Ero1α is a resident ER oxidase and is an important member of the oxidative protein folding machinery. It generates disulphide bonds de novo and donates them to protein disulphide isomerase (PDI), which in turn oxidises nascent substrate proteins within the ER. Ero1 activity must be tightly regulated for two key reasons: (i) it must maintain the balance of oxidised PDI to ensure oxidative protein folding can occur, but cannot be so active that the ER becomes hyperoxidising and dysfunctional, and (ii) Ero1 activity must be regulated to prevent the accumulation of hydrogen peroxide, a reactive oxygen species (ROS), within the ER. The regulation of Ero1α comes principally from 3 intramolecular disulphide bonds which are reduced by substrate upon activation, and re-oxidised upon inactivation by an unknown mechanism. Using an SDS-PAGE based assay we tested three hypotheses: that sulphenylation by Ero1α-produced hydrogen peroxide could induce re-oxidation; that an internal disulphide exchange mechanism could generate and distribute disulphide bonds within Ero1α; and that ER oxidoreductases could act to inactivate Ero1α. Having successfully expressed, purified and characterised a recombinant version of Ero1α, this was tested in a number of assays to address the above hypotheses. In vitro findings show that Ero1α is specifically and rapidly oxidised by ERp46 and PDI. Sulphenylation and internal disulphide exchange-mediated oxidation of Ero1α provided a comparatively slow and incomplete method of re-oxidation. In vivo results suggest that ERp46 and PDI may have implications in Ero1α activity regulation. Overexpression of several ER oxidoreductases had no effect on Ero1α re-oxidation after DTT challenge, whereas Ero1α oxidation was impaired slightly in PDI- ERp46 double knockdown cells. Depletion of PDI from cells results in the DTT-resistance of Ero1α, suggesting that Ero1α, PDI and glutathione are involved in an intricate mechanism of sensing and reacting to ER redox conditions. Two key ER oxidoreductases, PDI and ERp57, are oxidised in semi-permeabilised cells. Oxidation coincides with permeabilisation of the plasma membrane and the removal of cytosolic glutathione, directly implicating glutathione in the maintainence of the redox states of ER oxidoreductases. Oxidation during the permeabilisation of cells is an enzymatic process which is mediated in part by Ero1α. Semi-permeabilised cells harbour a more oxidising environment than do microsomes. This study contributes significantly to the research field by complementing several previously reported findings, as well as providing a novel investigation into the molecular regulation of Ero1α and its relationship with PDI and glutathione in the cell.

In addition to their role in fighting infection, mast cells have long been implicated in the pathogenesis of allergic and autoimmune inflammatory diseases and cancers. Increasingly, however, there is recognition that these cells may also play a part in protecting against development of pathologies. Indeed, there is increasing evidence that mast cells comprise heterogeneous phenotypes that exhibit functional plasticity to allow them to play both pro- and anti-inflammatory roles during an immune response. This plasticity appears to reflect that immature mast cells are tailored by their particular microenvironment not only to trigger protective inflammatory responses but also to limit pathology by resolving inflammation and promoting wound healing and tissue repair. Mast cells can be activated by a range of stimuli including (pathogen-derived) antigen/allergen-mediated crosslinking of antibody-bound to Fc receptors, most notably FcεRI, pathogen-associated molecular patterns (PAMP) such as bacterial lipopolysaccharide (LPS) acting on TLR4, inflammatory cytokines such as IL-33 (via the IL-1R/TLR-like receptor ST2) and tissue-derived signals such as SCF (via cKIT). During infection these stimuli provoke a response optimised for pathogen clearance however in autoimmune or allergic disease such responses can initiate and exacerbate host pathology. Thus the challenge for therapeutic targeting