The metabolic modifiers, which include growth hormone, beta-adrenergic agonists, and anabolic steroids, modify animal metabolism and affect the mechanism of nutrient absorption. This experiment aimed to investigate the effect of a beta-adrenergic agonist (BA) and growth hormone (GH) in sheep on processes involved in regulating lipid metabolism, in the tissues principally responsible for nutrient energy metabolism; adipose tissue (subcutaneous adipose tissue), skeletal muscles (longissimus dorsi (LD) and supraspinatus (SS)) and liver. In addition, the study sought to determine whether a transdifferentiated-myogenic cell culture system demonstrating myogenic and adipogenic characteristics would respond to these agents in a similar manner as in vivo. Male lambs (120 days old) given ad-libitum feed were exposed for 6 days to bovine GH (bGH, 3.75mg/kg body weight, POSILAC, Monsanto) (n=10), the BA cimaterol (at 10mg/kg in the feed) (n=10) or a control C group (n=11). BA treatment significantly induced muscle hypertrophy, whilst GH increased liver weight without any significant effect on muscle weights. The increase in plasma IGF-1 in GH-treated lambs suggested that the GH effect was mediated by the IGF-1. There was no effect of both treatments on fatty acid profile in the SS muscle, whilst in subcutaneous adipose tissue, BA tended to increase the unsaturated fatty acids proportion (P = 0.058). The metabolomics analysis suggested the strong effect of GH on significantly elevating plasma NEFAs and glycerol concentrations (P < 0.05). This was associated with significantly higher plasma concentration of the ketone body 3-hydroxybutyrate (P < 0.05) and glucose (P < 0.001). These changes were accompanied by reduced plasma glucogenic non-essential amino acid concentrations. Given the lack of increase in muscle mass, these have might contribute to the increase in plasma glucose level, in GH-treated lambs, in addition to the lipolytic effect of GH. This would suggest GH induced an insulin resistance. Similar effects were not observed in BA-treated sheep. In addition, in GH- and BA-treated lambs the reduction of muscle fatty acid synthase (FAS) mRNA (P < 0.01) indicated a lowered lipogenic potential implicating the effect of both agents in developing leaner muscle. Although, this effect was more pronounced in GH treated muscles with both acetyl CoA carboxylase (ACC) and FAS mRNA being reduced in subcutaneous adipose tissue. The cell culture experiment was carried out using transdifferentiated-myogenic C2C12 cell lines. Expression of genes with associated with myogenesis and adipogenesis were induced, and fat droplets were observed. This suggested the presence of both myotubes and adipocytes in co-culture and thereby the establishment of a cell culture system analogous in vivo where the muscle has an intramuscular fat deposition. Upon the treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) and Des (1-3) insulin-like growth factor-1 (Des (1-3) IGF-1, there was a strong stimulatory effect of dbcAMP in regulating genes associated with myogenesis (P < 0.05), which might contribute to the hypertrophic effect, whilst lipogenic gene expression was reduced (P < 0.05). This indicated that this cell culture system exhibited the similar characteristics as had been observed in vivo in the sheep trial. Des (1-3) IGF-1 appeared to induce adipogenesis, while not affecting lipogenesis or lipolysis, suggesting different pathways of IGF-1 induction between in vivo or in vitro. In conclusion, the BA strongly affects the muscle development and hypertrophy, whilst GH affects the lipid mobilisation and deposition. However, both agents were demonstrated to potentially affect metabolism which would impact of the production of lean muscle.

Clostridium autoethanogenum is an anaerobic, facultative autotrophic bacterium that was isolated from rabbit faces in the last decennium of the twentieth century. It is used to convert carbon monoxide rich waste gas in to compounds such as acetate, ethanol, 2,3-butanediol and lactate. Carbon dioxide reacts with water to form carbonic acid and bicarbonate. This reaction is catalysed by enzymes called carbonic anhydrases. It was unknown if these enzymes were present in C. autoethanogenum. Genes encoding putative carbonic anhydrases were cloned and heterologous expressed. One gene encoded an active enzyme of a novel sub-clade of β-carbonic anhydrases. This gene was disrupted in the genome of C. autoethanogenum. The mutant was unable to grow at low pH and low carbon dioxide concentrations. Production of ethanol and 2,3-butanediol by WT C. autoethanogenum in carbon monoxide fed chemostat cultures was improved by employing phosphate limitation. A pilot study on the effect of phosphate limitation on rhamnose based growth showed 1,2-propanol and 1-propanol as native products of C. autoethanogenum. Acetolactate is the metabolic branch point for both branched chain amino acid and 2,3-butanediol production. An acetolactate synthase gene was deleted. The resulting mutant shows a subtle growth difference in media containing amino acids. Finally the strength of a series of heterologous promoters was determined in C. autoethanogenum. The research presented in this thesis improves our knowledge on C. autoethanogenum’s metabolism and offers tools to optimise it for product formation. This will enable improved exploitation of this organism for a carbon neutral future.

