The insect Malpighian tubule is involved in osmoregulation, detoxification and immune function, physiological processes which are essential for insect development and survival. As the Malpighian tubules contain many ion channels and transporters, they could be an effective tissue for targeting with novel pesticides to control populations of Diptera. Many of the insecticide compounds used to control insect pest species are no longer suited to their task, and so new means of control must be found. The malarial mosquito, Anopheles gambiae, spreads the Plasmodium parasite which is responsible for over one million deaths each year, and is one of the species on which many current insecticides are no longer effective. Anopheles is notoriously difficult to study due to a lack of natural mutation stocks and transgenic capabilities, as well as the difficulties involved with maintaining a colony. The fruit-fly Drosophila melanogaster is a useful model organism for Anopheles, and previous studies suggest that the mechanisms of Malpighian tubule function are well conserved between the two species. Following microarray investigations to identify genes which were highly enriched in both the Anopheles and Drosophila Malpighian tubules, four homologous genepairs were selected, AGAP097752 and CG15406, AGAP012251 and Picot, AGAP009005 and ZnT35C, and AGAP002587 and CG8028. Analysis of the Anopheles Malpighian tubule microarray data-set showed ion channels and transporters to be highly expressed in the tubules, although similarly to Drosophila, very few of the renal up-regulated genes have been characterised. The gene-pairs chosen were all novel, but putatively predicted to be involved in sugar transport, phosphate transport, zinc transport and monocarboxylate transport respectively. These are functions which are likely to be essential, but so far remain unstudied in the insect renal system. The gene-pairs were chosen with two main purposes; to determine how closely expression of the genes was conserved between Anopheles and Drosophila, and also to determine which of the genes were essential, and could therefore be effective insecticide targets. The homologous gene-pair AGAP007752 and CG15406 have well-conserved expression in the Malpighian tubules, suggesting that they are functionally important genes. This was shown in Drosophila, where knockdown of CG15406 4 expression was lethal to the fly. A direct role in tubule fluid secretion was not found, and experiments to determine the sugars transported by CG15406 were inconclusive, possibly due to an abundance of highly-expressed sugar transporters in the tubules. The inorganic phosphate co-transporters AGAP012251 and Picot also show conservation of expression in the Malpighian tubules, and are likely to be involved in the transport of inorganic phosphate into the tubules for incorporation into metallo-organic concretions. In the Anopheles tubules the concretions are found in the main segment, in the Drosophila tubules they are located in the distal initial and transitional segments, where AGAP012251 and Picot are expressed. Picot is essential for Drosophila development through to adulthood, and for survival as an adult, although the transporter does not appear to be directly involved in fluid secretion. Expression of neither AGAP012251 nor Picot is confined to the tubules. The putative zinc transporters AGAP009005 and ZnT35C show a highly conserved expression pattern, and appear to be involved in the secretion of excess zinc from the Malpighian tubules. ZnT35C is essential early-on in Drosophila development, and for survival in the adult fly. Similarly to Picot and CG15406, there is no direct role for ZnT35C in fluid secretion from the tubules under normal zinc conditions. The putative monocarboxylate transporters AGAP002587 and CG8028 are not as well conserved, as AGAP002587 is highly upregulated in the tubules of female mosquitoes both before and after a bloodfeed, whereas CG8028 has no sex-specific up-regulation. CG8028 is not essential for Drosophila development or survival, and plays no discernable role in fluid secretion. The data collected during this investigation suggests that in general there is a high level of conservation of expression between homologous transport genes in the Anopheles and Drosophila Malpighian tubules. The three gene-pairs which show the greatest conservation of expression are also essential for development and survival in Drosophila. This suggests that cross-species studies are an effective way of finding essential and important genes. The data collected also suggests that Drosophila is a reliable model for Anopheles, and could be used as a high-throughput system of finding genes which could be effective insecticide targets in Diptera.

