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Results

An asymmetric and imprinted chromosome segregation mechanism organizes the bacterial nucleoid. 27 Jun 2006

Chromosome organization, replication and transmission to daughter cells underlie genome integrity and the life process. New microscopic methods have revolutionized understanding of chromosome dynamics in bacteria, and have revealed an unprecedented choreography of gene and macromolecular component position in time and in space. In recent work from the applicant s laboratory, an unexpected asymmetry in the chromosome segregation process has been revealed by studying the positioning and segregatio n of 20 different genetic loci in pairwise combinations; markers duplicate in the central region of the cell, where the replication machinery is located, with one sister segregating towards a cell pole and its sister remaining closes to midcell. This generates a translation symmetry [L-R; L?R ] organization of sister chromosomes. Underlying this asymmetric segregation is an imprinting process that directs the asymmetry in the same direction through generations. The purpose of this proposal is to provide RA1A funding for an ex-Wellcome Prize Student whose pioneering D.Phil work led to the demonstration of asymmetric segregation. By using a combination of genetics and biochemistry, in combination with microscopy, this work will reveal the molecular mechanisms responsible for chromosome organization and asymmetric imprinted segregation

Amount: £228,129
Funder: The Wellcome Trust
Recipient: University of Oxford

Role of KATP-channels in the control of glucagon secretion from human and rodent pancreatic islets. 11 Jul 2006

The control of glucagon secretion from the a-cells of the pancreatic islets ispoorly understood. Both paracrine (due to substances released from neighbouring cells) as well as intrinsic processes (i.e. within the a-cell itself) have been proposed to be of importance. Here we shall apply a combination of biochemical measurements of glucagon release, biophysical experiments (patch-clamp and capacitance measurements) and confocal [Ca2+]i-imaging to rodent and human pancreatic islets. We shall: 1) test the hypothesis that the intrinsic regulation of glucagon secretion depends on closure of a-cell KATP-channels; 2) establish the ion channel complement of the a-cell, which determines the electrical as well as secretory responses to the membrane depolarization resulting from closure of the KATP-channels; 3) assess the importance of the intrinsic mechanism(s) vs. paracrine processes inthe control of glucagon secretion by experimental interference with critical signalling pathways (e.g. insulin, somatostatin, Zn2+, GABA); and 4) investigate the relative roles of KATP-channels and the cAMP effector proteinsPKA and cAMP-GEFII in the modulation of glucagon secretion by adrenaline and GLP-1; both agonists acts by increasing cAMP and yet they have opposite effects (stimulation by adrenaline and inhibition by GLP-1) on glucagon secretion.

Amount: £196,104
Funder: The Wellcome Trust
Recipient: University of Oxford

Structural studies of the paxillin interaction network and its role in focal adhesion dynamics and signalling. 27 Apr 2006

We propose to extend our recent structural studies (Arold et al., 2002; Hoellerer et al., 2003) to generate a dynamic picture of the interactions of the focal adhesion (FA) proteins FAK, ILK and actopaxin with the adaptor protein paxillin. We will define the role these interactions play in recruitment and activation of proteins to FAs as well as their eventual exclusion during FA disassembly. This picture will be developed at resolutionsthat extend from the atomic to the cellular level. Atomic details will be provided by NMR and X-ray crystallographic imaging of individual functional domains in complex with different binding partners. Molecular interactions will be characterised by studying binding events by NMR, isothermal titration calorimetry and surface plasmon resonance, using different fragments of FAK, ILK, actopaxin, paxillin, and GRB2. In particular, we will define which interactions are mutually permissive or mutually exclusive, how they are affected by phosphorylation, and we will seek to relate this information to the dynamic process of FA turnover. Models of molecular interactions developedthrough these studies will be tested in a cellular context by expression of suitable constructs in cultured cells, followed by fluorescent microscopy and cell adhesion and migration studies.