of mast cells in inflammatory or malignant disease is to limit mast cells with pathogenic phenotypes whilst preserving those contributing to protective homeostatic and anti-pathogen responses. Thus, as a first step, it was a core aim of these studies to generate in vitro mast cell models representing the phenotypic and maturational heterogeneity of mast cells in vivo, as these are difficult to isolate and purify due to their limited numbers in tissue. Distinct murine mast cell phenotypes, namely mature serosal peritoneal-derived mast cells (PDMC), connective tissue-like mast cells (CTMC) and mucosal-like mast cells (BMMC), the latter two subtypes both derived from bone marrow progenitors, were found to differentially respond, in terms of cytokine production and degranulation, to important immunoregulatory receptors in health and disease, namely FcεRI and TLR4. Consistent with their distinct functional responses, these mast cell subtypes were also found to display differential calcium signalling profiles in response to FcεRI and TLR4 signalling, further highlighting the importance in investigating phenotypically relevant and microenvironment-specific (serosal versus mucosal) mast cells in drug discovery programmes. Recently there has been great interest in IL-33, a pro-inflammatory cytokine increasingly recognised as playing an important role in a variety of mast cell responses associated with allergic inflammatory disorders and tumour pathogenesis. Consistent with this, whilst IL-33 can stimulate mast cell cytokine production, but not degranulation, via the IL-1R/TLR-like receptor ST2, responses to this cytokine are amplified following IgE sensitization and/or exposure to SCF or serum. Such augmented responses reflect increased calcium mobilization, PLD, SphK, ERK and NF-κB signalling and mTOR activation and can be suppressed by existing therapeutics targeting the costimulatory signal, for example, Imatinib or Dasatinib for SCF/cKIT and potentially Omalizumab for IgE/FcεR1. Moreover, IL-33/ST2 signalling can modulate mast cell responses resulting from antigen-mediated crosslinking of FcεRI and LPS-TLR4 signalling. Indeed, ST2 signalling can differentially modulate LPS/TLR4 responses depending on the presence (enhances) or absence (inhibits) of IL-33, as in the latter case ST2 acts to limit LPS cytokine production, potentially by sequestering MyD88. This receptor crosstalk is likely to occur under pathological conditions, thus targeting of such cooperative signalling may allow the downregulation of hyper-inflammatory responses, whilst leaving protective and homeostatic mast cell responses intact. ES-62 is an immunomodulator produced by filarial nematodes to dampen immune responses in order to promote parasite survival and prevent tissue damage without immunocompromising the host to infection. As a serendipitous side effect of its anti-inflammatory actions, ES-62 exhibits therapeutic potential in both allergic and autoimmune inflammatory disease and thus to further explore the potential for safe, targeted downregulation of pathogenic mast cell responses, the parasite product was exploited in order to identify signals regulating mast cell activation. ES-62 was found to be able to induce hypo-responsiveness of all three mast cell phenotypes in terms of degranulation and cytokine production in response to stimulation of FcεR1-, TLR4- or IL-33/ST2, either alone or in combination. ES-62 mediated these effects, at least in part, by mechanisms involving downregulation of PKCα (and in BMMC, MyD88) expression and calcium mobilisation and, in PDMC, potentially by subverting the negative feedback interactions of ST2 on TLR4. The precise mechanism of modulation varies both with receptor usage and mast cell phenotype as ES-62 exhibits differential effects in PDMC and BMMC. Nevertheless, collectively these data support the role of calcium-, PKCα- and MyD88- as key regulatory intersection sites in the functional crosstalk amongst these important immunoregulatory receptors and importantly, suggest they are potential targets for therapeutic intervention in pathogenic mast cell responses.

