Native subtypes of NMDA receptors with distinct developmental and anatomical distribution are present in the brain. Knowledge of their function and pharmacology greatly helps in understanding the involvement of NMDA receptors in physiological and pathological processes in the central nervous system. Multiple patch-clamping methods were carried out in my experiments. First, the single-channel properties of native NMDA receptors were studied in outside-out patches excised from neuronal cell bodies in substantia nigra pars compacta (SNc) and subthalamic nucleus (STN) from 7-day old rats. The steady state channel activations produced by NMDA (100 nM or 200 uM) and glycine (10 uM) were studied at -60 mV. The results showed that both large and small conductance channels are present on STN and SNc neurons. These large conductance NMDA channels from SNc and STN display a high ifenprodil sensitivity, suggesting NR2B-containing NMDA receptors are present on SNc and STN neurons. In addition, direct transition analysis suggests that the small conductance channels may be due to NR2D-containing NMDA receptors. Second, whole-cell patch-clamp recording of bath application-induced NMDA receptor-mediated currents (Inmda) in dopaminergic neurons of SNc in brain slices from P7 rats was used to characterize the ifenprodil inhibition and voltage-dependence of Mg2+ block of NMDA receptors. The NMDA-induced whole-cell currents were evoked by NMDA (10 uM or 200 uM) and glycine (10 pM). There are two main findings from the whole-cell experiments. (1) The NMDA-induced whole-cell currents display a high ifenprodil sensitivity, suggesting the presence of NR2B-containing NMDA receptors on P7 rat SNc neurons. (2) The combined application of Mg2+ and ifenprodil reduced the voltage-dependent Mg2+ block, which is consistent with recombinant NR2D-containing NMDA receptors that have a lower affinity for Mg2+. This suggests that NR2D-containing NMDA receptors are present in P7 rat SNc. Third, in order to evaluate the deactivation kinetics of extrasynaptic NMDARs, outside-out patches containing multiple channels were obtained from P7 rat dopaminergic neurons of SNc and stimulated with a brief synaptic-like (l-4ms) pulse of ImM glutamate. The results suggested that the triheteromeric NR1-NR2B-NR2D rather than diheteromeric NR1-NR2D NMDA receptors are present on extrasynaptic sites of SNc neurons. In addition, in order to investigate voltage-dependent Mg2+ block and memantine block, several channel block models were developed to evaluate multiple Mg2+ effects on native NMDA receptors and competition between Mg2+ and memantine for block of NMDAR channels.

Size-exclusion high performance liquid chromatography was developed to study surface membrane proteins of malignant lymphocytes. The membrane proteins were separated in accordance to their molecular sizes or complexed sizes and displayed, in conjunction with SDS-PAGE, as two-dimensional matrices on which the location of proteins is determined by both retardation in the HPLC column and the mobility on the SDS-PAGE gels. Any association or induced-interaction between proteins are reflected by predictable changes of the retention times, thus by the location on the two-dimensional matrix. Individual membrane proteins in the mixture were identified or separated by the changes in their retardation in the column when mAb-Ag complexes formed by applying specific monoclonal antibodies to the extracts of vectorially labelled cells. A number of surface markers have been identified and several new components are characterized. This study has demonstrated that (1) SE-HPLC is a potent method for analysis of complicated mixtures, particularly when a complete separation is not necessary (2) the technique has advantages for the study of non-covalentinteractions between proteins, particularly valuable for antibodies of low affinity (3) the method is powerful for compositional study and initiative investigation of the complexity of mixtures. By using this technique in conjunction with SDS-PAGE, B-CLLs were found to have heterogeneous surface phenotypic presentations with respect to both quantity and quality. The HPLC-SDS PAGE study revealed that the expression levels of individual surface components in B-CLL patients were at a correlated fashion and, across the patient panel, present a continuous spectrum of such relationships, confirmed the earlier reports of immunological studies by Maddy et.al. that individual B-CLL patients can be ranked in a sequence according to their expression level of CD45 isoforms and that this sequential variation of CD45 is in correlation with levels of sIg and CD21. In addition, this study discovered a group of uncharacterized proteins, band 4.1 (160KD), band 4.3 (135KD) and band 2 (300KD) which are also correlated with CD45RA, CD21 and sIg. A comprehensive repertoire of B-CLL phenotypes has been established with respect to the membrane protein expression.

