The incidence of psychiatric disorders, neurodegenerative diseases, and pain is on the increase. In terms of therapeutic intervention, this is self-evidently an area that requires the identification of novel pathways and neuronal targets. The CB1 and CB2 receptors are being investigated as potential therapeutic targets, however there are also likely to be considerable opportunities for modulating the synthesis of the most abundant endocannabinoid in the adult brain, 2-AG. The molecule is generated from diacylglycerol by the sn1-specific DAG lipases (DAGL-alpha and DAGL-beta) that we recently cloned (Bisogno et al. 2003). To fully exploit DAGL as a target, we need to understand how its function is regulated by protein-protein interactions. These are often mediated by linear peptides sequences and we have used a comparative genomic approach to identify the most highly conserved sequences in the DAGLs. We have synthesised four peptides (as well as reverse control peptides) in tandem with the antennapedia peptide sequence that mediates uptake into the cytosol of live cells. We now have evidence that three of these sequences represent functional motifs that have the same biological effects on cells as two conventional DAGL inhibitors (RHC80267 and THL); in this context they specifically inhibit DAGL dependent neurite outgrowth and also DAGL dependent proliferation of neural stem cells. The student will test the hypothesis that the peptides are inhibiting the function of the DAGLs by measuring their effects on DAGL activity in collaboration with scientists at Wyeth. The student will next use alanine scanning to identify key residues required for inhibitory activity and then introduce mutations to these residues in full length constructs of DAGL and determine their effects on enzyme activity following transfection into a variety of cell types. The student will also use two approaches to identify proteins that the peptides, and by implication, the DAGLs interact with. In the first approach the DAGL peptides (both the functionally blocking sequence and the reverse control) will be coupled directly to a sepahrose column. Brain extracts will be run over the columns, and specifically interacting proteins eluted and separated by conventional gel electrophoresis. Proteins that interact with the blocking but not control peptide will be identified following silver staining of the gels. Mass spectrometry methods will be used to identify proteins that interact with the blocking peptide, but not the 'reverse' control peptides. In the second approach, mini-constructs encoding the active inhibitory peptides will be used as bait in conventional yeast two hybrid screens. We anticipate that antibodies will be available for some of the interacting proteins, but if they are not we will outsource the generation of antibodies. Finally, we will test the hits for co-immunoprecipitation with DAGLs in a number of tissues and cell types.

When a molecule is oxidised, it either loses electrons (increasing its 'oxidation state') or, more commonly in organic chemistry, it gains an oxygen atom from another molecule (the 'oxidant'). Oxygenated molecules are important intermediates for the preparation of complex molecules, including medicinally interesting compounds, and are thus important for phamaceutical production.However, oxidation reactions are often difficult to achieve on a large scale, due to the following reasons:(i) Many oxidants are either toxic, or are thermally unstable materials that are potentially explosive;(ii) Oxidation reactions are by nature exothermic and may involve induction periods - this makes a reaction inherently unsafe, as thermal runaway is unpredictable and thus difficult to control.(iii) Presence of oxidants in organic solvents may generate organic peroxides, which are explosive at a certain limit, and may also cause thermal runaway reactions;(iv) The reaction can be unselective, producing many products, which may be difficult and costly to separate.This project proposes to overcome these problems by designing a new equipment to perform these reactions safely and cleanly, using largely electricity and water to generate oxidants. As the oxidant is generated and consumed immediately, the effective concentration of the reactive oxidant is kept to a minimum during the process, thus eliminating explosive hazards and environmental exposure. We are interested in 'waste free' reactions that produces side products that are environmentally benign, such as water, or in a form that can be recovered and reused (recycled).

