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Enterococci are a leading cause of infective endocarditis, bacteraemia and urinary tract infections. Unfortunately, an increasing proportion of these organisms display resistance to front-line antimicrobial agents. The aim of the work described in this thesis was to assess the efficacy of antibiotics against clinical strains of enterococci and evaluate their ability to trigger adaptive mechanisms (stringent response) associated with drug tolerance and virulence. Enterococcus faecalis and Enterococcus faecium isolates grown in planktonic and biofilm states were assessed for susceptibility to vancomycin, rifampicin, linezolid and tigecycline. As a potential avenue to overcome resistance and preserve antibiotic activity, combinations of drugs were evaluated for synergy against sensitive and drug-resistant strains of enterococci using in vitro and in vivo models. Transcriptome analysis of parental E. faecalis OG1RF and mutants defective or void of stringent response activity were examined to determine if this response was induced by exposure to antibiotics and if virulence gene expression was modulated. The majority of enterococci tested planktonically were susceptible to all four antibiotics, while biofilm-associated cells were refractory to killing. Rifampicin in combination with vancomycin, linezolid and tigecycline demonstrated enhanced killing properties against enterococci in planktonic and biofilm growth states compared to killing by individual agents. When tested in vivo, synergistic combinations improved Galleria mellonella survival compared to monotherapy. Exposure of parental E. faeca/is OG1RF to vancomycin and linezolid triggered upregulation of stringent response genes (re/A and re/Q). In contrast, tigecycline did not induce modulation of these genes, which suggests that the stringent response was not activated. Virulence genes associated with biofilm formation were upregulated by all antibiotics during exposure, independent of stringent response induction, while increased modulation of an endocarditis-associated adhesin and proteases were only apparent in linezolid exposed cells. Comparison of stringent response deletion mutants of E. faecalis OG1RF revealed that expression of re/A was necessary for an antibiotic induced stringent response, while re/A and re/Q worked synergistically to enhance virulence gene modulation. Combination therapy is widely used when difficult-to-treat infections are identified, though the clinical outcome is not always predictable. Additionally, antibiotics have properties other than those related to their therapeutic use. Specifically, they act as signalling molecules that provoke adaptive responses in bacteria, including modulation of gene expression. The findings of this thesis suggest that tigecyclinebased combinations may prove useful in therapy against enterococci when antibiotic resistance is detected. Additionally, tigecycline did not appear to trigger a stringent response thorough modulation of associated genes, while vancomycin and linezolid induced expression of stringent response and virulence factor associated genes. The clinical relevance of an active stringent response in E. faecalis has not been fully elucidated, though it may underlie patient treatment failure.

Nrf2 (Nuclear factor erythroid 2-related factor 2), a redox-sensitive transcription factor, plays a critical role in the regulation of cellular defence and contributes to a number of cellular processes. Nrf2 is regulated through an interplay of complex transcriptional and post-translational mechanisms that modulates its activity during cellular perturbations or other biological processes thereby ensuring cellular homeostasis is maintained through the orchestration of adaptive responses. However, there is mounting evidence that constitutive upregulation of the Nrf2 pathway drives the enhanced proliferation and chemoresistance of various cancers. Therefore, an ability to modulate the activity of the Nrf2 pathway holds promise as a therapeutic strategy in certain disease settings. The work presented in this thesis showed that CDDO-Me provoked the induction of the Nrf2 pathway in C57BL6J WT and Nrf2 KO mice and CD1 WT mice. Analysis of CDDO-Me induced gene expression changes in both WT and Nrf2 KO mice showed a significant increase in the relative mRNA levels of ARE-dependent genes in the livers of CDDO-Me treated WT animals. Notably, CDDO-Me also provoked the accumulation of Nrf2 and NQO1 in human PBMCs and PHHs demonstrating its translational relevance. The mechanism of action of CDDO-Me as an inducer of Nrf2 is poorly understood. It was shown here that CDDO-Me post-transcriptionally evoked concentration and time-dependent, accumulation of Nrf2 protein in Hepa1c1c7 cells. Furthermore, CDDO-Me was shown to stabilize Nrf2 protein independently of the modulation of protein kinases and other signalling pathways that are purported to regulate Nrf2 activity. The work here also provides in vitro insights into the molecular mechanism of Nrf2 inhibition by the quassinoid brusatol. Brusatol post-transcriptionally evoked concentration- and time-dependent, yet transient, depletion of basal and inducible protein levels of Nrf2 in Hepa-1c1c7 cells. Furthermore, the ability of brusatol to inhibit Nrf2 was not affected by siRNA depletion of Keap1. In keeping with the latter observation, brusatol induced the depletion of Nrf2 independently of the proteasome and autophagic degradation machineries. Thus, these findings indicate that brusatol exploits a previously unknown mechanism of Nrf2 degradation. By examining the molecular mechanisms underlying the activation of Nrf2 by CDDO-Me and its inhibition by brusatol, this work reveals novel aspects of regulation within this important cellular pathway, and informs the design of new pharmacological inducers and inhibitors, which hold promise as therapeutic agents in a number of diseases.