Selenium (Se) is an essential nutrient for health. In mammals Se is incorporated into ~25 selenoproteins in the form of the amino-acid selenocysteine encoded by the UGA codon through a complex interacting with selenocysteine insertion sequence (SECIS) in the 3’Unstralated Region. The selenoproteins have functions in antioxidant defence and redox control, thyroid hormone metabolism and mitochondrial metabolism. Previous scientific work has found that Se also affects a group of downstream targets. The aim of my work is to investigate whether expression of selenoproteins or the downstream targets affected by Se is regulated through epigenetic mechanisms involving microRNAs, a small non-coding RNA species that regulates a gene or groups of genes by binding to the mRNA 3’UTR. Gut epithelial Caco-2 cells were grown in either Se deficient or Se-supplemented medium for 72h. RNA extracted and miRNA expression analysed using a custom-designed human genome V2 Agilent 8x15K array. In addition, global mRNA transcriptome expression was analysed using an Illumina HumanRef-8 v3 microarray. Se supply increased the expression of thirteen miRNAs and 53 mRNAs the observed differences were confirmed by real time PCR. miR-185 was selected as a further target of investigation because of its high sensitivity to Se. Bioinformatic analysis of the Se susceptible miRNAs and Se sensitive genes was carried out using miRWalk, MicroCosmo, microRNA.org, miRBase and microRNA.org algorithms and Ingenuity Pathway Analysis. This identified miR-185 recognition elements in the Se-sensitive mRNAs for Glutathione peroxidise 2 (GPX2), Glutathione peroxidise 3 (GPX3), and Selenophosphate Synthetase 2 (SEPHS2). Expression of GPX2 and SEPHS2 was altered by miR-188 with a specific anti-miR and affected differently according to miRNA exposure. The data suggests that these mRNAs are targets of miR-185. In conclusion, the experiment indicates that miRNA expression as regulation of target genes is regulated by Se supply in Caco-2 cells.

At present mammalian cell factories are being employed for recombinant protein production. However, the yields of proteins produced from such systems are often poor. This thesis describes experiments to study the effects of altering targeting signals (signal peptide) and 3’ untranslated regions (3’UTR) in an expression vector on protein expression. A variety of gene constructs containing Gaussia princeps luciferase as reporter were created using a seamless cloning method. In these constructs a variety of signal peptides, some with altered hydrophobicity, were combined with the Gaussia luciferase coding region and either the native Gaussia luciferase or human albumin 3’UTR. These were then transfected into CHO AA8 Tet-Off cells to measure how modification of the signal peptide/3’UTR affects protein expression. The results indicate that the Albumin 3’UTR, in conjunction with an appropriate signal peptide, boosts protein production by approximately 3 fold compared to the native Gaussia luciferase 3’UTR. Deletion analysis of the Albumin 3’UTR showed that deletion of regions 1-50, 1-100, 1-150, 101-150 significantly reduces protein production compared with deletion of regions 51-100, 51-150 and 1-50&101-150. Interestingly, mRNA abundance levels were significantly decreased for constructs containing deletions in regions 1-50, 1-150 and 1-50&101-150. UV Cross linking and electrophoretic mobility gel shift competition assays showed strong competition by RNA transcripts from the deletion construct 1-50, which was then used as bait for isolating bound protein/s from a CHO cell extract. Three proteins, including CUG-BP1 an RNA-binding protein involved in mRNA stability and translation were identified by mass spectrophotometry analysis. Knock down of CUG-BP1 expression using siRNA, led to impairment of complex formation between CHO cell protein extract and Albumin 3’UTR RNA transcripts, and in addition it led to an increase in the reporter activity and mRNA expression level in cells expressing the reporter gene with the full length Albumin 3’UTR and deletion variant 51-100. It is hypothesised that the differences in mRNA expression levels and secreted luciferase activity were due to CUG-BP1 binding to the Albumin 3’UTR. Further work is needed to explore the effects of CUG-BP1 on mRNA translation and stability.