Endosomal sorting complex required for transport (ESCRT) proteins are conserved between Archaea, yeast and mammalian cells. ESCRT proteins mediate membrane scission events in the downregulation of ubiquitin-labelled receptors via the multivesicular body (MVB) pathway and HIV budding from host cells. In addition, ESCRT proteins have an established role in the final stage of cytokinesis, abscission, although the functional mechanisms by which they mediate daughter cell separation have yet to be demonstrated biochemically in vivo. The ESCRT machinery is composed of four subunits: ESCRT-0, -I, -II and -III; and the modular composition of the ESCRT machinery is reflected in its various functions. ESCRT proteins are recruited sequentially to the endosomal membrane for MVB formation: first, ESCRT-0 sequesters ubiquitylated cargo destined for degradation; second, ESCRT-I and II deform the peripheral membrane to produce a bud; and third, ESCRT-III constricts the bud neck to form an intralumenal vesicle. Thereafter, AAA-ATPase Vps4 redistributes ESCRT-III subunits back into the cytoplasm to mediate further MVB formation; it is the association of ESCRT-III and Vps4 that forms the conserved membrane scission machinery in all ESCRT functions. At a precise time during late cytokinesis, ESCRT-I protein TSG101 and ESCRT-associated protein ALIX are recruited to the midbody where they localise to both sides of the dense proteinaceous Flemming body through interactions with CEP55; TSG101 and ALIX in turn recruit ESCRT-III components. Immediately before abscission, ESCRT-III redistributes outwards from the Flemming body to the abscission site; microtubules are severed and the daughter cells separate. Thereafter, ESCRT-III appears on the opposite side of the Flemming body and the process is repeated to produce the midbody remnant. How this selective and specific redistribution of ESCRT proteins is regulated in space and time remains unsolved. To this end, polo kinase and Cdc14 phosphatase were identified as potential regulators of ESCRT function, due to their significant functions in regulating cytokinesis. Homologues in the fission yeast Schizosaccharomyces pombe, Plo1p and Clp1p, are required for either formation or stabilisation of the contractile ring that drives cytoplasmic cleavage. Furthermore, human polo-like kinase, Plk1, maintains CEP55 in a phosphorylated state to negatively regulate its localisation to the midbody; and although Plk1 proteolysis facilitates abscission complex assembly, Plk1 re-emerges at the midbody late during cytokinesis. It was hypothesised, therefore, that polo kinase and Cdc14 phosphatase regulate members of the ESCRT machinery to mediate cytokinetic abscission. To address this, fission yeast was used to study interactions between Plo1p, Clp1p and ESCRT proteins. Initially, ESCRT function in fission yeast cytokinesis was examined by characterising formation of the specialised medial cell wall, the septum, in individual ESCRT deletion strains. ESCRT genes were shown to be required for cytokinesis and cell separation in fission yeast, implying a role for the ESCRT proteins in this process. A yeast genetics approach was then employed to investigate genetic interactions between ESCRT genes and plo1+ and clp1+. Double mutants were produced from crosses between ESCRT deletion strains and mutants of plo1 and clp1. Synthetic defective growth rates were observed in double mutants, indicating genetic interactions between plo1+, clp1+ and ESCRT genes. The effect of single ESCRT deletions on vacuolar sorting in fission yeast was characterised. Single mutants of plo1 and clp1 were also shown to affect vacuolar sorting, indicating novel roles for these proteins in fission yeast. Analysis of vacuolar sorting in double mutants provided further characterisation of observed genetic interactions: plo1+ was regarded to function upstream of ESCRT genes, and clp1+ downstream. The yeast two-hybrid assay was used to further analyse interactions. Physical interactions were observed between Plo1p and Sst4p (human HRS, ESCRT-0), Vps28p (VPS28, ESCRT-I), Vps25p (EAP20, ESCRT-II), Vps20p (CHMP6, ESCRT-III) and Vps32p (CHMP4, ESCRT-III). Clp1p was also shown to interact with Vps28p. Interactions were then investigated between human homologues of these proteins in HEK293 cells. Immunoprecipitation and co-immunoprecipitation methods revealed interactions between Plk1 and CHMP6, CHMP4B, CHMP3 and CHMP2A (all ESCRT-III). Furthermore, interactions were demonstrated between CDC14A and CHMP4B and CHMP2A. These results indicate that polo kinase and Cdc14 phosphatase have conserved roles in regulating ESCRT components. Characterising the nature and functional significance of this regulation may inform future approaches in disease prevention.

