Hypertension is a key factor in the progression of cardiovascular diseases, and vitamin D is now recognised as a significant factor affecting blood pressure. Low serum concentrations of calcidiol may be related to the pathogenesis and progression of hypertension. The effect of both calcidiol and calcitriol on the proliferation and gene expression of HUVECs and on the subsequent proteins produced, secreted, or regulated by endothelial cells is investigated. This was followed by studies on the thoracic and abdominal segments of aortas isolated from adult female Wistar rats to determine direct vasomodulatory effects of calcitriol using a wire myograph system. The result showed that calcitriol (10-13 M) significantly enhanced the proliferation of HUVECs whereas a relatively high concentration of calcidiol (10-9, 10-7 M) significantly inhibited cell proliferation after the same incubation period: 24 and 48 hours. Calcidiol had the greatest effect on the expression of genes, particularly those involved in regulating blood pressure, with decreased ET-1 mRNA expression, and there was a significant increase in eNOS mRNA relative to the control, particularly with 10-11 M of calcidiol. However, calcitriol had little effect on the expression of these genes so its effects on blood vessels may relate to non-genomic mechanisms. Ex vivo studies have shown that calcitriol acutely reduced endothelium-independent contraction in rat thoracic and abdominal aortas and does not involve NO. The acute effect of calcitriol reduced or inhibited the dose-dependent (10–9 to 10–5 M) contractions induced by PE, and there was a significant inhibition of the subsequent dose-dependent (10–9 to 10–5 M) relaxation induced by acetylcholine. The acute vasorelaxant effects of calcitriol do not appear to be mediated via the AC-cAMP-PKA signalling pathway but were similar to the effects of the Ca2+ channel blocker, verapamil. In summary, this thesis reports novel information regarding the effect of calcitriol on regulation of vascular tone through enhancement of a potential relaxation of arteries in an endothelium-independent manner, which is mediated by a non-genomic mechanism. Future studies need to confirm these results with in vivo studies. Hence, inducing relaxation in smooth muscle cells ex vivo may facilitate to use calcitriol as an antihypertensive drug.

The transient receptor potential vanilloid 1 (TRPV1) channel protein is activated by lipid metabolites synthesized in the cytosol of nociceptors in response to noxious stimulation. Lipid species include the endocannabinoid anandamide (AEA) and the linoleic acid metabolite 13 (S) hydroxyoctadecadienoic acid (HODE) which act as endogenous TRPV1 ligands (endovanilloids) by evoking TRPV1 mediated Ca2+ entry. Members of the fatty acid binding protein (FABP) family have been widely reported to act as intracellular lipid binding proteins for hydrophobic lipid species in aqueous cytosolic environments. The aim of this thesis was to identify which FABP isotypes could solubilize then shuttle AEA and 13(S)HODE to TRPV1 during nociception. Inhibiting FABP mediated transportation of endovanilloids could represent an alternative approach to analgesia by indirectly antagonizing TRPV1 activity during nociception while avoiding the widely reported negative side effects of direct antagonism. For the first time, it was found that FABP isoforms 5, 7 and 8 were expressed in rat dorsal root ganglia cell preparations. Furthermore, subsequent cell free competitive binding assays confirmed that FABP5, 7 and 8 could all physically bind to AEA and 13(S)HODE albeit with variable affinities. The ability of FABP 5, 7 and 8 to physically associate with TRPV1 and therefore deliver AEA was then assessed in live mammalian cell lines transfected with plasmid DNA constructs expressing recombinant TRPV1 and FABPs. Physical interactions between TRPV1 and FABP5, 7 and 8 were observed in COS-7 cells examined by fluorescence microscopy. However, when increases in intracellular Ca2+ levels were measured in COS-7 cells co-expressing TRPV1 and FABP, in response to treatment with 1µM AEA, the magnitude of AEA evoked Ca2+ influxes were not significantly different to those observed in COS-7 cells not co-expressing the FABPs. This suggested that the FABPs did not functionally associate with TRPV1 and did not deliver AEA to TRPV1 receptors. In conclusion, data in thesis showed that the FABP isoforms expressed in DRG cell preparations could physically associate with lipid species reported to activate TRPV1 during nociception.

