Improving cognitive function is a growing area of interest for pharmaceutical companies. With an ageing population, cognitive decline is becoming a greater problem. Understanding the physiological effects of nootropic drugs and the changes that occur during cognitive enhancement will enable the design of safer treatments to enhance cognition. In this thesis cognitive enhancing strategies are investigated using neurophysiological and proteomic approaches. The effects of two classes of putatively cognitive enhancing drugs on emergent network oscillatory activities in hippocampal slices are investigated. The effect of an enriched environment, which causes an improvement in cognitive function, on the expression level of proteins in the hippocampus is also investigated. The development of a new muscarinic acetylcholine receptor (mAChR) agonist that is selective for the M1 mAChR subtype, called 77-LH-281, has recently been achieved. 77-LH-281 binds to an allosteric site of the M1 mAChR which accounts for its increased selectivity over other mAChR agonists. This agonist causes gamma frequency oscillatory activity in hippocampal slices, a pattern of network activity that the in vivo equivalent of which is associated with cognitive processes. This gamma activity is dependent upon both excitatory and inhibitory networks. 77-LH-281 does not promote epileptiform-like activity in naïve slices as well as a range of models of epileptiform activity, unlike non-subtype selective mAChR agonists like oxotremorine-M. Oxotremorine-M changes the slow inter-ictal-like events following application of 4-AP and NBQX into continuous beta frequency oscillations. This action is not mediated by the M1 mAChR. Thus selective M¬1 mAChR display a preferable range of oscillatory activities compared to non-subtype selective mAChR agonists. Ampakines are a further class of nootropic drugs. Ampakines are positive modulators of AMPA-type glutamate receptors and they cause improvements in cognitive function of laboratory animals and humans. The ampakines investigated in this thesis are CX691, which increases the amplitude of currents through the AMPA receptor, and CX546, which increases the length of time the AMPA receptor is open. These ampakines do not induce oscillatory activity in naïve hippocampal slices, but they increase the frequency of inter-ictal-like epileptiform activity. CX546 also induces ictal-like activity in the 4-AP induced epileptiform event model. Ampakines may therefore promote epileptiform activity in individuals that are susceptible to epilepsy. Exposure to an enriched environment leads to improvements in cognitive performance. This behavioural change is mediated by changes at the level of the proteome. Exposure to an enriched environment changes the expression of many classes of proteins including signalling proteins and proteins that are involved in the structural changes that occur during cognitive enhancement. One of the proteins that significantly changes in expression is a protein that is associated with cognitive deficits, known as MeCP2. MeCP2 is a transcriptional repressor and increases in expression in the enriched environment. This thesis demonstrates the diversity of molecular, cellular and network level approaches that can be used to induce and investigate cognitive enhancement. A combination of these approaches enables the in vitro evaluation of current cognitive enhancing strategies and may lead to the the development of novel approaches to enhance cognitive function.

