- Total grants
- Total funders
- Total recipients
- Earliest award date
- 17 Oct 2005
- Latest award date
- 30 Sep 2018
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
We have demonstrated that mechanosensitive channels and the glutathione-gated potassium efflux systems play major roles in bacterial survival of osmotic and chemical stresses, respectively. These systems are widespread in the major Gram negative pathogens. New insights into gating have resulted from our recent work. The predictions from these studies have been tested by mutagenesis, specific assay systems and protein biochemistry. These analyses have identified new questions regarding the molec ular movements required for the formation of the open state. This analysis has also identified opportunities for the design of chemicals that interact specifically to effect either inhibition or activation, both of which perturb homeostasis. This increases their potential as drugs, either alone or in combination therapies. In the proposed research we will use crystallography, molecular genetics, protein biophysics, chemistry and electrophysiology on purified and reconstituted systems. Our spe cific goals are: elucidate the mechanism of communication between glutathione ligands and pore in KefFC determine how mechanosensitive channels interact with lipids and sense changes in membrane tension elucidate the structures of MscK and YbdG mechanosensitive channels and of KefC design, synthesis and screening of inhibitors and activators for KefC and mechanosensitive channels as research tools and potential drugs.
Helicases are molecular machines that unwind and remodel DNA and RNA in a wide variety of essential cellular processes. To understand helicase function, we need to understand their molecular structures and the dynamic conformational changes central to their catalytic mechanisms. The DNA helicases XPD and XPB are the enzymatic components of the transcription factor TFIIH and are essential for the fundamental cellular processes of Nucleotide Excision Repair (NER) and transcription initiation. Euka ryal TFIIH is difficult to study at a molecular level and the archaeal homologues of XPD and XPB have proven to be important model systems. Building on our long track record studying the archaeal enzymes, we have brought together a number of cutting-edge technologies including crystallography, biochemistry, genetics, single molecule assays, EPR and biophysics. We aim to determine the structural changes inherent in XPD and XPB action, define the function of domains such as the Fe-S cluster domain of XPD and Thumb domain of XPB and gain an understanding of their mechanisms at a molecular level. These studies will provide valuable new insights relevant to human health and disease and provide an enhanced understanding of an important class of enzymes fundamental to human biology.
The Bunyaviridae family are a diverse group of over 350 RNA viruses classified into five genera, namely Hantavirus, Orthobunyavirus, Phlebovirus, Nairovirus and Tospovirus. Many bunyaviruses are important pathogens of humans, animals and plants for which preventative or therapeutic treatments are unavailable. Bunyavirus pathogenesis depends on formation of the ribonucleoprotein (RNP) complex, in which the bunyavirus genome is entirely encapsidated by the virus-encoded nucleocapsid protein (N). O nly in the form of the RNP is the genome replicated, transcribed and packaged into new progeny particles. Using Bunyamwera orthobunyavirus and Rift Valley fever phlebovirus, we propose to perform a detailed quantitative analysis of the role of individual N protein amino acids in both gene expression and virus assembly. Critical to these analyses, we have generated the first high-resolution crystal structure of a complete bunyavirus N protein. We will pair this information with findings of our fu nctional analyses to reveal the structure-function relationship of the entire RNP complex for both viruses. Also critically, we will generate infectious viruses incorporating N protein mutations, which will allow us to validate our findings in the context of a live-virus infection, and relate how perturbation of molecular interactions between individual viral components affects the overall viral life-cycle
The mammalian spinal cord contains all the neural machinery required to generate locomotor activity, even in the absence of descending and sensory input. Neuromodulation is an important component of this system, allowing locomotor activity to be modulated to suit different states or behaviours. Nitric Oxide (NO) is a potentially critical neuromodulator, about which almost nothing is known in the context of mammalian locomotion. We therefore aim to elucidate the role of NO in the control of mamma lian locomotion. We will initially determine the neuronal sources of NO in the mouse spinal cord using NADPH-d histochemistry and nNOS immunohistochemistry to reveal nitric oxide synthase expression, and DAF-2DA labelling to reveal NO production. Next, using electroneurographic recordings from ventral roots and whole-cell patch-clamp recordings from neurons in spinal cord preparations which can elicit locomotor activity, we will examine the effects of NO-mediated signalling on locomotor networks and determine underlying cellular and synaptic mechanisms. Patch-clamp analyses will concentrate on nitrergic neurons, revealed by DAF-2DA labelling, and motoneurons, the output cells of motor systems. Data obtained will address an important gap in our knowledge of spinal motor networks and provide novel information regarding NO-mediated signalling which should be applicable to other neuronal systems.
