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Results

Insights into the molecular mechanisms and dynamics of translocation through SecYEG: an approach using ensemble and single molecule techniques. 22 May 2006

1.    Insights into the molecular mechanisms and dynamics of translocation through SecYEG: an approach using ensemble and single molecule techniques Signal sequence-bearing secretory pre-proteins, and integral membrane proteins are translocated either through the inner membrane in bacteria or the endoplasmic reticulum in eukaryotic cells. The main secretory pathway in Escherichia coli involves the SecYEG translocon, which consists of three integral inner membrane proteins: SecY, which forms the protein channel, SecE that forms a clamp around the complex and SecG. A plethora of studies demonstrated that SecYEG undergoes large and complex sequential intramolecular conformational changes upon binding of partner proteins (e.g. SecA or ribosome) and translocation of secretory proteins. However, the nature of the open and closed states, the oligomeric organisation, the regulation of channel gating and the dynamic behaviour of the reaction remain poorly understood. The emergence of fluorescence techniques at the single molecule level has already improved our understanding of such dynamic processes. In-house developments in single molecule fluorescence spectroscopy instrumentation, combined with the expertise of the Radford group in protein folding and the Baldwin group in membrane proteins, place us in a strong position to unravel the SecYEG dynamics and conformational changes involved in protein translocation through this protein pore. The aims of the project are to: (a) Design structure-based FRET labelled mutants of SecYEG, focusing on key domains of the translocon (e.g. "plug domain", "hinge region", dimerisation interface and the two halves of SecYEG, and (b) characterise the structure/function relationship of these labelled SecYEG mutants using ensemble techniques. Measure intra- and intermolecular distances within and between SecYEG and the model substrate proOmpA during translocation using single molecule FRET (smFRET). Determine the extent to which restricting the movement of key domains by "locking" the protein in specific conformations can influence SecYEG function and derive a detailed mechanistic model for protein translocation.

Amount: £130,015
Funder: The Wellcome Trust
Recipient: University of Leeds

Molecular biology of bunyavirus host cell interactions. 24 Apr 2006

The Bunyaviridae is a family of mainly arthropod transmitted viruses that contain a tripartite single-stranded RNA genome. Some members of the family are of medical importance causing encephalitis or haemorrhagic fever in man. My laboratory has used Bunyamwera virus, the prototype of the family, as the representative on which to perform molecular biological studies such as functional analyses of recombinant expressed viral proteins, and the development of a reverse genetic system to recover infe ctious virus entirely from cDNAs. This proposal exploits the materials and reagents we have developed to continue and expand our work analysing the replication processes of bunyaviruses at the molecular level, with the overall goal of understanding how the virus interacts with host cells of mammalian and insect origin. Our underlying hypothesis is that differences in virus:cell protein:protein interactions between these cell types determine the outcome of infection. Specifically, using a combina tion of molecular, biochemical and cell biological approaches we will address the following interrelated areas: (i) interactions of viral proteins with cellular proteins during bunyavirus replication; (ii) real-time microscopic analysis of bunyavirus infection using fluorescently tagged viruses; and (iii) functional analysis of the bunyavirus NSs protein in mammalian and insect cells.

Amount: £1,224,645
Funder: The Wellcome Trust
Recipient: University of St Andrews

Cytosolic phospholipase A2a association with the Golgi apparatus regulates vascular function. 11 Jul 2006

We have discovered that cytosolic phospholipase A2a (cPLA2a) is inactivated byassociation with the Golgi apparatus in confluent monolayers of endothelial cells. This inactive pool of enzyme can be released from the Golgi when the monolayer is wounded. It then can bind to target membranes and lead to prostaglandin synthesis. We want to define the biochemical basis of Golgi association. Our evidence (co-immunoprecipitation, co-localisation, siRNA) points strongly to binding of cPLA2 to annexin A1 and other proteins. We shalldefine peptide sequences responsible for cPLA2 binding to the Golgi apparatus.Such peptides will be tested for their ability to promote release of cPLA2 from the Golgi leading to elevation of prostacyclin levels and thus reduction of blood pressure and clotting.

Amount: £156,624
Funder: The Wellcome Trust
Recipient: University of Leeds

VALUE IN PEOPLE AWARD. 30 Aug 2006

Not available

Amount: £150,000
Funder: The Wellcome Trust
Recipient: University of St Andrews

VALUE IN PEOPLE AWARD. 30 Aug 2006

Not available

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

Vacation Scholarships 2008. 27 May 2008

Not available

Amount: £5,760
Funder: The Wellcome Trust
Recipient: University of St Andrews

Vacation Scholarships 2008. 27 May 2008

Not available

Amount: £10,080
Funder: The Wellcome Trust
Recipient: University of Leeds

Value in People Award. 17 Oct 2007

Not available

Amount: £250,000
Funder: The Wellcome Trust
Recipient: University of Leeds

Value in People Award. 17 Oct 2007

Not available

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

Open Access award. 31 Oct 2006

Not available

Amount: £20,000
Funder: The Wellcome Trust
Recipient: University of Leeds

RNA structure and its influence on the assembly of single-stranded RNA viruses. 18 Apr 2007