Ethanol is a poison that adversely affects the health of individuals and is detrimental to society as a whole. Analysis of ethanol in biological matrices is the most frequent test carried out in forensic toxicology laboratories with application across a range of cases types including fatalities, road traffic accidents, criminal investigations and workplace drug testing. The interpretation of ethanol concentrations in post mortem samples can be challenging in relation to medico-legal investigations. The source of ethanol can be as a result of the ingestion of an alcoholic beverage or it may have been formed after death. The stability of alcohol is also adversely affected by the presence of bacteria, high temperatures and unsuitable storage containers. A robust and sensitive method was developed for the analysis of common volatiles such as ethanol, methanol, isopropanol, acetone and n-propanol using headspace gas chromatography coupled with a flame ionization detector (HS-GC-FID). The method was validated in accordance with ISO/IEC 17025, and was used to investigate the stability of volatiles in blood when stored under different conditions, and to investigate the prevalence of volatiles in different biological matrices collected post-mortem. Storage of blood samples in the freezer within sample vials containing preservative and antioxidant improved the stability of all volatiles with the exception of methanol which remained stable under all conditions investigated. The identification of ethanol or acetone in vitreous humour was found to be a suitable alternative matrix in cases where femoral blood was unavailable or ethanol production was suspected. The concentration of ethanol in bile correlated well with femoral blood but to a lesser extent than vitreous humour. Urine was not a suitable matrix for estimating blood ethanol concentrations. Alcohol biomarkers, Beta-Hydroxybutyrate (BHB) and fatty acid ethyl esters (FAEEs) have been reported as useful markers for, investigating the role of alcoholic ketoacidosis (AKA) in post-mortem cases, and foetal exposure to chronic maternal alcohol consumption, respectively. Methods were developed and validated in accordance with ISO/IEC 17025 for BHB in blood and urine using gas chromatography - mass spectrometry (GC/MS), and for FAEEs in meconium by liquid chromatography - tandem mass spectrometry (LC/MS/MS). Post-mortem case samples submitted to the Forensic Medicine and Science (FMS) toxicology laboratory for routine tests were analysed for BHB using the validated method. The findings highlighted the importance of measuring BHB in blood in all cases where the deceased has a history of alcohol misuse and where the cause of death cannot be determined following the post-mortem. The role of alcoholic ketoacidosis in the cases analysed in this study was significantly under-reported. Meconium samples collected from infants born at the Princess Royal Maternity Hospital, Glasgow, were analysed for FAEEs, using the validated method to investigate the prevalence of each FAEE (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate). The study found evidence of chronic maternal alcohol consumption in approximately one third of the cases tested, in contrast to self-reported use and highlights the importance of screening for the presence of FAEEs in meconium. The identification of suitable biomarkers of excessive maternal alcohol consumption during pregnancy, carried out as part of screening program, in addition to clinical evaluation would help to diagnose and support newborns with Foetal Alcohol Spectrum Disorder (FASD). The method developed for the analysis of BHB in blood and urine was successfully adapted and validated for analysis of structurally similar drugs such as Beta-hydroxy-Beta-methylbutyrate (HMB), a legal dietary supplement, in plasma and urine collected from 8 subjects, pre- and post-administration of a 3g dose of HMB. A significant increase was observed in urine and plasma following administration of of HMB. The method was then applied to the analysis of post-mortem blood and urine to investigate the concentrations of exogenous and endogenous levels of γ-hydroxybutyrate (GHB).

SNARE proteins are essential components of the machinery that facilitates membrane fusion in eukaryotic cells. SNARE proteins are subject to multiple levels of regulation, one of which is imbedded in the syntaxin (Qa-SNARE) molecule. It has been demonstrated that some syntaxins adopt a closed conformation, whereby an autonomously folded N terminal domain (the Habc domain) forms intramolecular contacts with the SNARE domain; this conformation precludes complex assembly. The Sec1p / Munc18 (SM) family are a conserved group of proteins that regulate membrane fusion through interactions with their cognate syntaxins. Formulation of unifying hypotheses describing how SM proteins function has been problematic, primarily due to the multiple modes of interaction that have been characterised for different members of this family binding to their cognate SNARE proteins. The yeast SM protein Vps45p regulates membrane fusion through the trans-Golgi / late endosomal system, and interacts directly with the syntaxin (Tlg2p) and the v-SNARE (Snc2p) proteins. Vps45p also binds to the assembled SNARE complex of Tlg2p, Vti1p, Tlg1p and Snc2p. In this thesis I demonstrate that the Habc domain of Tlg2p has an inhibitory effect on SNARE complex formation. This is an important finding, as whether or not Tlg2p adopts a closed conformation has hitherto been controversial. Furthermore, I have demonstrated that the inhibitory effect of the Habc domain on complex formation can be alleviated by Vps45p in vitro. In addition to investigating the functional significance of Vps45p’s interaction(s) with Tlg2p, I have also investigated binding of the SM protein to the v-SNARE Snc2p. I have demonstrated that the affinity of Vps45p for Snc2p is weaker than either of the modes of interaction characterised between Vps45p and Tlg2p. Finally, I have developed an in vitro fusion assay to enable us to dissect the functional significance of the various interactions that Vps45p displays with its cognate SNARE proteins.