Plant derived terpenoids represent a diverse class of chemicals with important roles such as fragrances, flavours and pharmaceuticals as well as high value medicinal compounds such as the antimalarial Artemisinin and the anti-cancer drug Paclitaxel (Taxol). High volume production of these products can be difficult due to reliance on plant production or economic difficulties in synthetic production. These high value terpenoids represent an interesting challenge to metabolic engineering and synthetic biology, not only the requirement to produce these chemicals at scale, but also because novel products can be developed via the intermediates of these pathways. Research into methods of microbial production of Taxol and its derivatives have focused on the engineering of Escherichia coli and the precursor pathways leading to geranylgeranyl diphosphate (GGPP), since GGPP is the last non-dedicated precursor to Taxol. The work reported here has taken a different approach and has instead attempted to utilise natural diversity in search of a better host for production of these high value terpenoids, by selecting an organism that already produces a large amount of GGPP. As well as being a precursor to Taxol, GGPP can also be converted into carotenoid pigments such as lycopene, β-carotene and zeaxanthin. Therefore production of carotenoids can be used as a surrogate measure for predicting how well a species will perform in the production of heterologous isoprenoids. Genes for carotenoid biosynthesis from Pantoea species have been used in many heterologous expression experiments, however Pantoea have not been considered as a possible chassis for the production of isoprenoids. As a relatively close phylogenetic relative to E.coli, these Gram negative bacteria are easily transformable, genetically tractable and many tools which have been developed for E. coli function in Pantoea. The work reported here addresses the developments of tools and techniques to assess the ability of Pantoea agglomerans to produce terpenoids including lycopene and taxadiene. The tools developed included: 1) a range of promoters, which were characterised in E. coli and P. agglomerans for their regulation of lycopene biosynthesis, 2) methods for the editing of the genome of P. agglomerans and 3) a range of vectors for the expression of the first 3 dedicated steps in Taxol biosynthesis, taxadiene synthase (txs), taxadiene 5α hydroxylase and taxadiene-5α-ol-O-acetyl transferase (5α) and taxane 10β-hydroxylase (10β). These genes initially could not be cloned in an operon together due to apparent toxicity. However by designing measures to minimise the burden imposed it was possible to express all four enzymes, of the three step process in P. agglomerans. This work discusses the difficulties associated with working with a less well characterised organism (such as P. agglomerans), how we overcame these issues and the unexpected phenotypes exhibited by this genus.

Wild aquatic birds, particularly ducks are considered as the major reservoirs of low-pathogenic avian influenza A viruses (LPAIAVs). Occasional spill-over to susceptible hosts like chicken and turkeys results in severe disease especially when infected with high pathogenic H5N1 virus subtypes that evolve from their progenitors LPAIAVs in gallinaceous birds, gaining zoonotic significance. Disparate disease outcomes between chicken and ducks to avian influenza virus (AIV) infection suggest potential species differences in mounting antiviral response. Mammalian studies indicate that influenza A virus non-structural protein 1 (NS1) interacts with vital host proteins to interfere with and hijack cellular functions for viral replication advantage. NS1 has been shown to bind to PI3K/Akt (phosphatidylinositol-3-kinase) p85beta subunit resulting in pathway activation. The PI3K/Akt pathway is crucial in the regulation of cellular differentiation, proliferation and survival through inhibition of pro-apoptotic factors. However, there are few studies of regulation of PI3K/Akt signalling in avian hosts, and in particular of its role in AIV replication. Understanding the host cellular response to AIV infections is key for developing effective control measures. Therefore, the present study investigated the regulation of PI3K/Akt regulation in avian cells and its implications for influenza A virus replication. Infection of primary chicken or duck embryo fibroblast (CEF/DEF) cells with A/mallard duck/England/7277/06(H2N3) virus demonstrated activation of PI3K/Akt only in the chicken but not in duck cells. The virus induced PI3K/Akt pathway activation was further confirmed by infecting cells with different low pathogenic avian influenza (LPAI) virus subtypes. Chemical inhibition of PI3K/Akt by treating infected CEFs with LY294002 resulted in significantly higher apoptosis induction and reduced infectious virus production compared to uninhibited control cells. These results suggest that activation of the PI3K/Akt pathway in infected chicken cells results in improved cellular survival allowing for enhanced virus replication. Analysis of previously published transcriptome data from IAV infected chicken and