Autophagy is a major route for lysosomal and vacuolar degradation in mammals and yeast respectively. It is involved in diverse physiological processes and implicated in numerous pathologies. The process of autophagy is initiated at the pre-autophagosomal structure and is characterised by the formation of a double membrane vesicle termed the autophagosome which sequesters cytosolic components and targets them for lysosomal/vacuolar degradation. The molecular mechanisms that regulate autophagosome formation are not fully understood. The conserved oligomeric Golgi (COG) complex is a hetero-octameric tethering factor implicated in autophagosome formation which interacts directly with the target membrane SNARE proteins Syntaxin 6 and Syntaxin 16 via the Cog6 and Cog4 subunits respectively. The work presented in this thesis demonstrates direct interaction of the yeast orthologue of Syntaxin 16, Tlg2, with Cog2 and Cog4. In addition, I investigated binding of the COG complex subunits to Tlg1, Vti1 and Snc2, the partner SNARE proteins of Tlg2. Direct interaction of Tlg1, the yeast orthologue of Syntaxin 6, with Cog1, Cog2 and Cog4 were observed. Given that Tlg2 has previously been shown to regulate autophagy in yeast, these data support a conserved role for the COG complex in mediating autophagosome formation through regulation of SNARE complex formation. In addition to investigating binding of COG complex subunits to the endosomal SNARE complex, I have also investigated a role for autophagy in regulating Tlg2 levels. The SM protein Vps45 has previously been shown to stabilise Tlg2 cellular levels. Our laboratory has demonstrated a role for both the proteasome and vacuole in the degradation of Tlg2. Here I demonstrated a role for autophagy in the regulation of Tlg2 levels and show that Swf1-mediated palmitoylation may serve to protect Tlg2 from being selectively targeted for autophagy. I also investigated the effects of the yeast T238N mutation on Vps45 function. The analogous mutation in human Vps45 has recently been associated with congenital neutropenia. Vps45 function is best characterised in yeast where it associates with membranes via Tlg2 and is required for membrane traffic from the trans-Golgi network into the endosomal system. Cellular levels of Vps45 T238N were destabilised and a concomitant reduction in Tlg2 levels was also observed. Vacuolar protein sorting remained unaffected in yeast cells harboring Vps45 T238N but was subjected to increased apoptosis under hydrogen peroxide-mediated stress. This identifies a novel role for Vps45 in maintaining cell viability. Finally, I also investigated a role for endosomal trafficking and autophagy in C.elegans post-embryonic development and identified a role for these pathways in the clearance of the pre-moult increase in intracellular membranes and cuticular formation.

Malaria, caused by the Apicomplexan parasite Plasmodium is a deadly disease which poses a huge health and economic burden over many populations in the world, mostly in sub-Saharan Africa and Asia. To design new intervention strategies and to improve upon existing drugs against malaria, it is important to understand the biochemistry of the Plasmodium parasite and its interaction with the host. We used metabolomics to dissect the biology of the reticulocyte preferring rodent malaria parasite Plasmodium berghei and showed that metabolic reserves in the reticulocytes can aid in survival of malaria parasites when their metabolism is genetically or chemically disrupted, pointing towards a direct role of host cell metabolism in parasite survival. These results have implications for currently used ways of intermediation in malaria infections which target only parasite metabolism against the human malaria parasites, Plasmodium vivax which prefers to infect reticulocytes and Plasmodium falciparum which is capable of infecting all erythrocytes. We also used metabolomics to show the biochemical differences between the asexual and sexual stages of P. berghei parasites and our data gave additional insights into the preparatory phase of the gametocyte stage at the metabolic level with the discovery of a phosphagen system which plays a role in gametogenesis. Targeted metabolomics of P. berghei life stages using isotopic labelling showed that TCA cycle metabolism is predominant in the mosquito stages. Discovery of a reductive arm of TCA metabolism in reticulocytes pointed towards the existence of rudimentary mitochondria in young erythrocytes. Another surprising discovery was the presence of up regulated γ-Aminobutyric acid (GABA) metabolism in the ookinete stage in P. berghei which may act as an energy source during the ookinete to oocyst transition in the mosquito. This pathway presented novel candidates for transmission blocking.