Single-stranded RNA (ssRNA) viruses are one of the largest classes of viruses, including many human and animal pathogens: e.g. HIV, HCV, Norwalk virus1, picornaviruses like poliovirus1, rhinovirus1, equine rhinitis A virus (ERAV)2,3 and Foot-and-Mouth Disease virus4, as well as plant viruses such as Tomato Bushy Stunt Virus (TBSV)5 and Turnip Crinkle Virus (TCV)6-9 and bacteriophages like MS210-14. For many of these viruses novel routes towards anti-viral therapy are urgently required10. However, viral assembly and disassembly, both essential processes in the viral life cycle and therefore attractive potential drug targets, are poorly understood. To further the understanding of viral assembly processes, the following specific aims will be pursued in this project: 1) Investigating the degree of order in ssRNA when encapsidated by viral protein shells, and the influence of N-terminal arms on some coat proteins on this order. 2) Examining the role(s) of secondary structure elements in ssRNA molecules in the initiation of viral capsid assembly.

Amount: £135,946
Funder: The Wellcome Trust
Recipient: University of Leeds

Characterisation of MINA, an interaction partner of the Kaposi's Sarcoma-associated Herpes Virus ORF57 protein. 18 Apr 2007

Kaposi's Sarcoma-associated herpesvirus (KSHV) ORF57 protein is conserved in all herpesviruses and is an essential regulator of gene expression implicated in many roles during infection. We have recently identified a novel cellular interaction partner of ORF57: MINA (myc-induced nuclear antigen with 53kDa), however the function of MINA is poorly understood. Therefore to understand the role of the ORF57 - MINA interaction, an evaluation of MINA function is required. We hypothesise that MINA: I. Is a protein arginine demethylase. II. Influences the intracellular localisation, protein-protein and RNA-protein interactions. III. Is a regulator of KSHV ORF57 function. To test this hypothesis, this study has 3 distinct objectives: 1) Test whether MINA possesses demethylase activity. 2) Identification of proteins which interact with MINA in uninfected and KSHV-infected cells. 3) Determine whether MINA influences the subcellular localisation, RNA binding or protein-protein interactions of KSHV ORF57 or other identified MINA-interacting proteins.

Amount: £135,946
Funder: The Wellcome Trust
Recipient: University of Leeds

Genetic dissection of biomineralisation defects and links with retinal degeneration. 16 May 2007

We have ascertained patient cohorts with Amelogenesis Imperfecta and retinal degeneration. This proposal aims to determine whether there is a previously unknown common pathway critical to enamel biomineralisation and retinal function, through genetic investigation of these cohorts. Furthermore, as little is known about the genetics of AI, we will also investigate families with AI alone, to better understand enamel biomineralisation and highlight processes which may link it with retinal function. We therefore propose the following investigations; 1) We have DNAs from five pedigrees segregating recessive Cone-Rod Dystrophy and hypomineralised AI. Two link to chromosome 2q11 and share a common haplotype across 1.2Mb. We will analyse the seven genes within the shared region in linked families. 2) We have identified eleven families with recessive Usher Syndrome and hypomineralised AI. SNP mapping has implicated 14 chromosomal regions consistent with linkage. We will test further markers to identify the gene(s) involved. 3) We will use homozygosity mapping in eight families with recessive hypomineralised AI to identify new genes critical to biomineralisation. 4) For any disease-causing gene identified, we will determine pattern of expression, mutation spectrum and phenotype range, including clinical ophthalmic and dental assessments, laboratory analyses of teeth and bone mineral density assessment.

Amount: £192,191
Funder: The Wellcome Trust
Recipient: University of Leeds

Cognitive Systems Foresight: Human Attention and Machine Learning. 31 Dec 2006

Human observers move their eyes in order to direct their attention to important aspects of a visual scene. There are models called salience maps; they predict where the eyes will move to when looking at a scene. At present, there models do not deal with video input, nor do they predict how an observer's task will affect where they look in. In other words, there are no models for real-life viewing situations, where an observer has a specific task. We are proposing a new approach to this problem. We have access to video information from cameras used in urban surveillance, and to the operators whose job is to spot abnormal behaviour in such video inputs. We shall obtain (previously unseen) video recordings of events in UK urban streets, and display them in a simulated control room to operators familiar with the town in question. We shall monitor where they look on the bank of video screens, and also when they decide that an event is abnormal and/or requires some form of intervention, e.g. calling the police. We shall use the record of eye fixations to teach a computer system to distinguish between "normal" and "abnormal" events. In this way, we shall be able to learn what is important for humans to do such surveillance by observing their eye fixation behaviour, for a realistic (and difficult) task and set of real-life video sequences. The project is important for four reasons. First, this will be the first attempt to develop a model of human attention/eye movements which will be firmly based on realistic video input and a real task. Second, this will be the first time that a computer system is able to learn from human behaviour in this way. Third, we will learn much about the ability of trained observers to cope with a demanding task as the number of TV monitors increases. Finally, we will develop an automated system which will be able to analyse the input from any urban CCTV camera in order to alert operators to look at that video stream - at present, most CCTV video streams are not observed by anyone since there are too many cameras for the number of human observers. Therefore, an automated alerting system is greatly needed and this project constitutes the best attempt to date to produce one.

