetition of behaviours despite negative consequences, defines a range of psychiatric disorders from addiction to obsessive-compulsive disorder. Disorders of binge drinking and binge eating in obesity are prominent public health issues. Influential models of addiction, namely that of compulsivity and incentive motivation, define the field of addiction; yet, remarkably, there is a paucity of clinical research to support these models. We tend also to conceptualize habit an d motivation as related to reward and incentive; however, the relationship of the conditioned avoidance response to habit and motivation is not known. The role of dopamine to negative stimuli and this motivational escape response is also poorly understood. The proposal aims to address these questions. To achieve this, I shall adopt a highly innovative approach combining carefully designed novel cognitive paradigms and fMRI in clinical and high risk populations. I will also use combined c omputational modeling and single unit electrophysiology in awake human subjects to assess dopaminergic function to negative stimuli. Empirical data will be used to develop a mechanistic model of the construct of compulsivity and its relationship to factors that may influence its expression such as biological susceptibility, motivation, aversive stimuli and dopamine.

Amount: £698,954
Funder: The Wellcome Trust
Recipient: University of Cambridge

Interactome analysis of the structure of the trypanosome nuclear lamina and other complexes. 05 Oct 2010

Defining function and the role of proteins in biological processes requires information at many levels. Genome sequences facilitate a parts list. A level of functional definition is possible by gene knockdown or knockout strategies. In the African trypanosome these approaches are powerful, but a complete definition of function requires determining the physiological context in which specific proteins operate. Protein interactions cannot at present be reliably predicted by in silico approaches, an d direct evidence is essential. It is proposed to use a new set of techniques to examine protein-protein interactions in trypanosomes. Using a GFP-tagged version of the protein (integrated at the endogenous gene locus), coupled with a supercooled ball mill procedure at -186˚C to generate a fine grindate, protein complexes have been isolated in high yield from total trypanosome cells. It is proposed to use this technology to explore how generally applicable the technique will be by focusin g on several complexes involved in intracellular transport. Proteins will be identified by MS/MS. If, as anticipated, the method is general, this will provide a powerful addition to the analytic methodology available for study of trypanosomes and potentially other (pathogenic) protists.

Amount: £22,800
Funder: The Wellcome Trust
Recipient: University of Cambridge

Finding donor siblings: the family relationships of donor conceived offspring. 21 Sep 2010

The proposed research will contribute to an on-going Wellcome Trust Enhancement Award in Biomedical Ethics entitled "Bioethics, Assisted Reproduction and the Family" for which I am Pl. The Enhancement Award focuses on three family types; gay fathers, solo mothers and donor siblings. The proposed research is central to the study of the third of these family types- donor siblings (donor conceived genetic half siblings raised in different families). By producing empirical data on the experiences of donor siblings who make contact with each other, the study will inform bioethical analysis in relation to the creation of families by donor conception following the removal of donor anonymity. The aim of the project is to conduct an in-depth study of the impact on individuals conceived by donor insemination of finding and meeting their donor siblings. A travel grant would enable me to collaborate with Professor Miriam Steele in New York to finalise the methodology, train the researchers and initiate the sampling procedures. It would also allow us to meet with Professor Robert Klitzman from Columbia University, a Collaborator on the Enhancement Award, to ensure that the data to be obtained will maximise bioethical analysis regarding this emerging family type.

Amount: £3,179
Funder: The Wellcome Trust
Recipient: University of Cambridge

Transgenerational epigenetic inheritance in C.elegans. 13 Jul 2010

Transgenerational epigenetics is the study of changes in gene regulation, inheritable over generations that are not due to changes in the DNA sequence. Although a few examples of epialleles have been observed in nature, and are often associated with inheritable DNA methylation, the molecular mechanisms underlying this alternative type of heredity remain poorly understood. The soil nematode Caenorhabditis elegans exhibits a transgenerational memory of RNAi-instructed gene silencing although this organism does not exhibit DNA methylation. This project aims to determine the mode of transmission of epigenetic marks between generations. We hypothesize that small RNAs will play a role in this process. We will address this question in two ways: 1) We will study the mechanism of transgenerational epigenetic inheritance at a transgene locus, focussing on chromatin and small RNAs as likely candidates. 2) We will determine whether small RNA populations are sensitive to environment and whether this can affect offspring phenotype. A mechanism such as this would enable organisms to fine-tune expression of sensitive genes resulting in a fast, heritable, but reversible response to a changing environment.