Amount: £200,690
Funder: The Wellcome Trust
Recipient: University of Oxford

Staphylococcus aureus invasion of host cells: the role of multiple fibronectin-binding sites, integrin-clustering and caveolae in invasion. 24 Apr 2006

Staphylococcus aureus is a major human pathogen. With strains emerging worldwide that are resistant to all antibiotics, it is imperative that novel strategies are developed to combat infections. The ability of any pathogen to attach to its host is critical to a successful infectious cycle. S. aureus is well equipped for this, expressing several proteins that allow it to attach to and invade host cells. The fibronectin binding-protein A (FnBPA) is one of S. aureus' major adhesins that also facilitates invasion of host cells. This study will determine the molecular interactions that occur between FnBPA and host cells to facilitate invasion by this pathogen. Key Goals: (1) Determine the role of multiple binding sites within the FnBPA protein. (2) Detect whether integrin clustering occurs after the bacteria and host cells interact. (3) Observe whether these integrin clusters migrate towards caveolae on the host cell surface. (4) Determine whether invasion is dependent upon the presence of caveolae on the host cell. Our hypotheses will be tested from both the bacterial and host cell perspective, using molecular and cellular biological techniques with a view to identifying novel strategies for the prevention and treatment of invasive diseases caused by this pathogen.

Amount: £151,213
Funder: The Wellcome Trust
Recipient: University of Oxford

New assessment procedures for surgical treatments for presbyopia. 30 Aug 2006

Accommodation is the process by which images of objects at different distancesare brought into focus by a change in power of the crystalline lens. Accommodation amplitude declines with age leading to the condition of presbyopia, in which by the fifth decade of life accommodation becomes minimal. Various experimental surgical procedures to treat presbyopia have been recently proposed. Although these procedures all aim to increase the amplitude of accommodation in presbyopes, they seek to achieve this by a variety of means. This research investigates the relative effectiveness of these competing potential treatments. It also seeks to develop procedures to assist in the optimisation of any techniques that may be shown to be effective. Much of the research will be based on the use of finite element modelling. Recent work by the applicants highlights shortcomings in current data on lens stiffness. New lens stiffness data will therefore be collected. The literature abounds with poorly supported and contentious claims on the basis of which members of the public are undergoing ocular surgery. This project will provide a more secure basis on which to assess proposed presbyopia treatments. While there is considerable commercial interest in this area, our aim is to develop impartial assessment procedures; independent funding is therefore essential.

Amount: £92,427
Funder: The Wellcome Trust
Recipient: University of Oxford

Developmental trajectories of unimodal and cross-modal attention deficits: the case of fragile X syndrome. 10 May 2006

Fragile X syndrome (FXS) is caused by the silencing of a single gene affectingmultiple aspects of cortical development. In late childhood and adulthood, FXSis associated with striking inattention and hyperactivity. Our previous work revealed attentional deficits in toddlers and infants with FXS, highlighting very early onset of difficulties. Hitherto, deficits have been investigated using visual stimuli, but clinical evidence points to difficulties in other modalities, such as audition. Critically, attentional deficits in FXS may be exacerbated by the requirement of selecting multimodal real-world stimuli. Theprimary aim of the current proposal is therefore to investigate the impact of FXS on early developmental trajectories of visual and, for the first time, auditory and crossmodal attention, combining genetics, cognitive developmentaland psychophysical methods. More specifically, we aim to test the following hypotheses: i) developmental trajectories of unimodal and crossmodal attentionin fragile X syndrome will increasingly deviate from normal over early childhood (Goal A); ii) tasks resulting in conflict across modalities will

Amount: £341,196
Funder: The Wellcome Trust
Recipient: University of Oxford

Identification of CD8 epitopes in cattle pathogens by systematic synergistic analysis of peptide specificity and 3D structures of cattle MHC class I. 24 Apr 2006

Research into the immune systems of cattle and other economically important species focuses on disease control. Vaccines which can protect cattle against important pathogens are often only partially effective as they often do not generate a cellular immune response. Thus there is an increasing effort to determine MHC restricted T cell epitopes. Of farm livestock, cattle MHC has been relatively well-characterised, although at the molecular/genome level, this is still some way behind human or mous e MHC. Immunoinformatic epitope prediction tools allow us to use the wealth of pathogen genomes now available without the need for labour-intensive, time-consuming, and expensive large-scale in vitro assays. Our objective is to develop in silico tools which can accelerate cattle vaccine discovery through the rapid identification of epitopes. 1. To obtain crystal structures for at least two cattle MHC class I molecules, each bound to one or more self or pathogen-derived peptides. 2. To gene rate detailed peptide binding data for at least two cattle MHC class I molecules using several in vitro binding assays. 3. To develop robust in silico prediction methods using data generated in 1 and 2. 4. To verify our predictions using bovine TB, RSV and FMDV functional in vitro assays.