Trafficking of the facilitative glucose transporter GLUT4 is regulated by insulin in fat and muscle cells. Under basal conditions, GLUT4 is retained intracellularly by continually cycling through the endosomal system, but translocates to the plasma membrane in response to insulin stimulation. Intracellular GLUT4-containing vesicles fall into two categories: cellugyrin-positive (sortilin-free) and sortilin-positive (cellugyrin-negative). The former are the source of GLUT4 that cycles through the plasma membrane under basal conditions while the latter are the source of GLUT4 that translocates to the cell surface upon insulin stimulation. Fusion of GLUT4-containing vesicles with the plasma membrane is mediated by formation of SNARE complexes including the plasma membrane localized t-SNAREs Syntaxin 4 and SNAP23, and the v-SNARE VAMP2 present in the GLUT4-containing vesicles. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation, although its precise role remains controversial. Munc18c binds directly to both Syntaxin 4 and VAMP2 as well as to the assembled SNARE complex through a series of different binding modes. It has been suggested that SM/Syntaxin interactions facilitate SNARE complex formation by bringing about a conformational switch to release an inhibitory effect of syntaxins’ Habc domain. In this study I have used in situ Proximity Ligation Assay (PLA) to visualize the effects of insulin stimulation on interactions between Syntaxin 4, SNAP23, VAMP2 and Munc18c in 3T3-L1 adipocytes and fibroblasts. I find that insulin treatment results in an increase of the formation of assembled Syntaxin 4/SNAP23/VAMP2 SNARE complexes as well as recruitment of Munc18c to these complexes. These studies also reveal the existence of two pools of Syntaxin 4 under basal conditions: one in complex with SNAP23 (lacking VAMP2 and Munc18c); the other in complex with Munc18c and VAMP2 (lacking SNAP23). Additionally I have used in vitro binding studies to demonstrate that Syntaxin 4 binds directly to VAMP2 in a SNARE motif related manner and that this interaction is inhibitory to the rate of Syntaxin 4/SNAP23/VAMP2 SNARE complex assembly. Syntaxin 4 also binds directly to SNAP23, an interaction that enhances SNARE complex formation. Munc18c is phosphorylated on Tyr-521 in response to insulin-stimulation of 3T3-L1 adipocytes. I report here, that wild-type Munc18c inhibits SNARE complex formation, whereas a phosphomimetic version facilitates this process. Finally PLA studies reveal that the Syntaxin 4 pool in complex with VAMP2 and Munc18c associates with sortilin-positive vesicles, and that it is this pool which facilitates fusion of GLUT4 carrying vesicles upon insulin-stimulation. These studies also demonstrate the other Syntaxin 4 pool, that in complex with SNAP23, associates with cellugyrin-positive vesicles, and likely regulates the basal cycling of GLUT4 through the plasma membrane. I have used the data presented in this thesis to formulate a model whereby the two pools of Syntaxin 4 described are functionally distinct, and differ in their ability to mediate delivery of GLUT4 to the plasma membrane in response to insulin through the function of Munc18c.

Insulin resistance in skeletal muscle is of major pathogenic importance in several common human disorders including diabetes, hypertension and obesity, but the underlying mechanisms are unknown. In order to define mechanisms of insulin resistance, studies have been undertaken in skeletal muscle of the SHRSP, an animal model of hypertension, which exhibits insulin resistance in skeletal muscle and adipose tissue, when compared to the normotensive WKY control animal. Anti-peptide antibodies, directed against the phosphorylated or unphosphorylated residue Ser-1177 of human eNOS, were prepared and characterised, to allow the study of eNOS regulation in SHRSP skeletal muscle by phosphorylation at this residue. However, some doubt exists over the specificity of these antibodies and future studies have not been undertaken at this stage. Flotillin is a protein known to be involved in a P13-kinase independent pathway, which is required for GLUT 4 translocation and increased glucose uptake in response to insulin. Flotillin expression is increased in skeletal muscle from SHRSP and for this reason a yeast two-hybrid screen was undertaken using flotillin-1 as bait, the aim being to identify flotillin interacting proteins which have possible roles in insulin-stimulated glucose uptake. A number of interesting putative flotillin interacting proteins was identified in this screen, however one must be cautious as no duplicate clones were identified. Also, due to time constraints no biochemical studies have been undertaken to determine if the proteins identified do indeed bind flotillin. Studies of the 'classical' P13-kinase-dependent insulin-signalling pathway were also undertaken. It was demonstrated that key proteins involved in the insulin-signalling pathway (GLUT-4, IRS-1, IRS-2 and the P13-kinase p85 subunit) are expressed at similar levels and have similar subcellular distribution (on crude fractionation of skeletal muscle) in skeletal muscle of the SHRSP and the control WKY. Furthermore, levels and activity of PKB (a protein kinase downstream of P13-kinase) were similar in WKY and SHSRP, suggesting that the insulin-signalling pathway leading to activation of PKB in SHRSP skeletal muscle is intact.