Amount: £152,106
Funder: The Wellcome Trust
Recipient: University of Cambridge

evolutionary conservation of group B Sox function in Drosophila. 13 Jul 2010

The primary aims of the project are to: 1) perform a comparative in vivo analysis of the genomic activity of two closely related Sox domain transcription factors in four different Drosophila species; 2) determine the evolutionary conservation of the regulatory network driven by the Sox factor Dichaete in neural cells; 3) investigate the conservation in genome wide chromatin accessibility profiles across 4 species of Drosophila.

Amount: £152,106
Funder: The Wellcome Trust
Recipient: University of Cambridge

Elucidating the molecular networks that regulate polarized cell growth, using functional genomics. 13 Jul 2010

Almost every living cell must maintain some degree of polarity, whether permanent or transient, in order to live and function correctly. There are many obvious examples of cell polarity provided by the mammalian system, including epithelial cells, neurons and photoreceptors. However, the fission yeast Schizosaccharomyces pombe provides one of the simplest and most easily studied examples. The rod shaped cells of this organism have two distinct regions, the hemispherical growing tips - where cells expand, with the addition of components to the cell wall and cell membrane - and the non-growing cell sides. Many factors are known to localise specifically to cell ends and organisation of the growth domains is highly dependent on microtubule-dependent intracellular transport mechanisms. However, our understanding of the complex regulatory networks involved in polarized growth regulation are extremely limited, with many regulators and regulatory principles likely remaining to be identified. New semi-automated methods allowing for high-throughput/high-content (HT/HC) data collection and analysis can be applied to rapidly and systematically look for novel genes involved in the regulation of polarized growth, which can then be used as a starting point to more fully elucidate their entire network. Data has already been collected from one such screen providing several interesting candidates. Additionally, in previous work, the motor protein Klp2 involved in microtubule organization during growth was investigated, with some interesting findings with regards to its in vivo activity. This may produce significant results solving a long-standing puzzle in S.pombe research and clarifying how microtubules are organised during interphase. Objectives 1. To continue investigating the regulation of the microtubule organiser Klp2, in particular its interaction with other regulatory proteins 2. To validate and further investigate candidate genes suggested by the previous screen 3. To identify further genes affecting cell polarity by carrying out a HT/HC screen for regulators of microtubule dynamics

Amount: £152,106
Funder: The Wellcome Trust
Recipient: University of Cambridge

Glycosphingolipid catabolism and transfer in health, disease and immunity. 13 Jul 2010

1) To investigate the molecular mechanism of lipid binding and solubilisation by saposin molecules. 2) To reveal the molecular mechanism by which saposins A and C present substrate and activate GalC. 3) To fully investigate the consequences of known clinical mutations on GalC stability, activity, cellular location/trafficking and interaction with substrate, membranes and saposins. Can these parameters be affected in vitro and in vivo by inhibitor or chaperone binding? (In collaboration with Prof. Paul Luzio) 4) To reveal the molecular mechanism by which saposins load lipid antigens onto CD1d and CD1b molecules. To test the hypothesis that saposins mediate loading of specific myelin-related autoantigens, and determine whether autoreactive T cells or NKT cells exist in patients with Krabbe disease and Multiple Sclerosis (In collaboration with Prof. Timothy Cox).

Amount: £152,290
Funder: The Wellcome Trust
Recipient: University of Cambridge

Innate memory: What is the biological significance ofmemory NK cells in infection and cancer? 13 Jul 2010

Our goal is to study the newly discovered ?memory? NK cells. The three main aims are: 1. Define their lifespan and effector functions 2. Determine their biological significance in viral infections and cancer 3. Characterize their transcriptome

Amount: £152,290
Funder: The Wellcome Trust
Recipient: University of Cambridge

Characterisation of novel long non-coding RNAs in the biology of heart faliure. 14 Jun 2010