duck lung cells revealed higher expression of phosphatase and tensin homolog (PTEN) a potent PI3K/Akt negative regulator and down regulation of cytoskeletal actin remodelling associated genes like Cdc42 and RAC 1 in duck compared to chicken cells. Previous studies on AIV morphogenesis have shown roughly spherical budding virion morphology in CEFs while elongated in duck cells and speculated host factors responsible for virus morphogenesis. Based on these observations, repercussions of PI3K/Akt activation on cytoskeletal actin remodelling and virus morphogenesis in H2N3 virus infected CEFs and DEFs was studied. The results revealed loss of actin filament integrity and recorded perfectly spherical budding virion morphology upon PI3K/Akt inhibition in CEF cells. While duck cells still produced elongated viruses irrespective of PI3K/Akt inhibitor or mock treatment. Strikingly, a complete loss of cytoskeletal actin architecture was observed in virus infected duck cells irrespective of inhibitor or mock treatment. Analysis of chicken and duck PI3K/Akt p85β sequence revealed amino acid changes that may interfere AIV-NS1 binding and activation of PI3K/Akt. In particular, a highly acidic glutamic acid (E) amino acid residue at 475 position was observed across all major mammalian and avian hosts studied except ducks in which E475 residue was substituted with a non-polar valine (V) residue (E475V). However, these in silico experimental observations require further investigations. Infection of CEFs with LPAI viruses resulted in activation of PI3K/Akt for a shorter duration while recombinant H5N1 virus infection resulted in prolonged pathway activation. Infection of CEFs with chimeric(c) H9N2 virus carrying segment 8 (NS) from H5N1 (50-92) confirmed the potential contribution of H5N1-NS segment for prolonged PI3K/Akt activation. A comparative AIV NS1 sequence analysis from LPAI and HPAI viruses, revealed several amino acid differences that might contribute to prolonged PI3K/Akt activation. Interestingly, an additional SH3-binding motif-1 was observed in H5N1 (50-92) virus NS1 in comparison to other four viruses used in the study upon in silico protein binding site predictions. In conclusion, the present study revealed lack of PI3K/Akt activity despite AIV infection in duck fibroblast cells. There is an indication that there may be differences in the p85β subunit underlying these differences in the ducks conferring disease resistance compared to chickens. The study signifies host-specific viral replication strategy that may find potential use in disease control interventions in the future.

Cystic fibrosis patients endure serious lung infection caused by colonisation and persistent infection by a wide range of pathogens, most commonly Pseudomonas aeruginosa (PA). One of the factors that facilitates establishment of chronic lung infection is formation of biofilms which are structures resistant to antimicrobial drugs and immune attack. Biofilms are embedded within extracellular polymeric substance (EPS) that maintains the structure of biofilms. PA produces two important polysaccharides Pel and Psl, which have been implicated in promoting biofilm development and biofilm maintenance, respectively, as well as cell aggregation . To the best of our knowledge, there has not been any study showing the presence of specific immune receptors for the recognition of PA biofilms. C-type lectin receptors (CLRs) are pathogen-recognition receptors that contribute to the recognition of infectious agents through the detection of carbohydrates moieties representing a subset of pathogen-associated molecular patterns (PAMPs). We hypothesized that CLRs such as DC-SIGN (CD209) and mannose receptor (MR) (CD206) could play a crucial role in the immune recognition of PA biofilms through the binding to different carbohydrate-containing components. Investigating the CLR-PA cross-talk and the response of immune cells expressing CLRs to different PA components could lead to a novel strategy to eradicate infections. The main aim of this thesis is to determine the capability of CLRs, particularly MR and DC-SIGN, to interact with PA biofilms. We have shown that CTLD4-7, a region of MR, and DC-SIGN bind to PA biofilms with DC-SIGN binding significantly better than MR. Both lectins also recognised several independent preparations of EPS lacking Pel. Surprisingly, we found that DC-SIGN also binds to planktonic PA in the absence of Psl and Pel which indicates that DC-SIGN could recognize non-EPS carbohydrate-containing ligands in the bacteria. Further investigation unveiled that DC-SIGN requires the presence of the common polysaccharide antigen (CPA) which is shared among all PA serotypes to bind planktonic cells. These results indicate that CPA is a candidate ligand for DC-SIGN in PA. To determine the significance of these findings, assays incubating human dendritic cells with purified EPS and planktonic PA were performed but no definitive conclusion could be drawn. These findings shed light on the potential impact of PA Psl and CPA-LPS on the recognition of PA by immune cells expressing CLRs and might open new avenues for therapeutic approaches.