Under normal physiological conditions, insulin is released from the pancreas in response to an increase in blood-sugar concentration. The insulin signalling pathway culminates with the presentation of the glucose transporter GLUT4 on the plasma membrane of muscle and adipose tissue, leading to the uptake of glucose into these tissues and the subsequent lowering of blood-glucose concentration to basal levels. This system is faulty in patients with Type 2 diabetes. SNARE proteins have been identified as important regulators of membrane fusion in vivo. Formation of SNARE complexes has been shown to provide the energy required for two opposing membranes to fuse. The SNARE complex consisting of Syntaxin 4, SNAP-23 and VAMP 2 has been implicated in the fusion of GLUT4 Storage Vesicles (GSVs) with the plasma membrane of adipocytes in response to insulin, and thus unravelling the interactions involved in complex formation will allow a greater understanding into the translocation of GLUT4 in response to insulin. In this thesis I developed an in vitro fusion assay, which confirmed that the SNARE complex consisting of Syntaxin 4, SNAP-23 and VAMP 2 is able to sustain fusion of two vesicle populations. This assay was utilised further to investigate the role of the SM protein Munc-18c on SNARE-mediated membrane fusion. Using this method, Munc-18c was shown to have both positive and negative regulatory roles, depending on experimental conditions. Site-directed mutagenesis of the SM protein was also used in an attempt to dissect the interactions involved in binding of the SM protein to the assembled SNARE complex. Finally, I developed a second in vitro fusion assay which utilised isolated plasma membrane fractions from 3T3-L1 adipocytes. This assay was used to investigate the effect of insulin on the plasma membrane proteins found in these cells. Analysis of the fractions showed that insulin increased the rate of SNARE-mediated membrane fusion; however the levels of the t-SNAREs were unaltered in response to insulin, indicating that the hormone functions to alter protein structure or function, but not amount.

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

Insulin is an important regulator of glucose homeostasis. Insulin stimulation of fat and muscle cells results in the rapid translocation of the glucose transporter GLUT4 to the plasma membrane from its intracellular stores, allowing uptake of glucose into the cells from the blood. This tethering, docking and fusion event is driven by the formation of a SNARE complex at the plasma membrane consisting of the t-SNAREs Syntaxin4 (Sx4) and SNAP23 and the v-SNARE VAMP2. The formation of this complex is regulated by the SM protein Munc18c. It has been shown that Sx4 is phosphorylated at residues Y115 and Y251 following insulin stimulation; however the effects of these phosphorylation events have yet to be studied. In addition phosphorylation of Y521 in Munc18c is also increased following insulin stimulation. This study aimed to first elucidate the effects of Sx4 phosphorylation on SNARE protein interactions and GLUT4 trafficking, then to begin to examine the impact of phosphorylation of both Sx4 and Munc18c. I have used an in vitro approach to assess the affect Sx4 phosphorylation has on SNARE complex assembly. I have shown, using phospho-mimetic recombinant proteins, that the phosphorylation state of Sx4 affects the rate of SNARE complex assembly and its binary interactions with VAMP2 and SNAP23. Moreover, that this may be due to a conformational change in the protein. I have also shown, for the first time, that it is likely that Sx4 can be phosphorylated on Y115 and Y251 simultaneously. In addition I have used a HA-GLUT4-GFP expressing HeLa cell line to show that expression of phospho- mimetic Sx4 increases translocation of GLUT4 to the PM under basal conditions. Finally, I have begun to investigate the implications of both Sx4 and Munc18c phosphorylation on their in vitro interactions. This data provides insights into the direct regulation of membrane trafficking proteins by the insulin-signalling pathway. Increased understanding of the regulation of GLUT4 translocation could help to develop future therapies for type 2 diabetes, where GLUT4 trafficking is impaired.