Currently, the field of mammalian membrane protein structural biology is in its infancy. Existing technologies and experiences have shown that it is possible to obtain the structures of mammalian membrane proteins if sufficient work and thought has been invested. However, there is still an urgent need to develop new methodologies and approaches to improve all aspects of this important area of biological research. Here, a series of novel technologies for the overproduction, purification and crystallisation of human membrane proteins are described which have been tested with a representative member from each of the G-protein coupled receptor (adenosine 2a receptor (A2aR)) and membrane enzyme (sterol isomerase (SI)) superfamilies. The methylotrophic yeast Pichia pastoris is an excellent host cell for the overproduction of recombinant proteins including membrane proteins of mammalian origin. However, the commercially available expression vectors are far from what is required to maximise the production levels as well as simplify the detergent extraction and purification of human membrane proteins. Here, a series of related expression constructs were made that had different combinations of tags at both ends of the recombinant protein. The final optimised expression vectors had a C3 protease-iLOV-biotin acceptor-His10 (CLBH) tag fused to the C-terminus of the recombinant protein. The -CLBH vectors gave high level production of both test proteins (one Nin – hSI; one Nout – hA2aR) that could be rapidly purified to homogeneity using a generic protocol. The position of the His10 tag did not affect the expression level of the recombinant protein. In contrast, fusion of the biotin acceptor domain to the C-terminus of the recombinant protein increased its expression by a factor of between 2-4. The biotin acceptor domain could also be fully biotinylated in vitro using recombinantly expressed biotin ligase allowing purification/immobilisation of the target protein with streptavidin beads. Removal of the expression/ purification tags from the recombinant proteins with C3 protease occurred more efficiently than when TEV protease was used. An optimised protocol was developed that gave maximal production of our target proteins in fermenter culture at an induction temperature of 22°C. Care was taken to find a methanol feed rate that gave the highest levels of protein production without causing the accumulation of excess methanol in the culture (which is known to be toxic to the yeast). Using this protocol it was possible to make both hSI and hA2aR with a production level >10 mg of recombinant protein per litre of culture. As most MPs are colourless, target protein identification is usually performed by methods such as radioligand binding and/or Western blotting. However, these techniques can be time-consuming, use a lot of protein and do not give any information on the aggregation state of the protein in detergent solution. Previously, it has been shown that the processes of identifying and analysing membrane proteins in detergent solution can be accelerated by attaching green fluorescent protein to the C-terminus of the recombinant MP. Here, the potential of the recently described iLOV fluorescence tag for membrane protein applications was assessed. iLOV was shown to be an useful tool for optimising processes such as yeast clonal selection, protein production in fermenter culture, detergent and construct screening as well as tracking recombinant MPs through the purification process. Of note, the iLOV tag allowed a direct assessment of the stability and dispersity state of both target MPs in a range of detergents by fluorescence size exclusion chromatography (FSEC). Using this approach, it was shown that wild-type hA2aR solubilised using a combination of dodecyl-βDmaltoside (DDM) and cholesteryl-hemisuccinate (CHS) aggregated during purification on a Ni2+ column. Furthermore, it was shown that the hA2aR agonistconformationally-fixed mutant Rag23 is stable in DDM without any CHS present. Moreover, Rag23 was found to be monodisperse in a series of short-chain detergents (decyl-βD-maltoside, nonyl-βD-maltoside (NM) and β-octylglucoside) suggesting that this mutant is well-suited to structural studies. SI was remarkably robust in short chain detergents demonstrating a reasonable level of stability in the short chain detergent NM. The FSEC experiments showed that wild-type SI has considerably higher intrinsic stability than native hA2aR suggesting that membrane enzymes will prove to be more amenable to structural analysis than GPCRs. Rag23 and SI were both purified to homogeneity in a simple four-step procedure: i) Ni2+ purification, ii) cleavage with C3 protease, iii) reverse Ni2+ purification and iv) gel-filtration chromatography. A buffer/salt screen was devised that allowedthose conditions where SI had maximal thermostability in detergent-solution to be identified. SI was found to have