Interferons (IFNs) are cytokines which play a crucial role in the host defence against pathogens through the upregulation of hundreds of target genes. The binding of an IFN molecule to its specific receptor, results in the tyrosine phosphorylation of a member of the Signal Transducer and Activator of Transcription (STAT) protein family, a process often termed ‘STAT activation’. The first observation of this mechanism was the discovery of STAT1 and STAT2, which are both activated in response to type-I interferon and form a DNAbinding heterodimer which acts as a transcription factor. In the 25 years since this breakthrough, the establishment of these proteins as partners in signal transduction has been shown repeatedly. Whilst STAT1 has since be found to act as a signal transducer for many different cytokines, STAT2 appears to be almost exclusively driven by type-I IFN. Nonetheless, STAT2-deficient phenotypes suggest a broader and IFN-independent role for this protein. This thesis focused on the role of the STAT N-domain, which has been recently identified as an important site for cytokine-independent interactions. Using a variety of methods, including fluorescence microscopy as well as biochemical and biological assays, STAT2 was shown to be constitutively bound to STAT1 via their N-domains. The consequence of this binding was a strong inhibitory effect on the activity of STAT1. This was attributed to the formation of semi-phosphorylated dimers which were excluded from entering the cell nucleus. Furthermore, a single residue was found to abrogate this interaction and resulted in a hyper-responsive STAT1 phenotype in several immune responses such as parasitic immunity, antigen presentation and senescence. Therefore, STAT2 should now be considered an innate negative regulator of STAT1 activity in all cytokine signalling pathways with the exception of type-I interferon.

Campylobacter jejuni is one of the world’s leading causes of food-mediated bacterial infection, affecting millions of people every year. BamA, a highly conserved, essential membrane protein found in C. jejuni and other Gram-negative bacteria is responsible for folding a number of essential β-barrel outer membrane proteins making it vital for survival in this pathogenic organism. Along with essential roles in basic physiological functions, such as nutrient import and export, outer membrane proteins are also found in a number of multidrug resistance and virulence mechanisms in pathogenic organisms, making them and BamA popular targets for therapeutic intervention. BamA is well characterised in model organisms, however the conversed mechanism which facilitates assembly in different species remains unclear. In this work, the biogenesis of β-barrel proteins is investigated in C. jeuni NCTC-11168. A recombinant expression system is developed and used to express recombinant CjBamA (Cj0129) in an E. coli BL21 host, followed by subsequent purification and biophysical characterisation. A pelB leader sequence is used to target the recombinant protein for outer membrane localisation, with an extended 10x-polyhistidine tag and purified by immobilised metal affinity chromatography. Secondary structure analysis is performed using synchrotron radiation circular dichroism confirming the presence of a dominant β-sheet fraction. Furthermore, bioinformatics tools are used to investigate the structure of BamA homology models and explore the structural relationships between BamA and a previously unidentified accessory BamD protein found in the Campylobacter genus.