We will build an E. coli that can detect and kill S. aureus. The project will consist of three steps (Fig 2): detection, destruction, and enhancement. First, we will build E. coli to signal detection of AlP by producing green fluorescent protein (GFP). We will use the BioBrick BBa_1746220 created by Cambridge in 2007 to detect group I AlP in E. coli; Cambridge did not succeed in verifying this BioBrick (igem.org), thus we will first "debug" it to create a working copy. In particular, we note that BBa_l746220 does not include SarA, a general transcription factor believed to act in concert with AgrA to activate promoters P2 and P3 (Novick 2003). We will explore addition of SarA to the BioBrick, as well as other refinements, such as including the entire P2 to P3 promoter regions (Novick 2003). We will pair this with GFP (BioBrick BBa_K203103) to cause fluorescence upon detection of AlP. We will then create our S. aureus destroying E. coli by replacing the GFP with the "Kamikaze" BioBrick BBa_K284022 created by UNICAMP-Brazil in 2009: BBa_K284022 creates lysosyme, thus killing the cell and any nearby bacteria, and has been verified to work (iGem. org). Finally, we will enhance the sensitivity of both these constructs by inserting an E. coli quorum sensing loop in between the AlP detector and the output (e.g. verified BioBrick BBa_l15030, UT Austin 2005): this serves to amplify the signal from AlP to produce greater output (GFP or lysosyme) per cell and recruit other nearby E. coli.
Whilst much has been learnt over recent years about how viruses interact with the interferon (IFN) system, there are still significant gaps in our knowledge. By building upon our extensive experience on the interaction of paramyxoviruses with the IFN system we propose to address some of these questions. More specifically, we will continue to characterise the nature of (paramyxo)virus inducers of IFN and how the IFN-induction cascade is activated and controlled, with particular reference to obser ved heterocellular nature of IFN induction. We will also identify the IFN-induced genes that affect the replication of paramyxoviruses (in particular PIV5), and detail how these viruses deal with cells in a pre-exisitng IFN-induced anti-viral state. In this regards, the importance of virus cytoplasmic bodies to the life-sytle of these viruses and their ability to establish prolonged/persistent infections will be examined. In addition, we will develop generalised methods for the rapid selection o f viruses that are either good inducers of IFN or cannot block IFN signalling, both for our fundamental studies and because such viruses may be developed as potential attenuated vaccine candidates. Such procedures may be particularly valuable for newly emerging viruses that pose an immediate threat to human and animal welfare.
This project will study the production of medical literature in north-western Europe: England, Netherland, France and the Swiss Confederation, together with a more focused case study of ownership in 16th century England and Scotland. Together these countries made up one of the three main zones of book production in Europe (the others being Italy and Germany.) From Paris, Lyon, Geneva and Basle, they supplied a large part of the learned market for medicine. They also served three distinct vernacu lar communities, the English, French and Dutch. They therefore provide an ideal laboratory for studying the marketplace of medical books. This project will first assemble the extant corpus of books published. Medical books will all be tagged for content. Secondly, the project will investigate the relationship between production and ownership through a pilot study of collections in 16th century Britain. The investigation of ownership will show whether a complex scholarly medical collection co uld be assembled without recourse to the larger centres of production in Venice or Germany. It will demonstrate how widely ownership of medical books had spread into the general book buying population. It will cast light on the importance of medical publishing in the general marketplace of books.
Xenopus locomotor interneuron lineage tracing and functional interrogation using lights. 02 Jul 2009
Major advances have been made in understanding the neuronal control of locomotion in lower vertebrates, mainly by making electrical recordings from individual, or sometimes pairs of neurons and by studying the synaptic connections they make. Such work enables inferences to be drawn about likely events that occur in the different populations of neurons that control locomotion. We wish to use a new approach to test some of these inferences. The approach exploits the use of light-sensitive, and li gand-sensitive, ion-channels and ion pumps to control neuronal activity. Expression of the appropriate exogenous proteins in an identified population of neurones makes possible their selective activation or inhibition. Early embryonic blastomeres of Xenopus laevis are uniquely predisposed to develop into specific types of neuron in the tadpole spinal cord. Thus, injection of the required cRNAs into appropriate blastomeres results in the labelling of specified classes of neurons whose activity ca n then be controlled by light, or a selectively-acting ligand (see pilot work). Our major objective is to evaluate the roles of different interneurons more precisely during swimming activity in the spinal cord and caudal hindbrain of the Xenopus tadpole.
The body of research into FMDV is impressive; over 150 complete genome sequences, a map of polyprotein processing, full-length cDNA clones from which virus can be rescued ( infectious copies ), a non-infectious replicon cDNA system, characterisation of the virus receptor, atomic structures of the virus particle, L proteinase and 3D polymerase, characterisation of immune responses with detailed epitope mapping. There still remain, however, substantial and critical gaps in our knowledge as to ho w this virus replicates in two modes; (i) very rapid cell lysis (3-4hrs), or, (ii) establishment of persistent infections. Even a cursory inspection of the FMDV genome reveals a number of features which are unique to aphthoviruses. Surprisingly, perhaps, the function or contribution to RNA replication of these features is not understood and is not currently being investigated. Our proposed program of research focuses upon questions concerning the replication of virus RNA and the molecular interactions between virus RNA/virus proteins with host-cell proteins using the (non-infectious) FMD replicon system. An essential element of this proposal is to harness the knowledge gained for the design of new, recombinant, forms of the genome to produce attenuated phenotypes: candidates for a new form of disease control live, attenuated, vaccines.