Amount: £257,465
Funder: The Wellcome Trust
Recipient: University of Oxford

Molecular mechanisms underlying activation and regulation of ADP-ribosyl cyclases and their functional role in generating cADPR and NAADP. 23 Feb 2006

Intracellular calcium mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mediate many important physiological processes. Both cADPR and NAADP are believed to be generated by ADP-ribosyl cyclase (ARC) enzymes. However, information on how these enzymes are regulated by extracellular stimuli is almost completely lacking. Sea urchin egg homogenates represent a robust cell-free calcium release assay for biochemical reconstitution of endogenous signalling pathways. In this project we will characterize recently identified sea urchin ARC(s), enabling detailed biochemical dissection of ARC regulation and identification of ARC-associated proteins that mediate signalling cascades regulating ARC activity. By generating molecular tools that activate or inhibit ARC signalling components, we will identify ARCs' functional role during sea urchin fertilization and embryogenesis and gain general key insights into their mode of regulation. A central goal in this respect is to extend our findings to identify how mammalian ARCs are regulated during important physiological processes. We will identify those proteins that interact with mammalian ARCs in exocrine pancreatic cells and thus uncover the signalling pathways regulating them. We will also use a variety of approaches to functionally inhibit ARCs in these cells and thus uncover their functional role in the exocrine pancreas.

Amount: £257,375
Funder: The Wellcome Trust
Recipient: University of Oxford

Cognitive systems in the brain: modelling invariant object recognition in natural visual environments. 27 Feb 2006

We will build on recent neurophysiological discoveries on inferior temporal visual cortex neuronal activity to develop understanding at the computational neuroscience level of the mechanisms that underlie our ability to recognise objects in natural scenes, independently of their view, size, and exact position. Novel goals include: 1. We will incorporate our discoveries on the receptive fields of neurons in natural scenes into a refined model to show howinvariances can be learned, and how multiple objects can be recognised, in natural scenes. 2. We will incorporate a new 'continuous transformation' mechanism for invariance learning that we have recently discovered. 3. Becausethe spatio-temporal statistics of the world interact with the functional architecture and dynamics of the visual system, we will use 3D modelling toolsto generate training images with spatio-temporal statistics that are quantitatively defined but also increasingly approach those present when natural scenes are viewed. 4. We will correspondingly add realistic dynamics to the system by introducing integrate-and-fire neurons to some simulations.

Amount: £191,564
Funder: The Wellcome Trust
Recipient: University of Oxford

Pneumococcal vaccine escape: genome-wide characterisation of recombinants arising after vaccine implementation. 09 Feb 2006

Streptococcus pneumoniae (the 'pneumococcus') is one of the leading causes of childhood morbidity and mortality worldwide. A heptavalent conjugate vaccine (hereafter 'vaccine') is highly effective, but limited in its serotype coverage. There is evidence from large-scale national surveillance in the USA (the first country to implement the vaccine) that both pre-existing strains and vaccine-escape mutants of non-vaccine serotype 19A are emerging. These novel escape mutants, as determined by MLST, appear to originate from recombination between a vaccine serotype 4 and a non-vaccine serotype 19A. The expertise of the Centers for Disease Control (CDC) Atlanta, USA and Oxford University will be combined in this study. The CDC is conducting the largest ongoing surveillance of invasive pneumococcal disease post-vaccine implementation anywhere in the world; the Oxford investigators provide access to new high-throughput genome-wide sequence techniques, new methods for phylogenetic analysis and knowledge of pneumococcal molecular epidemiology. This investigation will use Affymetrix re-sequencing arrays and microarray analysis to compare pre- and post-vaccine genotypes with type 4 and 19A capsules to the 19A vaccine-escape recombinants. The data will be analysed to determine the genetic basis by which these recombinants have evolved and to predict possible future changes among pneumococci under vaccine selective pressure.