Diabetic neuropathy affects up to 50% of diabetic patients and commonly presents as neuropathic pain. Streptozotocin (STZ) injected type 1 diabetic rats exhibit thermal hypersensitivity and this is caused by the sensitization of transient receptor potential vanilloid 1 (TRPV1) on DRG neurons. Thermal hypersensitivity is ameliorated in STZ diabetic rats with the systemic treatment of vascular endothelial growth factor-A165b (VEGF-A165b). This thesis investigated the role of the activation of the receptor for advanced glycated end products (RAGE) on the sensitization of TRPV1 on DRG neurons and determined the effects of VEGF-A165b on this mechanism. Capsaicin-evoked TRPV1 activity was measured in DRG neurons from adult STZ diabetic rats to determine a sensitization effect and these effects were modeled in vitro by exposing DRG neuronal cultures from naïve rats to hyperglycemic conditions. STZ diabetic rats had sensitized agonist-evoked TRPV1 activity, increased RAGE expression on the DRG neurons, and increased expression of high mobility group box-1 (HMGB1, a RAGE agonist) protein around nociceptor terminals. In vitro it was determined that hyperglycemic conditions sensitize capsaicin-evoked TRPV1 activity and this effect was mediated by the activation of RAGE, indicating that RAGE activation in diabetes is likely to cause TRPV1 sensitization. It was determined that HMGB1 binds to RAGE on DRG neurons and this binding results in the sensitization of TRPV1 activity and this sensitization event was a PKC mediated effect. PKC is able to phosphorylate serine residues on the intracellular domain of TRPV1. This phosphorylation causes increased agonist-evoked TRPV1 activity. Here it was demonstrated that there was increased TRPV1 phosphorylation at serine 800, a PKC dependent phosphorylation site, on DRG neurons in hyperglycemia and this event is likely to contribute towards the TRPV1 sensitization effect induced by HMGB1-RAGE binding. In vivo VEGF-A165b did not alter the expression of HMGB1 around nociceptor terminals but it did reduce the expression of RAGE on DRG neurons. In vitro VEGF-A165b blocked TRPV1 sensitization in DRG neurons exposed to hyperglycemic conditions, blocked the PKC mediated phosphorylation event, and blocked the HMGB1 mediated sensitization of TRPV1. VEGF-A165b may, therefore, be preventing this sensitization effect by blocking by the PKC activation that occurs downstream of RAGE activation. These results demonstrate a novel mechanism of neuronal TRPV1 sensitization in vitro involving the activation of RAGE on the DRG neurons. This mechanism may contribute to the sensitization of nociceptors in diabetes and consequently the development of neuropathic pain. VEGF-A165b blocks this mechanism indicating that 1) VEGF-A165b has direct actions on the DRG neurons in preventing them from hyperglycemia mediated damage and 2) VEGF-A165b may be exerting its analgesic effects in STZ diabetic rats through this mechanism.

The delta opioid receptor (DOR) is a G protein-coupled receptor (GPCR) which is important in the regulation of neuronal function, predominantly via coupling to heterotrimeric Gi/0 proteins. The receptor has been shown to be a potential target for the treatment of chronic pain and affective disorders. Although a large number of opioid agonists exist, their properties vary widely, at least partly due to their differential coupling to post-receptor signalling systems, a phenomenon referred to as ligand-biased signalling or functional selectivity. The aim of the current project was to examine the signalling properties of a set of established and novel DOR agonists in an attempt to identify compounds that have biased signalling profiles. It was hypothesized that DOR agonists with partial efficacy regarding β-arrestin recruitment would be less liable to induce receptor internalization and desensitization of G protein-mediated signalling than full agonists. Chinese hamster ovary (CHO) cells, stably transfected with GFP-tagged human (h)DOR and CHO-K1 and U2OS cells over-expressing hDOR were exposed to a number of novel selective DOR agonists compared with the commercially available agonists, SNC80, ADL5859 and DADLE. The compounds’ potencies and efficacies were measured in four different assay systems; 1. Inhibition of forskolin-stimulated cyclic AMP accumulation, 2. Extracellular signal-regulated kinase (ERK1/2) phosphorylation using an immunocytochemical In-cell Western (I-CW) assay, 3. β-arrestin 2 recruitment and 4. hDOR internalization. The latter two assays employed DiscoverX Enzyme Fragment Complementation technology. An attempt to develop a secreted placental alkaline phosphatase (SPAP) reporter gene assay to measure DOR-mediated cyclic AMP inhibition was not successful. All of the ligands were nearly full agonists in relation to cyclic AMP inhibition although some were less efficacious than the standard SNC80 regarding ERK1/2 activation. Their absolute potencies and rank orders of potency in inhibiting cyclic AMP and activating ERK1/2 were quite different, although both signalling systems were apparently Gi/o- protein mediated. In contrast, the agonists exhibited a full range of efficacies and potencies in both β-arrestin 2 recruitment and hDOR internalization assays and there was a significant correlation between the maximum efficacies of the compounds in the two assays. A potential relationship between β-arrestin 2 recruitment/ hDOR internalization and desensitization of agonist-induced cyclic AMP accumulation was explored. Responses to the highly arrestin-recruiting agonists SNC80 and DADLE desensitized fully after extended exposure, whereas the novel partial agonists PN6047 and OPD00003 resisted desensitization. Bias factors were calculated for the agonist set and both PN6047 and OPD00003 were found to be significantly biased towards G protein-mediated cyclic AMP inhibition. In conclusion, this study reports, for the first time, a detailed characterization of signalling bias for a set of selective DOR agonists in cells over-expressing human DORs. The findings suggest that it is potentially possible to predict wanted and unwanted properties of agonists by determining post-receptor signalling profiles in vitro which will facilitate the discovery and development of novel therapeutics based on the DOR.