Gut hormones play an important role in beta-cell function, proliferation and protection from apoptosis. The recent discovery that glucagon-like peptide-l (GLP-l) and gastric inhibitory polypeptide (GIP) is locally produced in the alpha-cells of pancreatic islets has highlighted the potential importance of these locally produced incretin hormones, and changes in the processing of their prohomones by convertase enzymes, PC 113 and PC2. This thesis explores the roles of GLP-l, GIP and the cholecystokinin octapeptide (CCK-8) in the function and morphology of pancreatic islets in different metabolic states. Firstly, the effects of pregnancy and lactation on GIP secretion and related gene expression were studied in Wi star rats. This study indicated that changes in the secretion and action of GIP together with associated changes in mammary and adipose tissue gene expression play an important role in metabolic adaptations, especially during lactation. Islet adaptations to pregnancy were explored in mice lacking functional receptors for GLP-l and GIP. These data show that GLP-I but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-I. Diabetic states of different aetiologies, chemically induced by multiple low dose streptozotocin or hydrocortisone, were then investigated using incretin receptor deficient C57BLl6 mice. Absence of functional receptors for both the GLPI and GIP compromised the expansion of beta-cell mass seen in control mice treated with hydrocortisone, suggesting that GIP, as well as GLP-I, plays an important role in beta-cell adaptation to functional stress. Effects of Liraglutide, were evaluated in C57BLlKsJ db/db mice to ascertain whether the GLP-I mimetic is beneficial in maintaining islet architecture and alpha cell morphology in degenerative diabetes. Beneficial effects were observed on both xiv 'I the intra-islet distribution and numbers of glucagon-secreting cells, suggesting that such actions may be useful even in the treatment of more severe forms of diabetes. The final study determined the ability of enzyme resistant (pGlu-Gln)-CCK-8 to counter the development of diet-induced obesity-diabetes in high fat mice and Aston oblob mice. Beneficial metabolic effects were observed in both animal models treated twice daily with (pGlu-Gln)-CCK-8, including significantly reduced energy intake, body weight, circulating glucose and insulin and improved glucose tolerance and insulin sensitivity. Alpha cell numbers were increased but this was associated with strong staining for GLP-l. This study illustrates novel intra-islet effects of (pGlu-Gln)-CCK-8 and the potential of CCK-R activation for the treatment of obesity-diabetes. In summation, this thesis demonstrates that intra-islet and gut derived GLP- 1, GIP and CCK play important roles in the regulation of insulin secretion, beta-cell proliferation and protection from apoptosis. Further novel insights are provided for the role of GLP-l and GIP in islet adaptations to pregnancy, insulin resistance and beta cell insult. GIP also plays an important functional role in orchestrating metabolic adaptations in adipose and mammary tissue during lactation.

The ovarian tumour (OTU) domain present at the N-terminus of the nairoviral L protein has been shown to deconjugate ubiquitin and interferon-stimulated gene 15 protein (ISG 15) from cellular proteins, thereby interfering with innate immune cell signalling pathways. I have confirmed and extended these findings for the Dugbe OTU domain by showing that it has deubiquitinating and deISGylating activity and is able to effectively block the TNFα/NF-KB and interferon/JAK-STAT signalling pathways even at low doses. A larger OTU expression construct that included the downstream zinc finger domain had a reduced effect on ubiquitination and on the above-mentioned signalling pathways, but was equally effective at deISGylating cellular proteins. The effect of the OTU domain on ubiquitination, ISGylation and cell signalling was confirmed by generating point mutants of the catalytic site [C40A, H 151 A and a double mutant]. These mutations completely abolished the ability of the OTU domain to deubiquitinate and deISGylate cellular proteins. Unexpectedly, when used at higher doses, the C40A mutant still had a significant inhibitory effect on the TNFa/NF-lCB pathway, and all three mutants inhibited type 1 interferon action, suggesting that mutants can still block some cellular functions by binding to their target proteins even if they are no longer enzymatically active. This effect observed with the mutants was greatly reduced when the amount of each mutant OTU plasmid transfected was reduced by 10-fold; however the C40A mutant was still able to block type I interferon action even at low doses. Interestingly, infection of cells with the virus itself resulted in deubiquitination and delSGylation of cellular proteins, but had no effect on the cell signalling pathways.