Amount: £272,904
Funder: The Wellcome Trust
Recipient: University of Oxford

Functional characteristics and associated structural features of mammalian melanopsin photopigments 27 Apr 2006

Melanopsin (a member of the opsin family of G protein coupled receptors) is thought to be the photopigment of recently discovered inner retinal photoreceptors in mammals. To allow direct functional analysis of this protein we have lately developed methods for its heterologous expression in cell culture and shown that, under these conditions, it binds retinal to form a photosensory complex with the ability to regulate cell activity via G-protein signalling. Here we propose building on our heterologous expression breakthrough to conclude a detailed functional and structural analysis of this protein based around 3 goals.Goal 1. To consolidate our functional analysis of human and mouse melanopsins using spectroscopic, physiological and biochemical approaches to elucidate aspects of its photochemistry (especially its spectral sensitivity and photoactivation) and G protein coupling.Goal 2. To generate an accurate 3D structure of melanopsin, based initially upon an empirical computer model, and refined according to experimental examination (cf goal 3) and 2D/3D crystallography.Goal 3. To establish critical structure:function relationships for melanopsin by using our developing structural understanding to define suitable site directed mutants and exposing them to detailed functional analysis.

Amount: £73,220
Funder: The Wellcome Trust
Recipient: University of Oxford

Structural studies of the human Notch-1 ligand-binding region. 21 Feb 2006

During development it is essential that cells are guided to their correct location within the embryo and that they differentiate into the appropriate cell type. A key short-range signal which has been highly conserved during metazoan evolution involves the Notch receptor which regulates spatial patterning, timing and outcomes of numerous cell fate decisions. A structural understanding of the Notch receptor -ligand interaction, which is currently lacking, would greatly enhance our understanding of the signalling mechanism and may lead to the design of novel agonists and antagonists of the receptor. With the current interest in cell-based therapies, manipulation of the Notch signal represents a promising strategy to produce defined cell types in vitro for therapeutic use. In this application we aim to i) crystallise and determine the individual atomic structures of the ligand binding region of human Notch-1 (hN-1) and the receptor binding portions of its ligands human Delta-like 1 and human Jagged-1 ii) identify the structural basis for the observed inhibitory effect of hN-1 EGF10 on ligand binding and the mechanism by which post-translational modification overcomes this effect iii) identify the stability of a Notch-ligand complex by SPR, and determine its atomic structure by X-ray crystallography and/or NMR shift mapping.

Amount: £184,579
Funder: The Wellcome Trust
Recipient: University of Oxford

Dissecting the role of nucleo-mitochondrial interactions in mtDNA diseases. 17 Oct 2005

The key goals are to identify and characterise nucleo-mitochondrial interactions that are important in mtDNA diseases. Mitochondrial proteins encoded by nuclear DNA which interact directly or indirectly with mitochondrial DNA (mtDNA) can cause defects in mtDNA maintenance in both humans and yeast. MtDNA depletion syndrome (MDS) is one such severe disorder,affecting liver and muscle. MDS demonstrates that nucleo-mitochondrial interactions are central to normal cell function. We will test the hypothesisthat these interactions are important in diseases associated with aging, such as diabetes, that are linked to variants both in mtDNA and in nuclear genes causing MDS.We will:1. determine whether mtDNA replication is abnormal in tissues and cells from patients with MDS and with diabetes linked to a mtDNA variant. 2. investigate mtDNA synthesis and organisation in cell lines from MDS patients. 3. identify candidate genes, implicated both by proteomics and by their known phenotype in yeast, that can correct defects in organisation of mtDNA in complementation experiments. These studies will improve diagnosis and genetic counselling for MDS, and will pave the way to rational therapies for mtDNA diseases.

