Brain-selective kinases (BRSK1 and BRSK2) are serine/threonine kinase members of the AMPK-related family of protein kinases, the majority of which are regulated by the upstream kinase LKB1 whilst AMPK itself is regulated by CaMKK. The BRSKs are highly expressed in brain and have been implicated in neuronal polarization and the regulation of neurotransmitter release. They have also been shown to be involved in the basal phosphorylation of tau at the Alzheimer‟s disease (AD) related residue, serine 262, and are highly expressed in areas of the brain affected by AD, namely the hippocampus and the cortex. I have utilised the model organism Drosophila melanogaster to investigate interactions between transgenically expressed human tau, human BRSKs and upstream regulators Selective over-expression of 0N4R human tau in the Drosophila eye resulted in a disruption of eye morphology. In contrast, over-expression of human wild type BRSK2 (B-WT) had no obvious effect on the eye. However, co-expression of both tau and B-WT resulted in a neurodegenerative phenotype more severe than tau alone. This enhancement of phenotype was not observed when BRSK2 was expressed that either lacked the activating phosphorylation site (non-phosphorylatable, B-NP) or that is unable to bind ATP (kinase dead, B-KD). Co-expression of human tau and BWT significantly elevated tau phosphorylation at S262, suggesting that S262 is a key residue for tau-induced toxic phenotypes and the BRSK/tau interaction I observe. In support of this, no phenotype was observed in flies expressing the S262A variant of human tau with or without B-WT. To establish the upstream kinases responsible for activating human BRSK2 in Drosophila I removed endogenous Drosophila LKB1 by RNAi. This prevented the enhanced degeneration of the eye caused by tau/B-WT co-expression, demonstrating that LKB1 is a key upstream regulator of BRSK2. I also found that down regulation of the Drosophila CaMKK homologue, CG17698, by the same method, ameliorated B-WT induced eye degeneration implicating a calcium-dependent pathway in the regulation of BRSK. Over-expression of human CaMKKα in the CG17698 RNAi background prevented the rescue seen with CG17698 RNAi. Over-expression of cac1, a calcium channel subunit, in the presence of B-WT and human tau exacerbated the B-WT induced eye phenotype in a B-WT dependent manner, supporting the hypothesis that the human tau and B-WT interaction can be regulated in a calcium-dependent manner. Expression of total BRSK2, LKB1 and CaMKK were not altered in human postmortem AD brain tissue when compared to control. However, with the exception of LKB1, due to limited reagents and time constraints I was unable to investigate the proportion of phosphorylated (and thus active) to total kinase. This study defines a novel Ca2+ -dependent regulatory pathway to tau, which may contribute to AD and other tauopathies.

Protein disulphide isomerase (POI; EC 5.3.4.1) is a multifunctional enzyme which resides in the lumen of the endoplasmic reticulum (ER). It is approximated that over one-third of all human proteins fold in the ER. POI is one of the main folding catalysts, specifically facilitating native disulphide bond formation. POI has four domains, three of which have been solved and all possessing a thioredoxin-type fold consisting of the J3-a-J3-a-J3-a-J3-J3-a structure. Due to the intransigent nature of the b' domain of POI, it remains structurally unelucidated. The b' domain is vitally important as it holds the principal binding site and is essential for POI activity. The investigation of the two species which were produced during expression of most b' domain containing constructs led to the further biophysical analysis and identification of the monomer and dimer species. It was found that fractionation of the monomer and dimer species was vital in obtaining high resolution NMR data. An elaborate method using assignments from the b domain and b'x were used, and proved very effective in achieving the goal of full backbone assignment of the large 27.5 kOa bb'x protein molecule. The fruit of this labour is that it allowed further probing dynamic and more detailed molecular NMR analysis. NMR analysis identified evidence that the bb'x monomer species can exist in two forms, the closed form where the x-region binds to the b' domain and open form where the x-region is unbound. Chemical shift analysis revealed several key residues involved in protein flexibility and chemical shift mapping revealed the interface between the b domain and b'x. Defining the interface described here offers a method to model the full length POI domain structural organisation; which can be used to more clearly define POI function and how the domains of POI are coordinated in protein function. More detailed NMR analysis of relaxation dynamics (T1 and T2) revealed significantly differing motions attributed to the b, b' domains and x-region in the bb'x construct. The slower b' domain motion is believed to be related to the substrate binding function, where the b' domain appears to be gently flexing, in search for a substrate molecule.