Amount: £40,626
Funder: The Wellcome Trust
Recipient: University of Leeds

Engineering Solutions for an Ageing Population with Musculoskeletal & Cardiovascular Disease. 50 more years after 50. 23 Mar 2009

The   ageing population is increasing in number and life expectancy. The population   expects fifty more years after fifty with high levels of activity and quality   of life. However, the musculoskeletal and cardiovascular systems age and   degenerate, adversely affecting mobility, ability to work and quality of   life. Advances in engineering and bioscience have created opportunities for   novel devices and regenerative therapies, which utilise innovative   biomaterials or biological scaffolds to guide the patient's own stem cells to   repair degenerative tissues. Advances in patient imaging and diagnostics are   enabling earlier disease diagnosis with opportunities to intervene earlier in   the degenerative process and preserve healthy tissue, and potential to   provide patient specific continuum of care. WELMEC will deliver: - Longer   lasting joint replacements in the hip, knee and spine. - Novel regenerative   biological scaffolds for degenerative joint tissues, dental reconstructions   and cardiovascular surgery. - Advances in cell therapies using the patient's   stem cells. - Advanced medical imaging to facilitate earlier diagnosis and   intervention. - Novel protein biosensors for disease diagnosis and improved   patient targeting. WELMEC will integrate over 200 engineering, physical   science, life science and medical researchers with clinicians and   industrialists to develop and deliver innovative therapies and patient   services for the ageing population.

Amount: £7,456,503
Funder: The Wellcome Trust
Recipient: University of Leeds

Biomedical vacation scholarship. 29 May 2009

Not available

Amount: £7,200
Funder: The Wellcome Trust
Recipient: University of Leeds

Biomedical vacation scholarship. 29 May 2009

Not available

Amount: £4,320
Funder: The Wellcome Trust
Recipient: University of St Andrews

The identification and analysis of genes and proteins underlying recessive familial exudative vitreoretinopathy (FEVR). 07 Dec 2006

This project aims to identify and characterise the proteins underlying retinal vasculogenesis and angiogenesis by studying familial exudative vitreoretinopathy (FEVR), an inherited blinding disorder of the retinal vascular system. Genetic studies, in which my supervisor and collaborators have already played a major role, have identified three of the genes responsible for FEVR; NDP for X-linked FEVR, FZD4 for dominant FEVR and LRP5 for both dominant and recessive FEVR. However, these genes account for <50% of FEVR cases and in particular LRP5 accounts for <25% of recessive FEVR cases.In this project I intend to identify a new gene(s) responsible for the recessive form of FEVR by analysing a collection of consanguineous recessive FEVR families which don't harbour LRP5 mutations. A new locus will be mapped by identifying regions of autozygosity by microarray whole-genome SNP analysis, followed by traditional microsatellite genotyping and linkage analysis. A candidate gene approach will then be used to identify the mutated gene. Identifying further genes underlying FEVR will increase our understanding of this disease, facilitating its management and treatment. This work will also provide insights into retinal vessel formation, which should contribute to the study of other diseases involving abnormal vascular development.

Amount: £149,340
Funder: The Wellcome Trust
Recipient: University of Leeds

Student elective for Elizabeth Oxley. 18 Jul 2007

Burkitt's Lymphoma (BL) is a Non-Hodgkins lymphoma of B-cell origin, commonly presenting as a tumour of the jaw or abdomen. It is a high grade, rapidly progressing lymphoma which is invariably fatal within weeks or months if left untreated. However, response to intensive chemotherapy is good, resulting in a cure in the majority of paediatric cases in the west. An endemic form of BL exists in Sub-Saharan Africa, predominantly affecting children under the age of 15 years. Cases of endemic BL have been mapped to a geographical band 150 north and south of the equator, extending south-east into Mozambique. Previous research has identified a close association with both falciparum malaria and Epstein Barr virus.

Amount: £1,000
Funder: The Wellcome Trust
Recipient: University of Leeds

Value in People Award. 18 Jul 2007

Not available

Amount: £150,000
Funder: The Wellcome Trust
Recipient: University of St Andrews