Incretins are growth factors that have demonstrated neuroprotective properties in a range of studies. Here, we analyse the neuroprotective properties of the Glucagon like peptide-l (GLP-l) analogues in human neuroblastoma SH-SY5Y cells against methyl glyoxal (MG) stress. The results demonstrate a range of growth factor-related cytoprotective processes induced by liraglutide (Victoza®), currently used to treat type 2 diabetes. FUl1her, we study the comparative long-term effects of three different GLP-l analogues on cell viability, proliferation and cytotoxicity and demonstrate the long-term exposure of liraglutide and lixisenatide (Lyxumia®) to be more protective when compared to the exendin-4 (Byetta~ . We also report the absence of any additive effect on cell viability and proliferation, when using GLP-l analogues in combination, but a 3-fold increase in cytoprotection observed when comparing 100 nM doses of liraglutide and lixisenatide to exendin-4 (p<0.001). In addition, we demonstrate that lixisenatide (10 and 50 nM) is neuroprotective when compared to 50 nM liraglutide (p<0.05) and 50 and 100 nM exendin-4 (p<0.001) against MG post stress. Further, the most effective doses for native Glucose dependent insulinotropic peptide (GIP), (D-Ala2)GIP and (Pro3)GIP were 200, 100 and 1 nM exhibiting a 43 ± 3% decrease in LDH levels (p<0.001). Lastly, we show a novel mechanism wherein blockade ofMEK1I2 leads to the activation of AktIPKB (p<0.001). A decrease in the survival (p<0.05) and an increase in the cytotoxicity (p<0.05) during inhibition of MEK1I2 followed by liraglutide treatment, suggest a role ofMEK1I2 in protecting the cells. Our studies show that the liraglutide pre-treatment confers neuroprotection by increasing the cell survival and decreasing cytotoxicity but inhibition of MEK1I2 withdraws this neuroprotective effect, suggesting the involvement of MAPKIERK pathway in the neuroprotection conferred by liraglutide. Overall, incretins confer protection and have potential to be developed as possible drugs to treat neurodegenerative disorders.

The pro-inflammatory cytokine TNF -a. and the immunoregulatory cytokine IL-IO have been implicated in acute rejection of kidney allografts. Similarly, the pro-fibrotic cytokine TGF-IJ 1 has been reported to be involved in the development of chronic rejection. It has also been shown that polymorphisms in the TNFA gene promoter (position -308) and in the TGF-IJI gene (at codon 25) correlate with differential production of these cytokines in vitro. Gene polymorphisms in the IL-IO promoter were also identified previously. However, their function had not been determined. Therefore, the initial part of this study was to investigate the effect of the three single base substitutions (G/A, CIT and CIA) in the IL-IO gene promoter on IL-IO gene expression. The results show that the G/A polymorphism at position -1117 of the a- 10 promoter was associated with differential production of IL-IO in vitro. Homozygous inheritance of a 'G' at this position correlated with a significant increase in the production of IL-IO by Con-A stimulated PBMCs. In addition, a-to gene reporter assays performed in the U937 monocytic cell line demonstrated that the activity of IL-l 0 promoter constructs containing the '0' was significantly higher than constructs containing the 'A' at position -1117 when co-transfected with an Ets-l transcription factor expression vector. Results from gel shift assays suggest that this difference in gene expression may be due to altered binding affinities of transcription factors to the IL-IO promoter. The second part of this study was to determine whether the IL-IO gene polymorphism, together with the TNF-a and TGF-pl gene polymorphisms, could influence the incidence and severity of acute rejection in the first 6 months following renal transplantation. Their effect on chronic transplant dysfunction was also assessed. The cytokine genotypes of 115 consecutive first cadaveric kidney allograft recipients and their corresponding donors were screened. Acute rejection episodes (RE) and the incidence of chronic transplant nephropathy (CTN) were defined clinically and confirmed histologically where possible. RE were further classified according to severity (RS), namely steroid resistant or responsive RE. The patients were categorised as high or low producers of the respective cytokines according to their genotypes and these were then correlated with the RE, RS and CTN. The recipient TNF-a high and IL-IO high producer genotype was significantly associated with multiple RE (~) in HLA-DR mismatched transplants (p=O.0047 and p=O.04S respectively) while only the TNF-a high producer genotype was associated with steroid resistant RE (p=O.02S). Donor cytokine genotypes did not correlate with RE or RS. When cytokine genotype combinations were analysed in context ofHLA-DR mismatching, the recipient T'NF-ahigh/donor TGF-Pl high producer genotype was associated with an increase in the incidence of RE. The recipiem TNF-a higML-lO high producer genotype was associated with an increase in the incidence of multiple RE in HLA-DR mismatched transplants and was also associated with steroid resistant RE. There was no significant association between cytokine genotypes and CTN. In conclusion, cytokine gene polymorphisms are determinants of RE and RS following kidney transplantation. Routine screening of these gene polymorphisms may have a clinical role in identifying patients at risk of multiple RE and severe rejections.