ATP release from the surface of the ventro-lateral medulla (VLM) is integral to the hypercapnic response in vivo and can be seen in vitro. By employing horizontal slices of the ventral medulla containing the ventral chemosensitive nuclei, I have developed a model that consistently evokes hypercapnia-induced ATP release in vitro. Using this preparation I have studied CO2-triggered ATP release by means of microelectrode biosensors. I conclude that it is the change in PCO2 itself, and not associated pH changes that accompany it, that is directly responsible for eliciting ATP release from the surface of the VLM. In addition ATP release from this region may have a role in the response to hypocapnia as well as hypercapnia. Using pharmacological agents I have demonstrated that gating of connexin hemichannels mediates ATP release. The dorso-ventral distribution of Cx26 ascertained via quantitative PCR and immunofluorescence makes this hemichannel the most likely candidate. Dye loading the cells responsible for ATP release with carboxyfluorescein, which co-localised with Cx26, revealed these cells reside in the pia mater and subpial astrocytes. Application of gap-junction antagonists, with selectivity towards connexin 26, greatly reduced ATP release in response to elevated CO2 in vitro and in vivo and reduced the tone of ATP at the VLM surface. Moreover, by loading Cx26 expressing HeLa cells with ATP, I was able to recapitulate the entire in vivo response. Therefore I propose that ATP is released from sub-pial astrocytes and leptomeningeal cells through connexin 26 hemichannels in response to alterations in PCO2. Here Cx26 performs a dual role, as both the chemosensory transducer and the conduit for ATP release.

Helix-helix interactions between membrane-spanning transmembrane (TM) domains have been shown to drive the assembly of α-helical membrane proteins within biological membranes. However, the rules that determine these interactions are not yet fully understood, despite such interactions being found in an increasing number of proteins. Recent work has implicated TM domain interactions in the formation of the protein complex Ii-MHC, formed from the association of Major Histocompatibility Complex Class II (MHC) and the MHC-associated-Invariant Chain (Ii) proteins. Following biosynthesis, three MHC α/βheterodimers bind to the Ii homotrimer to form a nonameric Ii-MHC complex within the endoplasmic reticulum. This is a critical step in the export of MHC molecules to the antigen presentation system and hence the activation of an immune response to a pathogen. In this study we have explored the TM domain interactions within the Ii-MHC complex. Results from in vivo and in vitro experiments revealed the TM domains of the α- and β-chains of MHC have a propensity to self-associate into homo-dimers and to associate with one another to form hetero-dimers. Highly conserved GxxxG motifs (known to drive dimerization) were implicated in these interactions. The TM domain of Ii was confirmed to self-associate to form trimers by in vivo and in vitro methods, but surprisingly also displayed additional oligomeric states suggesting the interaction is not as specific as was previously thought. Furthermore, we show that in vivo, the TM domain of Ii can associate with those of the α- and β-chains of MHC, whilst in vitro methods suggested Ii preferentially binds to α-chains. Collectively, these findings strongly suggest that the TM domains of Ii and MHC have a role to play in the assembly of the Ii-MHC complex, and hence the very important process of antigen presentation. Additionally, in this study we have undertaken development of NMR spectroscopy methods that have the potential to increase our understanding of not only the Ii-MHC complex, but protein-protein interactions in general.