greatest stability in sodium phosphate buffer at acidic pH. Using this information, it was possible to purify monodisperse SI in DM suggesting that this protein may make an excellent candidate for structural studies too. Crystallisation trials with SI were performed using the commercially available sparse matrix screen MemSys/MemStart. In addition, a lipidic-sponge phase sparse-matrix crystallisation screen that was developed in collaboration with Prof. Richard Neutze (University of Chalmers, Sweden) was tested using SI. Cholesterol could be incorporated into all of the sponges that make up the screen upto a concentration of 10%. (This is important as the activity of many mammalian membrane proteins is cholesterol-dependent). To date, no diffracting crystals of SI have been obtained with either the conventional or lipidic-sponge phase crystallisation approaches. In short, a series of novel technologies/methodologies have been developed that will act as a platform for future efforts to solve the structures of a wide-range of human membrane proteins.

Epstein-Barr virus (EBV) is a human herpesvirus that has been associated over the past forty years with a number of malignancies both of lymphoid and epithelial origin. These include Burkitt's lymphoma (BL), Hodgkin's disease (HD) and nasopharyngeal carcinoma (NPC) to name only a few. One of the latent proteins of the virus, latent membrane protein 1 (LMPl) has been frequently detected in NPC biopsies and it plays a role in the initiation and development of the disease. Various strains of LMP1 have beed detected, but the LMP1CAO strain is the one most often encountered in endemic NPC cases. NPC tumours also show a deletion across chromosome 9p21which leads to loss of the tumour suppressor locus INK4a as well as deletion or hypermethylation of 3p21.3 that leads to loss of another tumour suppressor the Rassfl. The aim of the work presented in this thesis is to investigate the exact role LMPI plays both in the genesis and the development of epithelial malignancies such as NPC. In order to investigate this, transgenic mouse models expressing two different strains ofLMPl at their epithelium were used. Use of other transgenic and knock out mice was also involved, in order to investigate cooperative relationships that LMP1 may have with other oncogenes or tumour suppressor genes. Minimal skin chemical carcinogenesis was employed in order to determine the role of LMPI in initiation or progression of the tumourigenic process. LMPI CAO was found to be expressed in a wide variety of tissues in the transgenic mice, including both tissues of the epithelium as well as lymphoid tissues. LMPI CAO is a weak initiator as LMPI CAO transgenic mice develop lesions spontaneously and in some cases the ears of these mice progress from benign keratoacanthomas to malignant squamous cell carcinomas. LMPlcAO cooperates with loss ofINK4a locus to give an increased lesion load. Signalling pathways that were found to be activated by LMPI in lymphoid, epithelial cells or fibroblasts in previous studies, were investigated by Western blotting in order to determine whether they are activated by LMPI CAO in the epithelium in vivo. LMPI CAO in the epithelium in vivo, leads to activation of the p38, NF-KB, AP-I and MAPK pathways. Other proteins were shown to be upregulated or stabilised by LMPI including pS3, pI6INNK4ac,aspase-J and MMP9. Whether this is a direct effect of LMPI CAO or it is a secondary event due to the phenotype that LMPI causes is still unclear. The similarity between the LMPI transgenic mice and TGFa transgenic mice, as well as increased levels of the epidermal growth factor receptor (EGFR) in NPC biopsies and NPC cells in vitro, led us to investigate the possibility that LMPI may be acting via the TGFalEGFR pathway. Indeed, TGFa levels were found to be upregulated in transgenic affected tissues when compared to wild type sibling tissues. EGFR activates many signalling pathways including MAPK. Investigation of the MAPK pathway showed that LMPI does lead to its activation. In order to determine whether LMPI acts via upregulation of TGFa, LMPI transgenic mice were cross bred with TGFa null mice to create LMPI transgenic / TGFa null mice. The phenotype of these mice was observed and it was discovered that paradoxically, loss ofTGFa- a known oncogene- leads to a worsening of the phenotype. Further studies into the signalling pathways that may be affected by loss of TGFa showed that TGFa in this system may be acting as a tumour suppressor by upregulating Rassfl and also may be acting as a control of some of the signalling pathways activated by LMPI. The results show that LMP 1CAD is a weak initiator of proliferation but other cooperative events such as loss of tumour suppressors INK4a and/or Rassfl are needed for progression. This is consistent with previous studies performed in this laboratory as well as the facts that are currently known for NPC.