Microfluidics provides an exceptional environment for the generation of controlled droplet dispersions and their manipulation in prescribed flow fields. The spatio-temporal correspondence between microchannel position and reaction 'time' permits the study of kinetics of (chemical and physical) processes with unprecedented time resolution and dynamic range. Further, the combination of the small volumes of droplet 'reactors' and the precise formulation of their composition opens vast possibilities in chemical synthesis, including screening, discovery and optimisation. Monitoring reactions in real-time with non-invasive probes remains, hitherto, a major shortcoming of microchemical reactors due to the minute sample volumes (pL-nL) and fast travel speeds (1-1000 mm/s). This proposal seeks to develop, implement and validate a novel experimental approach to monitor microchemical reactions in real-time by coupling, for the first time, cavity ring-down spectroscopy and solvent-resistant microfabrication. This approach will permit the online study of model catalytic reactions, with unprecedented reproducibility and flow control. Cavity ring-down spectroscopy will permit the analysis of pL volumes, effectively eliminating the restriction of path length in microchannels, with nanosecond to microsecond time resolution, compatible with microreaction drops. In particular, we will elucidate individual and global reaction population outcomes and the effect of mixing and flow, with spatiotemporal resolution. This approach is applicable to a range of organic chemical reactions and, for this work, we will focus on selected model systems (detailed below) of fundamental and industrial relevance.

In vertebrates, the primary afferent neurons are specialized to detect chemical, mechanical and thermal stimuli. Much of our knowledge about the function of these primary afferent sensory neurons has been obtained by studying the properties of specific neuronal sub-types innervating soft tissues such as skin and muscle. The general properties of sensory neurons have also been investigated using isolated neurons. Little is known about the properties of sensory nerves innervating bone and joints. We have shown that it is possible to identify the cell bodies of neurons innervating either joint tissues or bone by dye (e.g. True Blue) injection into the target tissue (Fernihough et al. 2004, 2005). Back labelled neurons can then be studied immunohistochemically, electrophysiologically and with functional readouts such as imaging changes in intracellular calcium levels in response to various stimuli. Where possible mice will be used in our study as this offers the possibility to use genetically modified animals to probe the function of various receptors and ion channels expressed by sensory neurons as well as molecules expressed by associated cells in the target organ. The phenotype of identified neurons will be studied using markers (e.g. antibodies) that define sub-specific known sub-types of sensory neurons (e.g. neurofilament protein, sensory neuropeptides and neurotrophin receptors). The expression of specific receptors and ion channels, known or postulated to be involved in sensory transduction, will also be studied using immunolabelling and in situ hydridization of back-labelled neurons in sections of lumbar DRG. The types of receptors and ion channels studied will include those that define nociceptive neurons (e.g. TRPV1, TRPA1, Nav1.8), which signal noxious stimuli. The expression of molecules implicated in mechanotransduction will be explored. The functional properties of enzymatically isolated, identified joint neurons will be studied electrophysiologically (whole cell recording) or using calcium imaging methods. One area of specific interest is the mechanical sensitivity of the neurons. Low and high threshhold mechanically evoked responses have been reported in sensory neurons and we will identify which types of mechanosensors are associated with joint sensory neurons. These combined studies will provide key information about the normal joint sensory neuron phenotype. Sensory neuron properties are influenced by the environment. Increased mechanical sensitivity is seen with joint inflammation. The effects of inflammatory mediators such as TNF-alpha on mechanotransduction are poorly understood. Neurons from animals in which the knee has been experimentally inflamed by intra-articular injection of Complete Freund's Adjuvant will be studied. This results in increased responses to mechanical stimulation of the joint. We will determine if the increased sensitivity is due to a hypersensitivity of the primary transduction process. Identified joint neurons from normal animals will also be exposed to inflammatory mediators and growth factors in vitro (acutely or chronically) to determine if mechanosensitivity is increased. If so we can investigate the molecular processes that underlie changes in sensitivity. The cellular assays will be complemented by behavioural studies that will be carried out in the research organization. Wyeth has expertise in behavioural models that measure responses to mechanical stimulation of the knee. We will exploit information about the types of neurons innervating the joint and bone and use ablation methods to remove sub-types of neurons. Tracers which selectively bind to sub-populations of sensory nerves will be conjugated to saporin (a ribotoxin) and administered into the knee joint. The behavioural consequence of the treatments will be studied and correlated with histological examination to determine the degree and selectivity of nerve ablation.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.