The life history of a population of Lutraria lutraria in a depth of 7m at Hunterston, Ayrshire is discussed. Much of the present population Is thought to have settled in 1967. The functional morphology of Lutraria is described and related to its life as a large, deep-burrowing bivalve. Lutraria spawned in late spring and continued to do so through the summer in 1979 and 1980. Animals became spent in August and September. Unsuccessful attempts were made to induce spawning in the laboratory. Artificial fertilization was successful but development did not proceed beyond the ciliated gastrula stage. Larvae of Lutraria were not identified in plankton samples and young stages were not encountered in sieved sediment samples. The biochemical cycle of the total animal and five component parts (gonad and visceral mass, digestive gland, adductor muscle, siphon and 'other' tissue) is investigated. A marked increase in weight, reflected in an increase in weight of the component parts, was recorded in Autumn 1979. This is thought to be related to an exceptional increase in the phytoplankton at this time. Although a relationship between the biochemical cycle and reproductive cycle remains uncertain, definite seasonal changes were recorded in the respiration rate of Lutraria. At 10°C, the maximum rate of a standard 20g animal was 0.1283m1s 02/g. dry wt./hr. in May 1980 and the minimum rate was 0.O59mls 02/g. dry wt./hr. in October 1980. The effect of temperature on respiration rate was also investigated. Significant differences were recorded for five experimental temperatures (10°C, 15°C, 20°C, 25°C and 30 °C) in August and October but only between two temperatures (10 C and 30 C) in April. There was a decrease in respiration rate at 30 C in August and October, but an increase in April. Respiration rate is affected by a reduction in oxygen tension. A variety of responses were recorded with a small degree of regulation shown. Individuals of Lutraria were able to survive 48 hours under anaerobic conditions. In fully oxygenated conditions heart rate ranged from 4-15 beats per minute with an average of 8 beats per minute. Heart beat was markedly affected by changes in temperature and oxygen tension, increasing to a maximum 22 beats per minute at 25 C, and decreasing to a minimum 2 beats per minute in anaerobic conditions. Heart rate is reduced (12 beats per minute to 5 beats per minute) on exposure to air. Lutraria exhibits an intermittent pattern of pumping activity. Under normal conditions 35% of the time is spent pumping and this Increases as oxygen is reduced (3.00mls 02/litre) to 65% of the time spent pumping. 15. Under normal conditions the respiratory flow varies between 0.382 litres per hour and 1.023 litres per hxir. Adult Lutraria maintain their ability to burrow, albeit slowly.

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.

Biological indicator species can reveal consequences of changes in physical processes within the environment, through effects on their physiology, behaviour and population dynamics. Long-lived species tend to be positioned at the top of the food chain where they can act as indicators of environmental change occurring at lower trophic levels. During poor conditions, these long-lived top predators have been selected to prioritise their own survival above the current breeding attempt, in order to maximise lifetime reproductive success. Endocrine mechanisms involving corticosterone, the ‘stress hormone’, and possibly prolactin, the ‘parental hormone’, are involved in mediating the abandonment of breeding in response to environmental perturbations. This thesis aimed to assess what the breeding success of a top marine predator indicates about changes in the marine ecosystem and what mechanisms control changes in breeding success, using the black-legged kittiwake Rissa tridactyla as the model species. I combined population-level analyses of long-term datasets (1997–2010) of diet composition, adult body mass, breeding success and foraging behaviour from the Isle of May, National Nature Reserve, Firth of Forth, south-east Scotland (56° 11‘ N, 02° 33’ W) with an individual-level field experiment to simulate chronic stress. Kittiwakes breeding in the north-western North Sea depend primarily on adult (1+ group) lesser sandeels Ammodytes marinus at the start of the breeding season and subsequently switch to depend primarily on young of the year (0 group) sandeels. Analysis of the long-term data showed that the timing of the kittiwake breeding season has become later in recent years, whilst the timing of the switch from 1+ group to 0 group sandeels in the kittiwake diet has become earlier, which may suggest mismatches in the timing of prey availability and predator demand. Increasing proportions of clupeids (mainly sprat Sprattus sprattus) were seen in the diet and further years of study may reveal whether clupeids could be a beneficial alternative prey type for kittiwakes. Foraging trip duration was unrelated to diet composition, suggesting that the main prey types of kittiwakes do not differ in their distance from the colony. Whilst foraging trip duration during incubation was related to changes in adult body mass and hatching success, diet composition was unrelated. There was a weak effect of diet composition during chick-rearing on fledging success, mediated via changes in adult body mass. However, this effect was masked by a stronger, independent, negative effect of foraging trip duration during chick-rearing. To simulate chronic stress in kittiwakes, individuals were implanted with corticosterone, using Alzet® osmotic pumps, for a week at the end of incubation. The methodology applied to kittiwakes was based on a preliminary experiment carried out in Japanese quail Coturnix coturnix japonica. The body mass and prolactin concentrations of kittiwakes were unchanged after this treatment. Corticosterone concentrations had returned to pre-treatment values by the end of the treatment week, which may have been due to down-regulation or suppression of the stress response as a result of the treatment. Corticosterone-implanted males showed lower nest attendance than sham-implanted males but the opposite was true for females. Breeding success at the end of the season was lower in corticosterone-implanted birds, suggesting a prolonged effect of chronic stress. In order to investigate the effects of disturbance to a group or colony of birds prior to the capture of an individual, a preliminary experiment was also carried out to test the stress responsiveness of a captive bird, the Japanese quail. No increase in corticosterone concentrations was seen after a capture-restraint protocol and with increasing time since the group of birds was first disturbed. A suppressed stress response in this bird may be explained by long-term captivity or domestication. These results show that the breeding success of a top marine predator can indicate changes in the timing of prey availability and prey location, mediated through changes in adult body mass. I also found that changes occurring during the chick-rearing period contributed most to the outcome of the breeding season. Chronically elevated concentrations of corticosterone are important in the control of breeding success, whereas prolactin may only play a role close to the timing of breeding failure or after failure has occurred. This thesis demonstrates the need for continued long-term monitoring of wild populations and refining of experimental methodology to better understand the impacts of environmental change on top predators.