This thesis examines different variables affecting recruitment, and provides evidence of recent events affecting a seabird colony. Fieldwork was conducted during 2003-05 in the island of Foula, Shetland. Foula holds the largest colony of great skuas Stercorarius skua in the world; although numbers of breeding pairs in the colony increased rapidly from 1900-70, recently numbers have been decreasing. Data were collected by marking non-breeders and taking individual measures, individuals were followed in subsequent seasons to record their behaviour. An extended database was used to determine how long-term effects of variables such as hatching date, food availability and climate change affect the process of recruitment. The results show that food availability is related to breeding success and early hatching, as well as the probability of returning to the colony to breed. The variable used to quantify climate change (NAO winter index) was not related to recruitment, however it is suspected to influence food abundance. Contrary to expectation, individual quality did not have an effect on the probability of breeding for the first time, and there was no difference in body condition between potential recruits and established breeders; however historic data suggest a difference. The current situation faced by great skuas in the Foula colony may be a determined for the changes in recruitment rates as well as for the parameters that determine the recruitment process. Compared to two decades ago, numbers of pre-breeders have decreased substantially which may give evidence of density dependent effects preventing the addition of new recruits to the colony.

Glucose deprivation involved in hypoglycaemia has been associated with neurotoxicity and cell death. It is hypothesised that this neurotoxic process is initiated by a fall in cellular ATP concentration and a dysfunction of the Na+/K+ ATPase pump. Consequently the plasma membrane depolarises, opening the VGCC and allowing an excessive influx of calcium, which initiates glutamate release, ROS generation and the opening of the MTP. This intracellular activity subsequently triggers the apoptotic machinery necessary to promote irreversible cell death. In this study, primary cultures of embryonic rat striatal neurones were exposed to hypoglycaemia for periods between 1 hour and three days. Mitochondrial respiratory function and cytoskeletal integrity were affected. However several observations were found that conflicted with the general consensus of the mechanisms involved in hypoglycaemia-induced cell death. Evidence was obtained that there was :- 1. No calcium influx upon hypoglycaemia, indicating that the cell membrane does not depolarise 2. No glutamate toxicity 3. No ROS toxicity 4. No MTP involvement 5. DNA fragmentation independent of caspase activity 6. Reversal of cell damage upon the replacement of glucose 7. A decrease in intracellular calcium concentration upon glucose replacement. These data suggest that the removal of glucose from striatal cultures does not cause cell death but triggers the cell to enter a quiescent state with sufficient energy to maintain resting membrane potential but also with morphological, mitochondrial and DNA modifications. In conclusion striatal cells possess a neuroprotective mechanism against prolonged glucose deprivation and remarkably can recover metabolically with repaired DNA.