Amount: £368,767
Funder: The Wellcome Trust
Recipient: University of Oxford

Testicular function in mice lacking sertoli cell androgen receptors and FSH receptors. 07 Nov 2005

Testicular differentiation, development and adult function are critically dependent on the Sertoli cells. These cells line the seminiferous tubules and regulate and support germ cell proliferation and differentiation. While it is known that Sertoli cells and spermatogenesis are regulated by follicle stimulating hormone (FSH) and androgen, the relative contribution of each hormone to Sertoli cell activity has remained largely unknown. The recent introduction of relevant mouse models, particularly the FSH-receptor-null (FSHRKO) mouse and the Sertoli cell specific androgen receptor-null (SCARKO) mouse, have provided the potential to dissect out the hormonal mechanisms which regulate Sertoli cell function. We propose to develop mice lacking androgen-receptors on the Sertoli cell AND FSH-receptors or circulating gonadotrophins. This will allow us to determine the extent of Sertoli cell dependence on hormonal stimulation and will show the overall effects that hormones have on Sertoli cell function. Sertoli cell development, and function will be measured in these animals using morphometric techniques and

Amount: £265,649
Funder: The Wellcome Trust
Recipient: University of Oxford

A contribution to the refurbishment of laboratory space at the Sir William Dunn School of Pathology for Prof Robertson to facilitate her move from the WT Centre for Human Genetics. 20 Sep 2006

We are studying the complex cellular interactions underlying early mammalian development, and in particular the signals provided by members of the TGFß superfamily of secreted growth factors. We have previously shown that nodal activity is required for primitive streak formation and anterior patterning during gastrulation, and at later stages for establishment of the L/R body axis. We also described an essential function contributed by the Smad2 protein, the intracellular effector downstream of TGFß/activin receptors. The proposed experiments studies further dissect nodal contribution(s) during morphogenesis of the mouse organiser and establishment of the L/R body axis. We will use gene targeting techniques to selectively delete enhancer sequences controlling asymmetric and node specific nodal expression domains respectively. Using a novel in vivo cell marking system we plan to describe mesodermal subpopulations derived from the node and its progenitors. We will also test the role of the Smad2 pathway during node morphogenesis. Overall the proposed experiments should provide a clearer understanding of TGFß signalling pathways during mouse development.

Amount: £200,000
Funder: The Wellcome Trust
Recipient: University of Oxford

Analytical and empirical approaches in large-scale association studies. 29 Jun 2006

The proposed research will focus on design and power aspects related to the implementation and analysis of genome-wide association (GWA) studies. Initial goals will be (a) to evaluate the relationship between tag-set selection and downstream analysis methods, and (b) to identifyoptimal strategies for combining results and defining replication across GWA scans. Both represent unresolved issues of clear relevance to ongoing GWA studies. These studies, which extend my previous work in this field, will make use of both simulated and empirical large-scale datasets. A subsequent goal will be application of the findings to the analysis and interpretation of large-scale association studies being conducted in the sponsoring department (including the International type 2 Diabetes 1q Consortium and Wellcome Trust Case Control Consortium). In the later stages of the Fellowship, I would aim to address further related issues, including second-stage design and optimal analysis strategy selection. On a personal level, the aim of the Fellowship period willbe to establish myself as an independent researcher within the field of statistical genetics. To further my personal development, and to realise the research goals, I will spend part of the Fellowship with collaborating groups in the USA (at the Broad, and University of Michigan).

Amount: £492,390
Funder: The Wellcome Trust
Recipient: University of Oxford

Premotor networks in the human brain: anatomy, physiology and modulation in disease. 13 Dec 2005

This project aims to develop novel neurorehabilitation strategies to promote motor recovery after stroke. It is focused on the human premotor cortex (PMC), a region shown to be critically involved in recovery of movement ability after stroke. I propose to study the anatomy, physiology and functional connectivity of PMC circuits in the healthy human brain, to test for changes in these circuits after stroke, and to assess the long term effects of modulating plasticity in PMC. My hypothesis is that by modulating activity in PMC we will enhance the effects of experience-dependent learning and plasticity in healthy controls and the beneficial effects of rehabilitative therapies in patients after stroke.In the process of addressing this clinical question I will also investigate the basic anatomy and physiology of PMC in the healthy brain. In particular, I will characterise the regional anatomy of the human premotor cortex using information on brain connectivity derived from diffusion MRI. Further, I will test the functional connections between PMC and other brain regions using paired-pulse TMS and FMRI - in particular to look for hemispheric dominance effects, changes in functional connectivity of PMC after stroke, and task-dependent modulations in PMC circuits.