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.

The objective of this dissertation is to use modelling and statistical approaches to expand our knowledge of the immune responses against gastrointestinal nematode infections, to assess the impact of nematode infection, and to use our improved knowledge to examine novel means of selective breeding in farm animals (sheep) as a control strategy. To expand the knowledge of the host immune response against infection, Chapter 2 of this dissertation focuses on immunoglobulin A (IgA), an antibody that binds nematode molecules, and its transfer through the body from the abomasal mucus (i.e. site of infection, where it is produced) to the blood plasma, where it is typically measured. These findings have been published in Parasitology (Prada Jimenez de Cisneros et al., 2014a). The implications of low levels of infection in adult milking ewes, which are more resistant than lambs, were also studied. A relationship is generated between infection levels using parasitological data and production data. There were however limitations in the dataset, which are discussed at the end of Chapter 3. Parasite resistance in adults sheep at low levels of infection was also studied, especially since the most common parasitological marker of disease is the faecal egg count (i.e. number of nematode eggs in the animals faeces) which is subject to substantial measurement error, among other limitations. Chapter 4 analyses a dataset of adult animals with low infection levels using a zero inflated binomial model (ZINB) and extends the model by including other evidence of parasite resistance to discriminate between exposed and unexposed animals. To examine selective breeding, an individual-based data-driven immunogenetically explicit mathematical model was developed. One application of this model is to compare the efficacy of two selective breeding schemes, each based on a different marker for disease, namely faecal egg counts and plasma IgA. This work has been published in Journal of the Royal Society Interface (Prada Jimenez de Cisneros et al., 2014b). The model can be extended to create a distribution for the variation in larval intake that best fits the field data. This allows the partitioning of the variation in adult worm burden into different components. The purpose is to quantify the contribution of the immune response and larval intake to determine which of the two accounts for more of the variation in the level of infection. The model can be also extended to explore selection schemes in the two components of the immune response (i.e. namely the IgA mediated and IgE mediated immune response) and estimate animal size at the end of the grazing season.