MOZ (also known as KAT6A and MYST3) is a histone acetyltransferase that is implicated in the development of hematopoietic, heart and craniofacial tissues. Reciprocal translocations generating MOZ fusion proteins are associated with acute myeloid leukaemia (AML), a heterogeneous hematopoietic stem cell disorder where the myeloid cells do not enter into differentiation process, resulting in accumulation of blast cells and absence of a fully functional granulocyte lineage. Also, mutations that truncate the C-terminus of MOZ are associated with global developmental delay. Mechanistically, it is known that MOZ acts as a cofactor for certain transcription factors, particularly those with haematopoietic or neuronal specificity, such as acute myeloid leukaemia/Runt-related transcription factor 1 (AML1/RUNX1) and PU.1. However, at the outset of this project, it was relatively unknown which genes that are regulated by MOZ in leukaemia. Thus, this work investigates MOZ target genes in a leukemic cell line to fully understand its importance in cell function and disease conditions. Firstly, we performed siRNA silencing in K562 cell line, coupled with transcription profiling by RNA sequencing. We identified 406 deregulated genes under MOZ knockdown, of which 76 were downregulated. Many of the MOZ-target genes identified are implicated in haematopoietic development and cell proliferation. Secondly, we generated MOZ/KAT6A knockout K562 cells using CRISPR CAS9n nickase technology. It allowed further analysis of the function of MOZ in leukaemia. Our results confirmed the role of MOZ in maintaining levels of H3K14ac and H3K9ac and an apparent inverse relationship with the levels of H3K14 crotonylation. Furthermore, the results indicated that MOZ is important for leukemic cell proliferation and the gene dosage influences on the cell proliferation rate. Finally, we found that genes requiring MOZ for their expression are sensitive to a novel class of bromodomain inhibitors called iBETs. Furthermore, we found evidence for the existence of MOZ in complex with BRD4 protein. This provides a hitherto unknown link between MOZ, MLL and BRD2-4 complexes and insight into how these epigenetic regulators cooperate to control gene expression.