Amount: £473,189
Funder: The Wellcome Trust
Recipient: University of Oxford

Corticostriatal control over hippocampal and amygdala-dependent appetitive learning and memory systems. 13 Dec 2005

There is growing evidence that multiple, interactive learning and memory systems provide the basis of behaviour, and that disruptions or imbalances within these systems result in the manifestation of abnormal behaviour. Considerable effort is currently being directed at characterising the key neural structures comprising these systems, and this project will investigate the neural and neurochemical basis of the competitive interaction between hippocampal and amygdala-dependent learning and memory processes in rodents, using a systems and multidisciplinary approach. I hypothesise that this interaction occurs in regions of the corticostriatal system that receive converging inputs from the HPC and amygdala, and is modulated by dopaminergic and glutamatergic systems. The first set of experiments will validate the causal roles of the ventral and dorsal HPC in appetitive conditioning and establish the functional connectivity of specific limbic-accumbens pathways using selective permanent or temporary lesions and electrophysiological recordings. The neural loci of interactions between HPC and BLA-mediated information processing will then be investigated using reversible inactivationand in vivo voltammetry targeting the prelimbic-infralimbic cortical regions, orbitofrontal cortex, and rostral pole of the NAc. Finally, the neurochemical mechanism of the competition will be established using intra-cerebral infusions of neuropharmacological agents manipulating the dopaminergic and glutamatergic systems.

Amount: £465,427
Funder: The Wellcome Trust
Recipient: University of Oxford

Proliferation and death of T-cell subsets following vaccination. 12 Jul 2006

The kinetics of T-cell proliferation and death following vaccination in humansare poorly understood. There is an immense effort underway to develop a new generation of vaccines to generate protective T-cell responses against a number of key pathogens responsible for morbidity and mortality on a massive scale. A greater understanding of the processes that lead to effective and durable memory could have a major impact on the design of new vaccines and theimmunization regimes, perhaps allowing development of new adjuvants that enhance survival of memory T-cells following the initial 'burst' response.Labelling dividing cells with deuterium presents a new, safe, radiation-free method of measuring kinetics of antigen-specific T-cells and their subsets. I propose to use this technique to measure T cell responses induced by the tuberculosis vaccine MVA85A in eight healthy BCG-primed volunteers.I will also study apoptosis of vaccine-induced T cells following vaccination with new malaria and tuberculosis vaccines using an apoptosis assay and caspase activation assay. This research will utilise samples from twelve volunteers in upcoming studies in the Oxford clinical trials programme.I also plan to use anti-apoptotic agents in vitro to try modifying this process to increase the size of the resulting memory pool.

Amount: £242,111
Funder: The Wellcome Trust
Recipient: University of Oxford

The pathophysiology of rickettsial disease in Southeast Asia. 23 Mar 2006

The rickettsioses are clinically important but understudied arthropod-borne infections common in the Asia-Pacific region. Scrub typhus (caused by Orientia tsutsugamushi), and murine typhus (Rickettsia typhi) are the most common rickettsial illnesses in the region, although other rickettsial diseases are also seen. An estimated 1 billion individuals are exposed to scrub typhus and 1 million cases occur each year. This may be the most prevalent yet neglected treatable infection in the world. Scrub and murine typhus have different arthropod vectors, epidemiology, clinical manifestations and death rates, though intracellular growth within vascular endothelial cells is a feature common to both. Ongoing clinical treatment trials conducted by our unit provide a unique opportunity for studying the epidemiology and pathophysiology of these poorly understood diseases. The proposed study aims to facilitate the diagnosis of rickettsial pathogens causing disease in Thailand and Laos, and to elucidate the pathological role of the infected endothelial cell and its interactions with the immune system in the disease process. I will study host-pathogen interactions and their consequences in a large cohort of patients with rickettsial disease, predominantly examining the immunpathology both locally in the eschar with skin biopsies, and systemically, by using surrogate markers of endothelial cell dysfunction.

Amount: £338,790
Funder: The Wellcome Trust
Recipient: University of Oxford