Introduction: The expression profile of plasma membrane organic cation transporters OCT/Ns has been widely investigated in a variety of cell types including hepatocytes, enterocytes, and renal cells. Their functional characteristics in terms of translocation of a wide range of positively charged compounds have been widely examined as well. Recently, the focus has been on their role in the transportation of inhaled drugs through respiratory epithelial cells. The airway epithelium is considered as a protective barrier against inhaled particulates and infectious agents that may cause damage to epithelial cells resulting in the permeation of harmful agents to the underlying tissue. Therefore, restoration of the lost epithelial cells is essential for maintaining epithelial functions. It has been suggested that OCTs, and particularly OCT1, mediate the release of non-neuronal acetylcholine from the cytosol of airway epithelial cells to the extracellular space where it acts as an auto/paracrine molecule. Furthermore, the OCTN2 gene has been associated with respiratory airway diseases such as asthma. In the current study, we aimed to investigate whether the OCT1 protein plays a role in respiratory wound healing via the transportation of non-neuronal acetylcholine. This has indeed been suggested to play a role in the stimulation of cell proliferation through activation of either muscarinic or nicotinic receptors. Methods: The alveolar epithelial cell line A549 was used as a model of the airway epithelium as it had been reported to express cholinergic components such as acetylcholine, muscarinic and nicotinic receptors. In a first instance, OCT/Ns expression in the cell line was probed at pre and post transcriptional levels using normal polymerase chain reaction (PCR), quantitative PCR (qPCR) and In Cell Western™ ICW, respectively. The intestinal cell line Caco-2 and the broncho-epithelial cell line Calu-3 were used as positive controls. A scratch wound model was then establised to assess the role of OCT/Ns in epithelial repair following injury. Initially, the healing of wounded A549 monolayers was assessed in presence of a panel of OCT/Ns inhibitors using the PrestoBlue™ cell proliferation assay. In the second stage of the study, the OCT1 gene was transiently silenced using short interfering RNAs (siRNAs) and the proliferation of knocked down cells was monitored using the PrestoBlue and Cyquant® cell proliferation assays. Results: Traditional PCR showed that OCT1, OCT3, OCTN1 and OCTN2 were expressed in A549 cells, whereas OCT2 was absent. The levels of the OCT1 and OCTN2 proteins were then relatively quantified in these cells using ICW. Some of the OCT/Ns inhibitors employed exhibited proliferation inhibitory effects on unscratched monolayers, while the bronchodilator ipratropium decreased cell proliferation at high concentration without causing toxic effects. However, it was difficult to determine whether OCT/Ns were likely to be involved in wound repair because the drug exhibits other properties than only an inhibitory effect on these transporters. Following optimisation of the siRNA transfection protocol, the OCT1 gene expression was ultimately decreased to about 60% compared to control cells. Nevertheless, OCT1 knockdown had no effect on cell proliferation post injury as monitored using the PrestoBlue and Cyquant® assays up to 48 hrs post transfection. Conclusions: Decreased levels of the OCT1 protein to about 60% of control did not affect cell proliferation in the A549 cell line. This might be because the protein still exhibited normal functions in terms of translocation of non-neuronal acetylcholine despite a lower expression. A549 cell proliferation may also be governed by molecules present in the cell culture medium such as growth factors rather than by non-neuronal acetylcholine.

In eukaryotic cells, the shortening and removal of the poly(A) tail (deadenylation) of cytoplasmic mRNA is a key step in mRNA degradation. The Ccr4-Not complex is well-characterised as a major deadenylase enzyme involved in mRNA deadenylation. The complex contains two catalytic subunits: Ccr4 and Caf1. Currently, it is unclear whether the Ccr4 and Caf1 catalytic subunits work cooperatively, or whether the nuclease components have unique roles in deadenylation. To facilitate the biochemical analysis of deadenylase enzymes, we have developed a fluorescence-based deadenylase assay, which is sensitive, quantitative and suitable for micro-well plate formats. We demonstrate the utility of the new assay for the discovery of small molecule inhibitors of the human Caf1/CNOT7 deadenylase enzyme. These compounds may become useful tools to investigate the contribution of the Caf1/CNOT7 in deadenylation. Furthermore, to understand the requirement and relative contributions of the ccr4 and Caf1 catalytic subunits, we therefore developed a method to express and purify a minimal human BTG2•Caf1•Ccr4 nuclease sub-complex from bacterial cells. By using chemical inhibition and well-characterised inactivating amino acid substitutions, we demonstrate that the enzyme activities of Caf1 and Ccr4 are both required for deadenylation. We propose a mechanism, in which the Caf1 and Ccr4 subunits cooperatively participate in mRNA deadenylation by the Ccr4-Not complex.