Background and purposes: Citicoline, also known as CDP-choline (cytidine-5-diphosphocholine), is a naturally-occurring endogenous nucleoside, which is one of the neuroprotective drugs that have been used as a therapy in stroke patients. However, the mechanisms through which it acts are not fully understood. In terms of analysis of the signalling mechanisms associated with citicoline-induced protection, it has been previously shown that citicoline may protect the ischemic neurons by suppressing caspase apoptotic pathway activation. Moreover, preliminary in vitro studies have shown that citicoline induces angiogenesis (the formation of new blood vessels from pre-existing capillaries). However, the possible beneficial effects of citicoline treatment on revascularization and angiogenesis after stroke have not been fully examined. The present study was designed to investigate the key signalling mechanisms through which citicoline modulates apoptosis and angiogenesis-associated with stroke recovery. Methods: An analysis of citicoline signalling pathway was studied from phospho-protein screening array done by Kinexus. In vitro angiogenesis assays: migration, proliferation and differentiation into tube-like structures in Matrigel™ ™ assays, have been used in human brain microvessel endothelial cells (hCMEC/D3). Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. Analysis of apoptosis by flow-cytometry in hCMEC/D3. A hypoxia induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. Quantification of apoptotic cells were carried out under fluorescence microscopy using a combination of propidium iodide and DAPI stain solution. Apoptotic pathways in hCMEC/D3 stimulated with citicoline were examined by indirect immunofluorescence and real time PCR. Pharmacological inhibitor of Her2 (GW2974) was used to investigate the angiogenic signalling pathway by western blotting and Matrigel™ assay in hCMEC/D3 in the presence or absence of citicoline. Results: Kinexus results showed an over-expression of ASK-1, HER2, IRS-1 and Jun and inhibition of Hsp27, Integrin alpha4, MEK1 (MAP2K1) and Histone H2B Ser14 proteins. Citicoline induced EC migration and differentiation in poly-l-lysine and Matrigel™ . Using microarray screening, the Histone H2B (Ser14) appeared to be the main phosphor-protein expression blocked by citicoline in hCMEC/D3, and the expression of tyrosine-protein kinase erbB-2 receptor (Her2) appeared to be induced by citicoline in hCMEC/D3. Treatment with the Her2 inhibitor (GW2974) totally blocked citicoline induced endothelial tube formation in EC, whereas treatment with GW2974 in combination with FGF-2 did not affect FGF-2 induced endothelial tube formation. In cultured hCMEC/D3 treatment with GW2974 inhibited citicoline induced phosphor-Erk expression, whereas treatment with GW2974 in combination with FGF-2 did not affect FGF-2 induced phosphor-Erk expression. However, Citicoline had no mitogenic effects on hCMEC/D3. Phspho-Caspase-3 and phosphor-H2B (Ser14) expression were inhibited by citicoline in hCMEC/D3 whereas the expression of phosphor-Her2 and phosphor-Erk expression were increased. Moreover, citicoline treatment showed a decrease in number of apoptotic cells (positive PI staining) in hypoxia induced apoptosis compared to untreated cells. In cell migration assay, treatment with citicoline significantly increased cells migration in hCMEC/D3 compared to untreated cells on hypoxia conditions. Detection of apoptotic cells by flow cytometry showed inconclusive results in both treated and untreated hCMEC/D3. Results from indirect immunofluorescence showed a significant increase in active Caspase-3 and H2B (Ser14) expression in citicoline treated cells in comparison with untreated cells in hypoxia conditions. Results from PCR showed inhibition of pro-apoptotic genes including BNIP3, BNIP3L, caspase 4, caspase 8, caspase 9, CIDEB, DFFA, LTBR, TP53BP2, TRADD, and TRAF3 with citicoline treatment. They also show an over-expression of a number of anti-apoptotic genes including NAIP, NOD1, TNFRSF25, and TP53. Conclusions: A screening of phosphor-protein expression revealed that citicoline specifically over-expressed Her2 which demonstrated that citicoline plays a key role in Her2 induced angiogenesis. Blocking of Her2 pathway inhibited the formation of tube-like structures in citicoline treated cells and therefore citicoline induces angiogenesis through Her2 pathway, and that is important in terms of understanding the molecular pathway in which citicoline acts as a pro-angiogenic molecule in tissue remodelling after stroke. Citicoline decreased active caspase-3 and H2B (Ser14) expression, and positive PI staining which demonstrates a protective effect of citicoline against endothelial cells apoptosis. Citicoline also improved cell surivial by decreasing the expression ofBNIP3, BNIP3L, caspase 4, caspase 8, caspase 9, CIDEB, DFFA, LTBR, TP53BP2, TRADD, and TRAF3 genes and inducing the expression ofNAIP, NOD1, TNFRSF25, and TP53. Citicoline treatment significantly promotes wound healing in stroke mimicking conditions (hypoxia). Thus, the therapeutic properties of citicoline has the potential to promote vessel formation whilst reducing the risk cell death from hypoxic stress following ischemic stroke.