In this proposal we will use multi-disciplinary approaches to investigate choline phospholipid biosynthesis and metabolism in Trypanosoma brucei as a source of potential chemotherapeutic targets. Emphasis will be on understanding choline metabolic pathways, so that we can genetically and chemically validate them as drug targets. This project aims to answer three key questions; 1) T.brucei are auxotrophic for choline, so, how do they obtain choline? 2) Are T.brucei vulnerable to inhibit ion of de novo phosphatidylcholine synthesis? 3) Is catabolism of choline containing species essential to the parasite? Thus our key goals are to: genetically validate the choline branch of the Kennedy pathway (phosphatidylcholine de novo synthesis) as a drug target. understand the uptake and usage of choline containing species by bloodstream T.brucei. develop high-throughput assays for enzymes involved in choline metabolism. obtain kinetic, specificity and structu ral information about the enzymes involved in choline metabolism, that will allow the construction of structure-activity relationships to facilitate the design of more potent parasite-specific inhibitors. ultimately allowing development of potent inhibitors with activity against live trypanosomes compliant with Lipinski s rules, thus suitable for future development as novel anti-trypanosomal drugs.
Biomedical Science Research Complex (BSRC) New World Class Biosciences at the University of St Andrews. 18 Jul 2008
St Andrews University seeks £7.6 M in funding from the Wellcome Trust and will match this investment with over £10.7 M of its own capital. Beyond capital commitment the University will make six new appointments and make permanent three independent research fellows. This is a clear and significant commitment from St Andrews. A new laboratory building will physically link two existing buildings (Purdie/Chemistry and Biomolecular Sciences), creating a world leading and sustainable Biomedical Sciences Research Complex (BSRC). The BSRC will promote pioneering activity at the discipline interfaces of Chemistry, Biology, Physics and Medicine to address key problems in human health. According to the World Health Organisation infectious diseases account for 25% of all human morbidity and mortality. A UK Foresight report identified key global factors that could drive future threats from infectious disease: increasing travel, migration and trade (e.g. spread of infection); increasing risk from zoonoses (SARS, avian influenza and Escherichia coli O157:H7); the rise of drug-resistant organisms (MDRTB, malaria and MRSA) and climate change (distribution of disease). The report concluded that in order to prepare adequately for the future, 'traditional divides - e.g. between virology, bacteriology, mycology and parasitology, or between medicine, veterinary medicine and plant science need to be bridged.' The key objective is to cluster active researchers from different disciplines working in virology, bacteriology, model systems of human biology and parasitology, to allow rapid interchange of expertise, ideas and people so facilitating the use of state-of-
Understanding the Role of VPS35 in the Intracellular Movement of Alpha-Synuclein in Neuronal Cells 31 May 2018
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease affecting 10 million people worldwide. A key protein linked to the aetiology of the disorder is alpha-synuclein for which misfolding is linked to the pathogenesis. Importantly alpha-synuclein misfolding can be passed from cell-to-cell by mechanisms which are poorly understood. One proposed mode of transfer is via exosomes, small extracellular vesicles that are released from cells when multi-vesicular endosomes fuse with the plasma membrane. Trafficking in the endosome system therefore has potential to modulate exosome production and affect the transfer of misfolded alpha-synuclein. One gene implicated in late-onset familial PD is vacuolar protein sorting-35 (VPS35). VPS35 is a component of the retromer complex which is a coat protein involved in retrograde trafficking from endosome to the Golgi apparatus. It has the potential to modulate multi-vesicular body (MVB) formation/biogenesis and thereby influence the quantity of exosome-associated alpha-synuclein released from neuronal cells. Our hypothesis is that knockdown of VPS35 will modulate MVB/exosome biogenesis, augmenting the release of exosome-associated alpha-synuclein from cells. We suggest exosomes from VPS35 knockdown cells will be more potent in transfer of alpha-synuclein between cells. This will be observed by using immunoelectron microscopy, Nanocount technology, and immunofluorescence.
By 2050 10 million lives could be claimed a year by drug resistant infections. We must develop new strategies for antimicrobial drugs. Often in infections bacteria form biofilms, requiring concentrations of antibiotics up to 1000 fold higher to be treated. Cyclic dipeptides are molecules produced by organisms in all domains of life, and their function is unknown. They can inhibit bacterial growth and/or biofilm formation, albeit by undetermined mechanisms. The majority of the biological effects caused by cyclic dipeptides are inter-species and in some instances inter-kingdom, mediating host pathogen interactions. I will study enzymes from gram-positive and gram-negative bacteria involved in the production of different cyclic dipeptides. I will characterise each enzyme biochemically and structurally and determine their substrate scope. I will produce novel molecules, which will be used to disrupt growth and biofilm formation in Pseudomonas aeruginosa and Staphylococcus aureus growing alone and in bacterial co-cultures. I will combine genetic and chemoproteomic approaches to determine the molecular targets of cyclic dipeptides in P. aeruginosa and S. aureus. I will validate targets using bacterial mutants and biochemical assays. The identification of molecular targets of cyclic dipeptides will unveil crucial pathways for inter-species interactions and identify novel antimicrobial targets and molecules.