Emerging infectious diseases are, and have always been, an important threat to human health, animal health and the global economy. Emerging and re-emerging pathogens are opportunists, responding to changes in the host or environment. Therefore, diagnostic tests used for the detection of pathogens either of animal or human origin, must be just as adaptable in utilising new technology. In complex biological situations where multiple pathogens may be present or when using wildlife samples that may be rare or precious, DNA microarray technology is particularly valuable as hundreds or thousands of targets can be screened simultaneously in a single sample. This thesis investigated the potential of a DNA microarray as a screening tool for over 20 pathogens as part of the Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife project (WildTech), an FP7 EU funded project (www.wildtechproject.com). The pathogens included zoonotic viruses, bacteria and parasites. Publicly available software was used for design of oligonucleotides for the array. Evaluation of the oligonucleotides was carried out using reference samples from a variety of sources. Different rodent species were screened including the principal commensal species Rattus rattus, Rattus norvegicus, and Mus musculus. Nucleic acid extracted from samples of liver, kidney and lung was screened for the presence of pathogens. The array successfully identified Leptospira and Seoul hantavirus positive samples from animals trapped in the United Kingdom, France and Canada. These results were confirmed using previously established methods. It is likely that, over the next few years, diagnostic microarrays will become relatively inexpensive research tools. Molecular testing for emerging pathogens is increasingly being utilised and use of this technology will result in timely, accurate and inexpensive diagnosis to enable effective control of these infectious diseases with important implications for human health. The data contributed here can aid in the steps required to design microarrays for screening purposes.

The heart is the first functioning organ to develop during embryogenesis to maintain the growing embryo with oxygen and nutrients. However, cardiogenesis is a complex but well-regulated process, and any changes to this regulation can result in detrimental defects of the heart. For many years sarcomeric proteins have been associated with a range of cardiomyopathies and in more recent years are known to been involved in congenital heart defects (CHDs). To date, tropomyosin 1 (TPM1) and cardiac troponin T (TNNT2) have been associated with cardiomyopathies but never with CHDs. These two genes are important regulatory proteins of the thin filament of the sarcomere and vital for correct contraction and force generation within cardiomyocytes. To investigate a role for TPM1 and TNNT2 in the early developing heart, using the chick as an animal model, antisense oligonucleotide morpholino technology were utilised to manipulate both genes in ovo. The gross anatomical structures, ultrastucture and molecular functions of the treated hearts were analysed to determine if the morpholino treatment resulted in any developmental abnormalities. In addition, the TPM1 gene, including introns, was sequenced in a cohort of 380 patients with a range of congenital heart anomalies. In the TPM1-morpholino treated hearts, atrial septation and ventricular chamber maturation via the production of trabeculae were affected. Stereological analysis of these hearts revealed a reduction in the proportion of myocardium in the ventricular chamber along with increased luminal size. In addition, TPM1-morpholino treatment had an effect on myofibril maturation in vitro, as well as causing increased apoptosis in the developing ventricle and atrial septum. Four genetic variants of TPM1 were identified in the patient cohort; I130V, S229F, IVS1+2T > C and GATAAA/AATAAA in the polyadenylation signal. In silico analysis predicted the missense mutations to be disease causing. In vitro functional analyses of the IVS1+2T > C mutation that the IVS1+2T > C mutation resulted in abnormal splicing of the TPM1 pre-mRNA. TNNT2-morpholino treatment affected the growth of the atrial septum. However, the sarcomere appeared normal in this treatment group. Stereological analysis also revealed normal cardiac proportions except for the atrial chamber, which was reduced in size. The abnormal phenotypes observed in the TPM1 and TNNT2 treated groups may be a result of altered haemodynamics within the developing heart. Further studies such as in situ hybridisation of markers of haemodynamics may elucidate this role in the future. The abnormal splicing observed in the IVS1+2T > C may be a contributing factor to CHD in man and therefore, indicates that sarcomeric proteins are important for the future screening of potential contributing factors to CHDs.