Initially, it was thought that just 5% of the human genome was transcribed, around one fifth of which can be accounted for by the 20,000 or so known protein-coding genes, or the messenger RNAs (1 ). However mapping and quantification of the mammalian transcriptome has shown that the number of transcripts encoded by the human genome is at least 10 times as great as the number of identified 'genes' (2, 3) that up to 70% of the mammalian genome is transcribed (4). Much of the uncharacterised transcriptors appears to be composed of long non-coding RNAs (lncRNAs), defined as RNA transcripts greater the 200nt (5), 34 of which have so far been associated with human disease (6). To begin the genome-wide search for candidate lncRNAs associated with heart failure, the Foo lab carried out RNA-seq on RNA isolated from four human control hearts and four diseased hearts. Diseased he are from male subjects, aged 40-60 years with a well-documented medical history. RNA-seq is a high throughput sequencing technique in order to obtain information about the biological system's total RNA repertoire (7). The resulting information is a summary statistic- a form of 'read count' that is linearly related to the abundance of the transcript in the sample. Figure 1 shows a diagrammatical outline of a typical RNA-seq experiment. Verify differential expression of transcripts in a larger sample of human heart tissue. Obtain complete sequence of the non-coding transcript. For the RNA-seq derived RNAs, does the transcript reside in myocytes or fibroblasts of cardiac tissue? Ensure that the transcript of interest does not yield a product of translation. What happens when the transcript is overexpressed and repressed in wild type cardiomyocytes or fibroblasts in culture? Is the Inc RNA of interest upregulated in response to stress triggers? What are the interactors of a novel Inc RNA?

Amount: £152,006
Funder: The Wellcome Trust
Recipient: University of Cambridge

The relationship between lineage and connectivity in the mouse cerebral cortex. 15 Feb 2010

The mechanisms by which cortical circuits are assembled remain largely unclear. Recent evidence has suggested that the excitatory neurons of the cortex preferentially form synapses with clonally related cells, hinting at a potential mechanism for circuitry formation. The goal of my project is to answer the following questions: 1. Do clones of cortical neurons form ordered synaptic connections in vitro? 2. Do clones of cortical neurons respect areal or columnar boundaries in vivo? 3. Do clonally-related cortical neurons preferentially form connections with sibling neurons in vivo? 4. Time permitting, test candidate cellular and molecular mechanisms that underlie this phenomenon.

Amount: £36,245
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of plasmalogenphospholipids in obesity and insulin resistance. 15 Feb 2010

General aims: To investigate the pathogenic role of plasmalogen depletion in the development o he Metabolic Syndrome. Soecific aims: 1) To elucidate the effect of plasmalogen depletion on energy balance, lipid and carbohydrate metabolism. 2) To characterise the metabolic effects of tissue specific plasmalogen depletion in key metabolic organs such as adipose tissue. 3) To determine the therapeutic value of replenishing plasmalogens in models of obesity and insulin resistance. ' 4) To identify the metabolic pathways altered in the context of plasmalogen depletion using a systems biology approach.

Amount: £53,487
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of DNA methylation in heart failure 18 Jan 2010

To investigate if hypertrophic stimuli alters Dnmt3b expression, in vitro and in vivo. To investigate in vitro, the role of altered DNA methylation on cardiac myocy1e and fibroblast survival, cell cycle, proliferation and senescence. To investigate in vivo, the role of altered DNA methylation on cardiac hypertrophy, dilatation and angiogenesis.

Amount: £29,934
Funder: The Wellcome Trust
Recipient: University of Cambridge

The epidemiology of glaucoma. 21 Sep 2010

Glaucoma is one of the leading causes of blindness worldwide, second only to cataract. However, unlike cataract, visual loss from glaucoma is irreversible.Uncorrectable loss of vision is associated with threats to independent living,safety and emotional well-being, resulting in increased dependence on social and community services. It has been estimated that if 10% of glaucoma sufferers received earlier treatment that arrested significant visual loss, the United Kingdom economy would benefit by £555 million. Currently, intraocular pressure (IOP) is the only known modifiable risk factor for glaucoma, and all treatment modalities are aimed at reducing IOP. Identification of lifestyle factors associated with glaucoma may enable publichealth strategies as well as new treatments to help prevent this disabling condition. EPIC-Norfolk is an established population study which this project will form part of. Extensive lifestyle data from multiple time points are available and the last health check included a comprehensive ophthalmic examination. The primary goals of this project are to: - Update the definition of glaucoma- Describe IOP, optic nerve measures and glaucoma in the EPIC-Norfolk cohort -Determine nutritional, lifestyle and biological measures associated with the presence of glaucoma and associated physiological measures.