Ribavirin, 1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide, is a broad spectrum antiviral agent active against both DNA and RNA viruses. A range of 1,2,3-triazole, 1,2,4-triazole and benzotriazole analogues of ribavirin was synthesised using a crude extract of N-deoxyribosyltransferases from Lactobacillus leichmannii in the chemoenzymatic synthesis of nucleoside analogues with potential antiviral activity. N-Deoxyribosyltransferase catalyses the transfer of the 2-deoxyribose sugar between purine and pyrimidine bases. Of thirty two bases synthesised, twelve were found to act as acceptors in the N-deoxyribosyltransferase reaction. Nine 2'-deoxyribofuranosyl-1,2,4-triazole nucleoside analogues were isolated and characterised. Only one nucleoside showed any significant antiviral activity. 2'-Deoxyribavirin was found to possess antiviral activity against the influenza virus. Attempts were made to synthesise the nucleoside chemical from ribavirin on a larger scale, but without success. However, 5'-iodo-5'-deoxyribavirin and 5'-deoxyribavirin were synthesised, the former showing slight antiviral activity. The specificities of the N-deoxyribosyltransferases for purine, pyrimidine and triazole bases are discussed, gathering together the findings of previous workers with the results presented in this thesis.

G protein-coupled receptors (GPCRs) allow cells to respond to extracellular stimuli and are involved in virtually all major physiological processes in eukaryotes. Consequently, aberrant GPCR signalling can lead to disease, making them attractive candidates for research and the development of pharmacological interventions. However, GPCR signalling responses in higher eukaryotes are complex, with the presence of multiple signalling pathways hindering the isolation and study of specific signalling components. The model organism, Schizosaccharomyces pombe, provides a simplified system with which to investigate GPCR signalling in the pheromoneresponse pathway. Pheromone stimulation in Sz. pombe causes the production of proteins necessary for the mating process, including the carboxypeptidase Sxa2. This study utilises Sz. pombe reporter strains in which sxa2 has been replaced with the bacterial reporter lacZ, thus enabling quantification of the level of signalling through the pheromoneresponse pathway. Using this system, the pheromone-responsive Mam2 receptor was investigated, with specific focus on the large intracellular C-terminal tail. Truncating Mam2 to remove the C-terminal tail altered signalling by increasing the level of ligand-independent signalling and decreasing the level of maximal signalling. Similar effects were observed when a regulator of G protein signalling, Rgs1, was removed from strains containing the full-length Mam2 receptor. This suggested a relationship between these two signalling components, which was confirmed through yeast 2-hybrid analysis. Using this approach, an 8-residue section of the Mam2 C-terminal tail was found to be necessary for a direct interaction with Rgs1. Further characterisation of the Mam2 tail revealed an additional role for this region in receptor sensitivity to pheromone stimulation. Mutational analysis implicated three serine residues in receptor sensitivity, suggesting that the C-terminal tail of Mam2 may contribute to receptor internalisation from the plasma membrane therefore enabling desensitisation to prolonged pheromone stimulation. Similar techniques applied to the study of Rgs1 revealed that two conserved DEP domains in the N-terminus of Rgs1 are important for the interaction with the Mam2 C-terminal tail. Fluorescent tagging of Rgs1 and subsequent microscopic analysis indicated that Rgs1 localises to endomembranous structures surrounding the nucleus, contrary to predictions made by mathematical models developed by this group, which suggest that Rgs1 requires plasma membrane-localisation in order to function. A better understanding of the action of Mam2 and Rgs1 in the Sz. pombe pheromone response can inform mathematical models and future studies involving the more complex mammalian signalling cascades.