This thesis describes the syntheses and characterisations of heterobimetallic dithiolate complexes of NiII, PdII and CoII with FeII that derive inspiration from the chemistry and structures of the active sites of the [NiFe] hydrogenase family of metalloenzymes. These studies provide insight into the electronic structures of the Ni-C and Ni-L states of the [NiFe] hydrogenases and the complexes have potential as new catalysts for the production of dihydrogen. Chapter One outlines the role of dihydrogen as a potential fuel and replacement for energy sources derived from fossil fuel feedstocks. The Chapter describes the roles offered by synthetic analogues of the active sites of the [NiFe] hydrogenases as routes to meet the demand for H2 production. An introduction to enzymes and the roles of transition metals in biology, together with a description of the structure and function of the [NiFe] hydrogenases, are presented. A proposed catalytic cycle for the [NiFe] hydrogenases and a review of the coordination chemistry relevant to the active site of the [NiFe] hydrogenases is presented. Key questions about the [NiFe] hydrogenases and the aims of the work described in this thesis are summarized. Chapter Two describes the synthesis and characterisation of a series of bimetallic [NiIIFeII] complexes that incorporate a four coordinate NiII centre ligated by the N,N'-diethyl-3,7-diazanonane-1,9-dithiolate (L1)2- ligand bound to a pseudooctahedral FeII centre to give a Ni-(S2)-Fe bridging arrangement. The coordination sphere of the FeII centre is completed by a range of isocyanide ligands, R-NC (R = tBu, iPr, nBu, cyclohexyl, benzyl, 4-methoxyphenyl, 4-fluorophenyl) that act as surrogates for the π-acceptor CO and CN- found at the active site in the [NiFe] hydrogenases. Electrochemical and EPR studies on these complexes provide insight into the role of the isonitrile R group on the redox chemistry of the [NiIIFeII] centre. Our electrochemical and spectroscopic studies, supported by DFT calculations, show that these [NiFe] complexes possess a formal NiII/IFeII redox couple that is localized around the Ni unit. These complexes are compelling models of the Ni-L state of the [NiFe] hydrogenases and show promise as potential electrocatalysts for proton reduction. Chapter Three describes the synthesis and characterisation of a pentacoordinate NiII complex in which NiII is bound by the 1,1'-(1,4,7-thiadiazonane)bis(2-methylpropane-2-thiol) (H2L3) ligand. We show that this unit can coordinate to a pseudo-octahedral FeII centre to generate a bridged Ni-(S2)-Fe structures in which the coordination sphere of the FeII centre is completed by four isonitrile ligands. These complexes we show that the heterobinuclear [NiIIFeII] system supports a formal [NiIII/IIFeII] redox couple. X-ray crystallographic, and electrochemical and spectroscopic studies, supported by DFT calculations, provide insights into the nature of the [NiIIIFeII] species that represents analogues of the Ni-C state of the [NiFe] hydrogenases. Chapter Four describes the synthesis and characterisation of a range of novel four-coordinate [M(N2S2)] (M = Pd, Co, Zn) complexes in which the metal centre is bound by N,N'-diethyl-3,7-diazanonane-1,9-dithiolate (L1)2-. We describe attempts to coordinate these centres to a [FeII(CNtBu)4]2+ fragment to yield the corresponding heterobimetallic [MFe] complexes. We characterize these compounds by X-ray crystallography and investigate their electrochemical properties for direct comparisons with their [NiFe] counterpart described in Chapter Two to provide insight into the effect of substituting NiII for other transition metal centres. Chapter Five provides a summary of the principal conclusions of the research described in this thesis.

The P2Y2 receptor (P2Y2R) has been implicated in a range of clinical conditions, including but not limited to: cystic fibrosis, dry eye syndrome and cancer. However, a lack of high quality, drug-like modulators and tool-like compounds means there is scope to develop ligands that can further probe P2Y2R function in vitro and in vivo. Assessment of the reported P2Y2R antagonists led to the conclusion that 5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide (AR-C118925, 38) was the most drug-like P2Y2R antagonist and was chosen as a chemical starting point. Elaboration of the 2,8-dimethyl-5H-dibenzo[a,d]cyclohepten-5-yl moeity to a 7-chloro-2-methyl-4H-benzo[5,6]cyclohepta[1,2-d]thiazol-4-yl), led to the development of two potent, novel P2Y2R antagonists: MSG204 105 (pKB = 6.73 0.25, n = 3) and MSG249 119 (pKB = 7.06 0.02, n = 3), both of which exhibit improved physicochemical properties to AR-C118925 38 and provide more drug-like alternatives for future in vivo work. Structure activity relationships derived in developing these novel P2Y2R antagonists, has ultimately led to the development of multiple fluorescently-labelled P2Y2R ligands. Most notably, BODIPY 630/650 conjugate MSG260 193 (pKd = 6.99 0.04, n = 3) and BODIPY FL conjugate MSG262 195 (pKd = 6.88 0.01, n = 3), which have enabled the use of a novel P2Y2R BRET ligand-binding assay and provide a robust platform for future drug discovery programs.