The research described in this thesis examined whether concentrations of maternal steroids in the egg at fertilisation can influence ecologically important physiological and behavioural traits in juvenile salmonids. I have addressed four questions: Can experimental manipulation of egg steroid concentrations influence offspring physiology and behaviour? What is the extent of natural variation in the steroid content of a female's eggs at fertilisation? Does natural variation in egg steroid content influence offspring phenotype? Does maternal social status influence maternal and egg steroid concentrations. Experimental elevations of the cortisol and testosterone content of brown trout eggs (Chapter 2) indicated that concentrations of these hormones may influence juvenile size, resting metabolic rate and social status. However, there was great inter-family variation in the effects of treatment, and the possibility that the variation observed is due to differences in rearing environments rather than treatment is an equally plausible hypothesis. Thus, maternal steroids in the eggs at fertilisation may be able to influence aspects of juvenile physiology and behaviour that are associated with early competitive ability and survival, but so too many undetected variation in the rearing environment. To determine the scope for an effect of maternal steroids in the eggs, I then investigated the degree of intra-female variation in egg steroid content, both before (Chapter 3) and after ovulation (Chapters 3 & 4). Before ovulation, follicle cortisol content and weight varied between different regions of the ovary, but patterns of variation were not consistent between females. In some cases when females were allowed to spawn naturally, egg steroid content varied between nests deposited by the same female, but patterns of inter-nest variation were not consistent between the eggs of different females. I suggest that the steroid content of ovulated eggs can change while eggs are retained in the body cavity, resulting in inter-nest variation.

Salt stress is one of the most threatening environmental stresses reducing the global food production. Understanding mechanisms of salt tolerance in halophytic plants is a requirement for developing crop species with increased salt tolerance. This study focused on investigating ion transplant features in a halophytic relative of Arabidopsis, both at physiological and transcriptional level. A comparative approach was adopted in this study using the glycophytic model plant Arabidopsis thaliana, and its halophytic close relative, Thellungiella halophila. Net ion uptake and unidirectional Na fluxes during salt stress were analyzed in the two species. Furthermore, transcriptional profiles of ion transporters under control and high-salt conditions were compared between the two species. The considerable amount of data produced in this study provide important information for future physiological and molecular studies of both Arabidopsis and Thellungiella. The main results can be summarized thus: 1. After salt stress Thellungiella accumulates less Na in the shoots than Arabidopsis. Net uptake of Na into both roots and shoots was slower in Thellungiella than in Arabidopsis. 2. Lower unidirectional Na influx into root cells is the main reason for the lower Na accumulation in Thellungiella than in Arabidopsis. 3. Voltage-independent cation channels (VICs) are likely to be the Na uptake pathway in both Thellungiella and Arabidopsis. 4. Microarray analysis showed that after salt stress both species showed a tendency to reduce Na uptake by decreasing the expression of possible pathways for Na influx. However, transcriptional control of putative Na transporters occurred in Arabidopsis in the shoots, whereas it occurred in Thellungiella in the roots. 5. CNGC8 is a likely candidate for a Na uptake pathway in both Arabidopsis and Thellungiella. Transcript levels of CNGC8 decreased during salt stress in Thellungiella roots and Arabidopsis shoots.

Tumour suppressor p53 is frequently mutated in cancers. While wild type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilised and accumulate to high levels in tumour cells. Several studies have shown that mutant p53 acquires oncogenic properties and actively contributes to tumourigenesis. It is therefore important to understand how the stability of mutant p53 is regulated. This thesis shows that mutant and wild type p53 are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild type p53, this study suggests that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. The contribution of Mdm2 to the degradation of mutant p53 may reflect an ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome. Ubiquitination does not efficiently target mutant p53 for the proteasomal degradation, however ubiquitinated p53 mutants localize to the cytoplasm. This thesis suggests the role for the chaperone-associated ubiquitin ligase CHIP in ubiquitination of mutant p53, although other unidentified ubiquitin ligases appear to contribute. Interaction of mutant p53 with its family member p73 decreases ubiquitination, suggesting p73 can play a role in regulation of stability of mutant p53.