The release and clearance of adenosine are reasonably well-documented in the mature CNS but relatively little is known about how adenosine signalling changes during postnatal development. The activation of presynaptic A1 receptors (A1R) at cerebellar parallel fibre terminals is known to inhibit synaptic transmission and the expression of A1R has been observed in mature rat cerebellar slices. However its distribution during development or in relation to parallel fibre–Purkinje cell (PF-PC) synapses has not previously been described. In the mature cerebellum blockade of presynaptic A1R at PF-PC synapses enhances synaptic transmission suggesting an inhibitory adenosine tone and an extracellular purine tone is detectable with microelectrode biosensors under basal conditions. The active release of adenosine can be stimulated with trains of activity in the molecular layer of mature slices although this does not appear to be a source of the basal extracellular adenosine tone. This study used immunohistochemistry to determine the distribution of A1R at PFPC synapses in cerebellar slices at postnatal day 3 prior to PF-PC synapse formation, postnatal days 8-14 and postnatal days 21-28. This study also used cerebellar slices from rats at postnatal days 9-14 to investigate the pharmacological profile of the immature rat PF-PC synapse with electrophysiology and microelectrode biosensors. The immunohistochemistry suggests that A1R are widely distributed across Purkinje cell bodies and their dendrites and within the granule layer of the cerebellum and that its expression does not change during development. The same staining patterns were also observed prior to PF-PC synapse formation. Application of adenosine resulted in a variable A1R-mediated inhibition at immature PF-PC synapses. This did not appear to be gender-specific or correlated with age of rat and the synapses otherwise appeared identical in their properties. The comparison of log concentration-response curves generated for an A1R agonist suggested that some A1R may have a lower efficacy at this stage of development. Blockade of presynaptic A1R at immature PF-PC synapses suggested that an inhibitory adenosine tone is low or absent at this stage of development and is not the result of a low A1R expression or developmental differences in A1R efficacy. Inhibition of adenosine clearance via adenosine deaminase, adenosine kinase and equilibrative transporters had little effect on synaptic transmission suggesting that little adenosine is moving between the intracellular and extracellular spaces under basal conditions in immature slices. Active adenosine release measured by electrophysiology and microelectrode biosensors could be stimulated with hypoxia in immature slices but this was delayed and slower in comparison to the release observed in mature slices. Adenosine could not be actively released at immature PFPC synapses in response to electrical stimulation in the molecular layer.

Ras signalling is vital to many cellular processes. Ras proteins mediate a vast array of cellular signalling networks, and are conserved from humans to unicellular eukaryotes. The study of ras signalling in higher eukaryotes presents a number of technical challenges, due to the presence of multiple ras isoforms, regulatory proteins and activators. The fission yeast Sz. pombe represents an ideal system for the investigation of ras signalling, as it contains a single, nonessential ras protein (Ras1). In addition, Ras1 is involved in the regulation of a number of downstream pathways. A number of studies in recent years have highlighted the role of subcellular localisation in ras signalling output. The localisation of Ras1 in Sz. pombe has also been described as key in effector selection, with Ras1 at the plasma membrane regulating mating and Ras1 at the endomembranes regulating cell morphology. This thesis describes a series of studies utilising Ras1 mutants and chimeric Ras1 proteins which display differing localisation patterns to determine the role of Ras1 localisation in signalling. The data presented herein support the notion of a revised model for the role of Ras1 localisation in signalling, suggesting that the localisation of Ras1 to the plasma membrane is key to all signalling events downstream of Ras1. This thesis also describes the characterisation of oncogenic mutants of Ras1, demonstrating the importance of signalling magnitude in functional output. In addition, the importance of Ras1 regulation in cell viability and chromosome stability is also demonstrated. Finally, the functional expression of three human ras isoforms is described, validating the use of Sz. pombe as a model system for the heterologous expression of human ras signalling components.