Amount: £361,924
Funder: The Wellcome Trust
Recipient: University of Cambridge

Wellcome Trust PhD Programme for Clinicians at the University of Cambridge: Investigating rare copy number variants (CNVs) associated with severe early-onset obesity. 14 Jun 2010

Body weight and fat mass are highly heritable traits. I plan to investigate copy number variants (CNVs) that have been identified in a cohort of patients with severe early onset obesity established by Dr Farooqi at the University of Cambridge. My first goal is to identify CNVs of interest by prioritizing those present in multiple affected individuals and enriched in cases versus controls. Bioinformatic databases will be used to identify any prior information known about whether the genes may be associated with obesity. Multiplex ligation-dependent probe amplification (MLPA) will validate the CNVs and a fine-mapping custom array will attempt to identify the breakpoints. Co-segregation in families may suggest potential pathogenicity. In addition, working with Dr Barroso's team at the WTSI, I will sequence potential genes contained within these CNVs to identify whether rare variants are also associated with severe early onset obesity. I intend to establish whether the zebra-fish can be used as a model organism to study new candidate genes for obesity. Mutations in the melanocortin 4 receptor (MC4R) gene can cause severe obesity in humans. In collaboration with Dr Stemple's group at the WTSI, I plan to characterise the phenotype of an MC4R mutant fish that has been identified and in doing so, develop the techniques for studying other obesity candidate genes in this model organism.

Amount: £4,373
Funder: The Wellcome Trust
Recipient: University of Cambridge

Wellcome Trust PhD Programme for Clinicians at the University of Cambridge: 'Natural and modified variations in human induced pluripotent stem cells'. 14 Jun 2010

Human induced pluripotent stem cells (hiPSCs) have revolutionized stem cell biology and have renewed aspirations of moving the field towards clinical therapies. By genetic manipulation, fibroblasts have been shown to re-program to a pluripotent state similar to human embryonic stem cells (hESCs). This would allow for patient specific hiPSCs, generating models for disease as well as immune compatible cell therapies. A major problem in the field is heterogeneity in the derived hiPSCs both in derivation as well as differentiation. Additionally it is unknown whether hiPSCs may be made using cells other than fibroblasts and if these hiPSCs retain an ?epigenetic? memory following differentiation. The knowledge gained from the basis of hiPSC variability will be used to optimise strategies towards clinically relevant therapies. Key goals: 1) Generate hiPSCs from different human tissues 2) Compare hiPSC lines derived from the same individual and so same genetic background 3) Identify new lineage-specific markers through gene expression arrays 4) Explore the differentiation capacity and bias of the different hiPSC lines 5) Investigate epigenetic ?memory? in derivation of hiPSCs and subsequent differentiation 6) Use knowledge of the basis of variability to optimise differentiation strategies 7) Gain robust scientific training in stem cell biology and genetics

Amount: £30,392
Funder: The Wellcome Trust
Recipient: University of Cambridge

Signalling in T-cell immunity 10 Jun 2010

An immune response against opportunistic infections, foreign transplants and in autoimmune disorders is critically dependent on the generation of intracellular signal by the antigen-receptor (TcR?/CD3) and the co-receptors (CD28, CTLA-4) on the surface of T-cells. Key mediators of signalling include CD4/CD8-p56lck complexes, the tyrosine kinase ZAP-70 and a novel family of immune specific adaptor proteins. In this application, we propose to extend our studies on the nature of protein-tyrosine kinases and adaptors that regulate cytokine production in T-cells. Specifically, we propose to focus on the adaptors FYB and SKAP-55, their phosphoryation sites, interactions with other proteins, nuclear vs. cytoplasmic function and their regulation of thymic differentiation and peripheral T-cell function. Likewise, we propose to elucidate the molecular mechanisms that regulate CD28 positive vs. CTLA-4 negative signalling, and their connection with the TcR?/CD3 complex. Ultimately, we hope to provide insights that will allow for the development of therapies designed to enhance immune responses against infectious agents, and dampen inappropriate responses in autoimmune disorders.