The development of genome-wide histone modification mapping technologies has provided a significant body of evidence implicating histone modifications in gene regulation, cell specialisation and differentiation processes. Monomethylation of lysine 4 of histone H3 (H3K4Mel) and the presence of the histone variant H2A.Z have previously been shown as a feature of enhancers, defined simply as points enriched in the HAT p300, but it was unclear whether these potential enhancers were linked to inactive, active or poised genes or whether developmental changes were reflected in the occupancy of H3K4Mel. The aim of this study was to document the spatio-temporal distribution of H3K4MeI at developmentally regulated genes and investigate proteins specifically binding to this modification so as to unravel possible control mechanisms. H3K4Mel distributions were determined by native chromatin immunoprecipitation (nChlP) experiments, analysed by real-time PCR, using chicken haematopoietic cell lines representing different erythroid and myeloid differentiation stages. Mapping at the lysozyme locus showed enrichments of H3K4Mel at the gene enhancers in myeloid cells independently of expression status, even in multipotent myeloid progenitor cells where the gene is not expressed. In contrast, in none of the erythroid cells studied - where the gene is never expressed - is there any H3K4Mel found at the lysozyme locus. At the β-globin locus, the β-adult enhancer is strongly enriched in H3K4mel in early erythroid progenitor cells where the globin genes are not expressed but the modification is lost from this enhancer in IS-day embryonic erythrocytes - which strongly express the adult and hatching globin genes - despite a general rise in H3K4Mel levels elsewhere in the locus, including the inactive embryonic globin genes. In none of the myeloid cells studied - where globin genes are never expressed - is there any high H3K4Mel enrichment found at the β-globin locus. At the folate receptor gene, the promoter carries a high level of H3K4Mel when the gene is inactive but this decreases when the gene becomes active. Taken together, the data suggest that H3K4Mel is a pioneer modification that participates in establishing the enhancers of genes poised for future activation. Pull-down experiments using immobilised peptides or reconstituted nucleosomes containing H3K4Mei and extracts from HeLa cell nuclei, followed by tryptic mass spectroscopy of bound proteins, were used to identify possible binding partners. Five potential binding partners were defined and have been shown to associate with chromatin.

Glycosyltransferases (GTs) are a large enzyme family that are involved in the biosynthesis of complex carbohydrates and glycoconjugates. These glycosylation reactions are essential to many fundamental biological processes, including cellular adhesion, cell signalling and bacterial cell wall biosynthesis. The aim of this project was the development of operationally simple assays for GTs and their application for the identification and characterisation of novel GT inhibitors. The main focus was on the bacterial GT, α1,4-galactosyltransferase C (LgtC) which transfers galactose from uridine-diphosphate-galactose to the terminal lactose on the cell wall lipooligopolysacharide of Neisseria meningitidis. A fluorescence-based ligand displacement assay (LDA) was optimised and validated for compound library screening. Additionally, the applicability of the LDA was investigated with three other GTs: bovine β1,4-galactosyltransferase, TcdB from Clostridium difficile and NGT from Actinobacillus pleuropneumoniae. The validated LDA was used for screening two compound libraries (c. 400 compounds in total) against LgtC for the first time. Positive hits were identified from both screening campaigns and selected compounds from two structural classes were characterised. One of these inhibitor classes was identified as molecular aggregators, which inhibit LgtC non-specifically. Results from the enzymological characterisation of the second class of hit compounds suggest that these inhibitors were mixed-type inhibitors and inhibit LgtC in micromolar range with considerable potential for further development. Development of a novel biochemical assay was attempted, in order to characterise potential inhibitors from the compound screening. This assay was based on an unnatural fluorescent acceptor and coupling of the GT reaction to a glycosidase reaction. During the development of this novel biochemical assay, unexpected enzymological features of LgtC were discovered. LC- MS/MS was used to investigate unknown products originating from the LgtC-catalysed reaction. The results suggest LgtC transfers more than one sugar moiety to the unnatural acceptor. Additionally two existing biochemical assays: HPLC-based assay and phosphatase coupled assay were successfully optimised and validated for determining the kinetic parameters of the LgtC catalysed reaction.