Factors affecting catecholamine levels in vitro were studied using the modified high pressure liquid chromatography method with electrochemical detection. Differential centrifugation showed that platelet-rich plasma contained significantly higher catecholamine levels than platelet-poor plasma. Serum samples had significantly higher catecholamine levels than plasma. Plasma or serum samples clotted with glass beads had significantly higher catecholamine levels than those without. Therefore, consistent results can only be obtained when catecholamine samples are spun at the same speed, either plasma or serum can be used, but not both in a single study. Post-dated blood for transfusion was used to study stability of catecholamines, and showed that catecholamines are stable. Hence, the collection of blood samples for catecholamine measurements was modified. Blood samples were collected in Vacutainer tubes containing lithium heparin without antioxidants and not pre-cooled, samples were spun at the convenient time. This was welcomed by the clinicians who did not have to interrupt clinical assessment to care for blood samples as with the former method. The three clinical studies showed no significant differences in catecholamine levels in patients undergoing laryngoscopy with and without tracheal intubation, whether or not the patients were beta blocked or had received topical tracheal analgesia. The mean catecholamine levels were within the normal range. No relationships between baseline catecholamine levels and the baseline blood pressures or heart rate nor between the changes in catecholamine levels from the baseline and the corresponding changes in blood pressures or heart rate. The Injury Severity Score in minor injured patients had no relationship with plasma catecholamine levels, and no significant rise in noradrenaline levels when the ISS<30 and adrenaline levels when the ISS<17. The studies suggest that catecholamine levels are of no value in assessing the severity of minor injuries, or changes in blood pressures or heart rate during anaesthesia.

Adverse reactions to drugs and environmental chemicals are a serious problem with up to 30% of hospital patients experiencing such problems (Venning, 1983; Ludwig and Axelsen, 1983). There is evidence that many adverse reactions arise as a result of genetically controlled sensitivity (Festing, 1987). Large genetically determined differences in response to chemicals have also been recorded in laboratory animals. However, most toxicological screening involves a single strain and fails to detect genetically determined sensitivity. Should some animals show an adverse reaction, this is usually attributed to "biological variation". As the pedigree of such animals is not normally known at the time of use there is no way of showing whether these adverse reactions were inherited (Festing, 1975, 1979). The initial aim of this project was to develop a technique for studying genetic variation in sensitivity to treatment with drugs using in vitro screening methods. The techniques should not require hazardous or expensive chemicals and equipment, should require a small number of animals and should be reliable and easy to perform. Several end-points were studied, and a protocol for detecting genetic differences which included four end-points and two cell types was developed. In the second part of the project, the aim was to study genetic variation in sensitivity to Aspirin, Ethanol and Coumarin as model compounds, using the previously developed techniques in conjunction with suitable genetically-defined strains of mice. Two cell types (macrophages and hepatocytes) were studied and several end-points were used including neutral red uptake, total protein concentration, rate of phagocytosis and LDH activity in cells and supernatant. The study involved nine strains of mice. Although statistically significant differences among inbred strains were detected, in no experiment did strain distribution pattern suggest single-locus Mendelian control. There was no evidence that response to coumarin depended on the coumarin hydroxylase (Cyp2b) locus nor that response to alcohol depended on the alcohol dehydrogenase locus. It is concluded that further development would be necessary to develop these methods as a way of identifying genes associated with their type of genetic variation.

The cellular mechanisms by which opioid and general anaesthetic drugs produce their clinical effect remain unclear, but modulation of stimulus-secretion coupling may be important. The human neuroblastoma cell line, SH-SY5Y, is a model of a human sympathetic neuron. The uptake and release (depolarization and receptor mediated) of [3H] noradrenaline ([3H] NA) in SH-SY5Y cells has been characterized, and the role of cAMP and calcium in evoked release examined. Further studies examined the effect of opioids (including fentanyl), a volatile anaesthetic agent (halothane), and intravenous anaesthetics (thiopentone and propofol) on uptake and release of [3H] NA. SH-SY5Y cells are capable of the uptake and evoked release of [3H] NA. K+ evoked release is dependent on extracellular calcium but carbachol evoked release appears to be extracellular calcium- independent. cAMP does not have a role in the immediate evoked release of [3H] NA, but an effect on long term secretion cannot be excluded. Fentanyl inhibits the uptake and release of [3H] NA, but this effect is not opiate receptor mediated. Morphine, DAMGO, met- and leu- enkephalin had no effect on [3H] NA release, and these results indicate that opiate receptors on SH-SY5Y cells are not coupled to neurotransmitter release. Halothane inhibits K+, but not carbachol evoked [3H] NA release. In addition, halothane inhibits [3H] NA uptake but only at relatively high concentrations. Thiopentone, but not propofol, inhibits [3H] NA uptake, and both drugs inhibit [3H] NA release. The results of these studies suggest that inhibition of voltage sensitive calcium channels by general anaesthetic agents may contribute significantly towards the state of anaesthesia.

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