Amount: £320,175
Funder: The Wellcome Trust
Recipient: University of Cambridge

Pharmaceutical ethics beyond distributive justice. 18 Jan 2010

The aims and objectives of this project are threefold. Firstly, it aims to convey the conclusions of my current WT-funded research on Needs, Rights, and Preferences in Pharmaceutical Ethics to key stakeholders in healthcare, including policy makers. Secondly, it seeks to inform policy and practice and to stimulate further discussion. Thirdly, it pertains to a broader project of both reengaging academic philosophy with timely social concerns and making philosophical arguments accessible to wide r audiences. The project involves two stages: 1. preparing and disseminating outreach materials, e.g. a concise summary of key arguments, terminology guides, and annotated bibliographies; and 2. organising and running a series of approximately 8 seminars and two half-day workshops for relevant non-academic audiences both at Cambridge University and on the premises of interested organisations. These dissemination activities will draw on the outputs of my current research on the philosophic al presuppositions that underlie conflicting distributive claims in pharmaceutical ethics. The proposed methodology is Socratic engagement. It helps participants expand their own critical reflection and does not require prior knowledge of philosophical terminology. The anticipated impact is to further pertinent policy and practice, philosophically inform key healthcare stakeholders, and encourage further debate within the larger community.

Amount: £29,908
Funder: The Wellcome Trust
Recipient: University of Cambridge

Foetal testosterone effects on brain development. 01 Jul 2010

In this application we test if foetal testosterone (fT) (produced at significantly higher levels in males) exerts organisational effects on human brain development. Whilst fT has been demonstrated to exert organisational effects on brain development in other animals, this hypothesis has never been directly tested in humans. We have conducted a unique study where we measured fT prenatally in amniotic fluid and followed the children from birth to predict behavioural development longitudinally. Our research thus far has shown that fT is a key predictor of various social and communicative behaviours. We now have a valuable opportunity to employ neuroimaging with this sample to examine the underlying effects of fT on brain development. Our primary aims are: (1) To test if foetal testosterone (fT) and/or current testosterone (cT) correlate with volume of the whole brain and/or with volume of specific sexually-dimorphic brain regions, using structural MRI. (2) To test if fT and/or cT co rrelate with brain activity in these sexually-dimorphic brain regions, during social-emotional processing using functional MRI. (3) To test if fT and/or cT correlate with measures of neuroanatomical connectivity using diffusion tensor imaging (DTI).

Amount: £289,602
Funder: The Wellcome Trust
Recipient: University of Cambridge

TRC8, a novel ERAD ubiquitin E3 ligase in the regulation of MHC class I and other substrates. 08 Feb 2010

We will characterize the role of TRC8, a novel ERAD E3 ligase. Our recent identification of TRC8 as the E3 required for the US2-mediated dislocation of MHC I implies a role for TRC8 in the ERAD pathway. We showed TRC8 associates with signal peptide peptidase (SPP) and these enzymes form the core components of a novel ERAD complex. Together with the observations that loss of TRC8 leads to the development of renal tumours, our findings show TRC8 is an important human ERAD ligase which requires fu rther investigation. Our characterization of TRC8 will determine the functional requirement for the TRC8 sterol sensing domain, identify additional cellular components of the TRC8/SPP complex and determine whether SPP s protease activity is essential for protein dislocation. Preliminary data from mass spectrometry analysis identified the multifunctional FKBP38 protein as a new TRC8 substrate. FKBP38 regulates the PHD2 oxygen sensor, and we will therefore determine whether TRC8 regulation of FKBP 38 affects PHD2 stability, activity of the hypoxia-inducible transcription factor (HIF) and explains how loss of TRC8 causes renal cancer. Additional TRC8 substrates will be identified using SILAC-based quantitative mass spectrometry from different cell types. These studies will provide insight into the functions of this clinically important ligase.

Amount: £205,398
Funder: The Wellcome Trust
Recipient: University of Cambridge