Our group reported upregulation of a novel group of genes was associated with beta-adrenergic agonist (BA)-induced muscle hypertrophy in pigs. The aim of this PhD was to investigate the expression of these genes, and particularly the role of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2) and phosphoglycerate dehydrogenase (PHGDH), in muscle cell growth. A significant (p < 0.01) increase in mRNA transcript abundance was detected at day 2 of differentiation in C2C12 cells for PEPCK-M, PHGDH, phosphoserine aminotransferase-1, phosphoserine phosphatase, asparagine synthetase, sestrin-2 and activating transcription factor-5. This novel peak coincided with the peak in myogenin mRNA, connecting these genes with a crucial point of myogenic differentiation. Hypertrophy was induced in C2C12 myotubes treated with dibutyryl-cAMP (dbcAMP), mimicking the BA response in vivo, however mRNA expression of these genes were unaffected. The porcine myosin heavy chain (MyHC)-IIB promoter-reporter C2C12 cell assay demonstrated similar in vivo responses to known anabolic and catabolic agents. Thus, C2C12 cells were utilised to determine the role of PEPCK-M and PHGDH in myogenic differentiation. Firstly, C2C12 cells were treated with a PEPCK inhibitor, 3-Mercaptopicolinic acid (3-MPA). 3-MPA induced differentiation, resulting in a hypertrophic response comparable to dbcAMP treatment. However, it was unclear whether 3-MPA inhibited PEPCK-M enzyme activity as 3-MPA interfered with the in vitro assay. Next, C2C12 cells were transfected with either PCK2 or PHGDH overexpression construct. No obvious phenotype was observed, but PHGDH and PEPCK-M overexpression both increased MyHC-IIB mRNA. The reoccurring induction of the same group of genes along with MyHC-IIB supports the hypothesis that co-ordinated upregulation of these genes may drive hypertrophic growth. To conclude, PEPCK-M, along with other genes upregulated with BA-induced hypertrophy and C2C12 differentiation, show co-ordinate regulation in times of high biosynthetic demand. PEPCK-M appears to sit at an intersection that allows metabolic flux to be largely altered by diverting intermediates during energy metabolism.

Metabolism is an essential chemical process and pathway involving multiple reactions. Oxidative or functionalisation reactions dominate phase 1 metabolism and is predominately controlled by the Cytochrome P450s (P450s). Phase 2 reactions are frequently referred to as the detoxification, or elimination, phase. Several families of enzymes are involved, and the largest of these are the Uridine diphosphate 5’-glucuronosyltransferases (UGTs). The purpose of this project was to initiate the development of an equine in vitro toolbox, concentrating on the UGTs. This required identification of UGTs in the equine genome. The first step used syntenic analysis which enabled us to utilise relative gene order conservation between species to determine whether the predicted gene encoded a member of the UGT superfamily. Further analysis of sequence relationships provided confidence that the genes under investigation were UGTs, but also allowed us to determine which UGT orthologue we were investigating. PCRs were performed to isolate the genes, and subsequent sequencing enabled the UGTs to be investigated for key features, including signal peptides, signature sequences, transmembrane domains, and dilysine repeats, which are characteristic of this family of membrane-bound proteins. We isolated and characterised five putative equine UGTs. Subsequent analyses indicated these to be orthologous to human UGT1A6, UGT2A3, UGT2B17, and two UGTs orthologous to UGT3A2. Three equine UGT genes were cloned into a vector for the development of functional recombinant proteins. UGT1A6, UGT2A3 and UGT3A2 expression constructs were transfected into Human Embryonic Kidney 293 cells and stable cell lines generated for analysis. Four drugs were assayed to determine the functionality of the recombinant enzymes and individual substrate specificities. Whilst these studies were inconclusive, further work is required to establish function and substrate profile in order to take the first steps towards creating an in vitro toolbox for equine drug metabolism. Expression of UGT1A6 and UGT3A2 was measured in four tissue samples from 12 horses. For both genes, expression levels in the liver were greatest whilst the brain showed negligible expression. Expression levels of both genes in the kidney and lung were similar and lower than levels detected in the liver.