It is widely recognised that both nutrient-driven processes acting from the “bottom-up” and predator-driven processes acting from the “top-down” are important drivers of population change. However, studies that examine how these joint forces influence the population dynamics of oceanic species are lacking. In the Antarctic and Sub-Antarctic ecosystem, human-mediated changes have driven biological change at both ends of the food chain; rapid regional warming at the bottom and heavy exploitation of apex predator populations at the top. Consequently, many populations of marine predators have rapidly changed in size over the last 50-years. Unravelling the effects of bottom-up and top-down forcing on these open ocean ecosystems, has thus been highlighted as an immediate priority for polar scientists. The overall aims of this study were to use demographic, environmental and diet data to unravel the processes that contributed to a population of macaroni penguins at South Georgia declining rapidly between 1985 and 2012. I use mark–recapture modelling to examine the survival rates of macaroni penguins. Over 10 years, birds were marked with subcutaneous electronic transponder tags and re-encountered using an automated gateway system fitted at the entrance to the colony. These findings were combined with a 28-year time series of population counts and productivity measurements in an age-structured state-space population model to disentangle the processes underlying the observed population decline. Finally, I combined stable isotope analysis and tracking data to investigate the individual strategies macaroni penguins might employ to mitigate the effects of density-dependence during the breeding season. Macaroni penguins at South Georgia declined at 6% per year between 1985 and 2000, stabilising thereafter. This study indicates that the population declined in response to recruitment rates being lower than adult mortality. This trend was potentially accelerated by three large mortality events that were possibly associated with top-down predation pressure from giant petrels. Survival rates were low and variable during the fledging year, increasing to much higher levels from age 1 onwards. Year-to-year variability in demographic rates was induced by a combination of individual quality, top-down predation pressure and bottom-up environmental forces. The relative importance of these covariates on survival rates was age-specific, whereby predation pressure had a considerably greater effect during the fledgling year compared with birds older than 1-year. The population trajectory stabilised after 2000 in response to an increase in survival, as well as density-dependent feedbacks upon productivity. In order to minimise the effects of density-dependence during the breeding season and optimise daily energy expenditure, macaroni penguins appeared to make distinct dietary choices that remained highly consistent from year-to-year. Individually specialised foraging strategies occurred in response to seasonal variations in foraging range, conspecific density and prey availability at the foraging sites. The future stability of this population will depend on the carrying capacity of the environment supporting productivity rates at their present level, and the population size and breeding success of giant petrels not increasing so that adult survival rates can remain stable. More broadly, this study highlights the importance of considering multiple causal effects across different life-stages when examining the demography of seabirds, and demonstrates the additional insights that can be gained by using models with increased precision.

Human embryonic stem cells are derived from the pre-implantation blastocyst, residing in an extremely low oxygen environment. Application of this in vivo oxygen concentration to an in vitro setting has previously shown to be essential for driving the self-renewal and directed differentiation of human pluripotent stem cells. However, studies on hESCs at low oxygen have been frequently contra-indicatory. Here, comparative analysis of a hESC and iPSC line at a low oxygen concentration resulted in divergent effects across the two cell lines. Augmented TGFβ signalling was observed in conjunction with up-regulated transcription of pluripotency markers NANOG and POU5F1 in hESCs but not iPSCs. IPSCs also shifted to a state of increased proliferation whereas hESCs did not. It was also observed that exposure of hESCs and iPSCs to light throughout low oxygen culture induced large amounts of apoptosis, highlighting the requirement for careful selection of cell culture equipment for environmental oxygen control. Both the embryonic and adult brain retain tissue specific oxygen concentrations far below that at atmospheric oxygen concentrations. Previous studies showed that embryonic silencing of factors responsive to low oxygen caused a range of embryonic abnormalities, including deformation of the neural plate and tube. Here, differentiation of hESCs to an early neuroectodermal identity at low O2 did not definitively augment production of NPCs but did additively suppress BMP signalling above that at atmospheric O2. A concordant rise in apoptosis was also observed in a manner both independent of, and augmented by, exogenous BMP inhibition. Subsequently, neurogenesis at low oxygen produced terminal neurons with accelerated and augmented synaptic and induced excitability. This was characterised by a rise in the rate of membrane depolarisation, increased action potential overshoot and accelerated expression of pre-synaptic marker Synaptophysin. These results highlight a novel, critical role for low oxygen in augmenting the excitability of hESC derived neurons.

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.