NSP4, encoded by rotavirus genome segment 10 has been shown to be a transmembrane, endoplasmic reticulum (ER) specific N-linked glycoprotein. Consistent with its localization to the ER membrane, NSP4 was first shown to have a role in the morphogenesis of the infectious virion. The protein has also been reported to have cytotoxic activity when applied extracellularly to cells. Consequently it has been earmarked as an enterotoxin being secreted from virus-infected cells to cause early cellular pathology in the gut. The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions. Site-directed mutagenesis was used to block N-linked glycosylation at both of the known glycosylation sites near the amino terminus of NSP4. This revealed that the NSP4 induced formation of the cytoplasmic extrusions was dependent on the protein’s ability to become fully glycosylated. The cytoplasmic extrusions seen in cells expressing glycosylated NSP4 were also evident in virus-infected cells. Using real-time confocal microscopy a dynamic elongation of the cytoplasmic extrusions with a growth speed of 2 μm/min was observed in virus-infected cells. The cytoplasmic extrusions were found to contain β-tubulin and F-actin. Inhibiting their polymerization prevented the formation of the extrusions from virus-infected cells. Functional studies using Cell Tracker dyes showed that the cytoplasmic extrusions could disseminate vesicles from virus-infected cells onto the plasma membrane surface of uninfected cells. The vesicles were then found in the interior of the uninfected cells. Mono-specific antibody to NSP4 revealed the presence of the protein in the vesicles suggesting that the cytoplasmic extrusions facilitated the direct cell-cell spread of NSP4. The effect of NSP4 expression on the microtubular network of cells was analysed. It was found that NSP4 de-polymerized the microtubular network from the centre of cells and promoted the assembly of microtubules at the periphery of the cells in a glycosylation independent manner. Similar de-polymerization and re-assembly of the microtubules was observed in the virus-infected cells. Interestingly in the presence of nocodazole, tubular structures containing tubulin and viral proteins excluding NSP4 were found in virus-infected cells. A YFP-PCA assay was established to screen for cellular partners of NSP4. The functionality and the sensitivity of the assay were examined, but only two false positive colonies were isolated in the first screening. In conclusion, the function of glycosylated and unglycosylated NSP4 was examined with the former possessing the ability to promote the formation of the cytoplasmic extrusions from cells and both being capable of disrupting the microtubular network indicating that two forms of NSP4 play different roles in NSP4 function. The cytoplasmic extrusions seen in our studies may be relevant to rotavirus infection and pathogenesis.

An interruption of the blood supply to the brain, as occurs during ischemic stroke, results in a rapid decline of ATP levels and a subsequent loss of neuronal function and viability. Under physiological conditions the brain reuses ATP degradation metabolites, such as hypoxanthine, via the purine salvage pathway, to restore its ATP pool. However, the massive degradation of ATP during ischemia results in the accumulation and loss of diffusible purine metabolites and thereby leads to a reduction in the post-ischemic ATP pool size, leaving the brain more vulnerable to secondary ischemic insults (recurrent strokes) and less able to deploy reparative mechanisms. The aim of this study was to improve the recovery of post-ischemic ATP levels by enhancing the purine salvage pathway, with substances that are already known to be tolerated in humans. Using acute hippocampal rat brain slices, I found that 1 mM Ribose (Rib) and 50 μM Adenine (Ade), two main metabolites of the purine salvage pathway, significantly increased the tissue ATP levels under basal conditions. Rib/Ade pre-treatment results in accelerated decline of synaptic transmission after onset of oxygen/glucose deprivation (OGD), due to increased adenosine release. However, this intervention does not delay the onset of anoxic depolarisation, or improve the recovery of synaptic transmission after prolonged ischemic periods. Pre-treatment of brain slices with 1 mM creatine, which increases phosphocreatine levels and thereby buffers the rapid decline of ATP levels upon energy shortage, significantly delays the onset of AD and helps to improve the recovery of synaptic transmission. By using cultured cerebellar granule cells, for more protracted studies on cell viability after OGD, I show that addition of Rib/Ade after ischemia helps to improves cell viability. Therefore my results suggest that both, delaying the decline of ATP upon onset of OGD (pre-treatment with creatine), or enhancing the post-ischemic recovery of ATP (post-treatment with Rib/Ade) are useful strategies to improve cell survival and function after in vitro ischemia.