The emergence and spread of antibiotic resistance hampers effective treatment of bacterial infections. This is particularly the case for infections involving a biofilm component, as the activity of existing antibacterial drugs against these surface-attached communities is limited. The work presented in this thesis sought to identify and characterise compounds with antibacterial and antibiofilm activity against the important pathogen, Staphylococcus aureus. Antistaphylococcal activity was assessed for 16 antioxidants that are used in cosmetics, traditional medicines or as food additives, and which have been reported previously to have some antibacterial activity. Initial experiments with tert-butylhydroquinone (TBHQ) showed that activity that had previously been ascribed to the antioxidant, was a consequence of its conversion to tert-butylbenzoquinone (TBBQ) under culture conditions. TBBQ displayed innate bactericidal activity against S. aureus that was effected through perturbation of the bacterial membrane. The other antioxidants also inhibited staphylococcal growth through perturbation of the cytoplasmic membrane, and compounds that displayed selective action against bacterial membranes were identified. Of the agents with bacterial specificity, TBBQ, celastrol and nordihydroguaiaretic acid (NDGA) also eradicated staphylococcal biofilms; a rare property amongst antibacterial agents. Although these antioxidants exhibited a similar membrane-damaging mode of action, their mechanisms of antibiofilm activity differed. TBBQ eradicated preformed biofilms through sterilisation of slow-growing and persister cell populations, whilst celastrol and NDGA caused physical disruption of the biofilm. All three antioxidants acted synergistically with gentamicin against biofilms, eradicating surface attached populations at concentrations that did not cause irritation or visible damage to a human skin equivalent. The potent and selective antibacterial activity, and low resistance potential upon extended subculture, suggest that these compounds could be used topically in combination with gentamicin to treat infected wounds.

The increasing prevalence of infections attributed to antibiotic-resistant bacteria has prompted renewed interest in exploiting the antibacterial properties of Ag+ to treat such infections. However, the antibacterial mode of action (MOA) and bactericidal activity of Ag+ are poorly understood, and there are concerns that the prolific and unrestricted use of Ag+ in consumer products will select bacterial Ag+ resistance, thus limiting the clinical utility of Ag+. Ag+ resistance already exists, although aspects of the molecular basis of Ag+ resistance, and the current prevalence of Ag+-resistant pathogens are unclear. This thesis sought to address these issues. Ag+ was found to be bacteriostatic in culture media and bactericidal in buffer, and was unable to eradicate Staphylococcus aureus biofilms in vitro. MOA studies indicated that the primary antibacterial target of Ag+ is the cell membrane. Evidence was obtained suggesting that Ag+ does not interfere with the phospholipid component of the membrane, but instead probably damages integral membrane proteins to produce an antibacterial effect. A survey of hospital staphylococcal isolates (n=1006) found universal susceptibility to Ag+, and Ag+ resistance could not be selected in S. aureus and several other pathogens in vitro. However, in Escherichia coli, high-level Ag+ resistance arose rapidly and was not associated with a fitness cost likely to prevent its emergence in the clinical setting. Ag+-resistant strains contained mutations in genes regulating expression of an Ag+ efflux mechanism and outer membrane porins. A detailed characterisation of a known Ag+-resistance determinant (the sil operon), was also conducted to provide further insights into the mechanism of Ag+ resistance conferred by this determinant. Collectively, these studies provide further insights into the MOA of Ag+ and the mechanisms of Ag+ resistance, which could potentially be applied to optimising the future uses of Ag+ as an antibacterial agent.

In 2004, the FDA launched the Process Analytical Technology (PAT) initiative to support product and process development. Even before this, the biologics manufacturing industry was working to implement PAT. While a strong focus of PAT is the implementation of new monitoring technologies, there is also a strong emphasis on the use of multivariate data analysis (MVDA). Effective implementation and integration of MVDA is of particular interest as it can be applied retroactively to historical datasets in addition to current datasets. However translation of academic research into industrial ways of working can be slowed or prevented by many obstacles, from proposed solutions being workable only by the original academic to a need to prove that time invested in developing MVDA models and methodologies will result in positive business impacts (e.g. reduction of costs or man hours). The presented research applied MVDA techniques to datasets from three scales typically encountered during investigations of biologics manufacturing processes: a single product, dataset; a single product, multi-scale dataset; a multi-product, multi-scale, single platform dataset. These datasets were interrogated in multiple approaches and multiple objectives (e.g. indictors/causes of productivity variation, comparison of pH measurement technologies). Individual project outcomes culminated in the creation of a robust statistical toolbox. The toolbox captures an array of MVDA techniques from PCA and PLS to decision trees employing k-NN. These are supported by frameworks and guidance for implementation based on interrogation aims encountered in a contract manufacturing environment. The presented frameworks ranged from extraction of indirectly captured information (Chapter 4) to meta-analytical strategies (Chapter 6). Software-based tools generated during research ranged from translation of high frequency online monitoring data as robust summary statistics with intuitive meaning (Appendix A) to tools enabling potential reduction in confounding underlying variation in dataset structures through the use of alternative progression variables (Chapter 5). Each tool was designed to fit into current and future planned ways of working at the sponsor company. The presented research demonstrates a range of investigation aims and challenges encountered in a contract manufacturing organisation with demonstrated benefits from ease of integration into normal work process flows and savings in time and human resources.

All clinical opioid analgesics target the MOP (Mu Opioid Peptide) receptor. While these drugs provide analgesia, long-term treatment leads to tolerance and dependence. By targeting MOP and another member of the opioid receptor family, such as DOP (Delta Opioid Peptide receptor) or NOP (Nociceptin Orphanin F/Q Opioid Peptide receptor), these adverse effects are attenuated. Furthermore, solely targeting DOP or NOP may produce analgesia without the adverse effects associated with MOP. Three groups of variably mixed ligands have been developed; i) Fentanyl-based DOP and NOP bivalents, ii) peptide based MOP and NOP bivalents iii) tetrabranched NOP and DOP monovalent ligands. The pharmacology of these ligands has been investigated in a range of intracellular signalling assays. All compounds were tested in Chinese hamster ovary (CHO) cells expressing human MOP, NOP, DOP or KOP (Kappa Opioid Peptide receptor) receptors. Initial work with Fentanyl-based DOP bivalents resulted in a loss of functional activity at the MOP receptor. Further Fentanyl-derivatives conjugated with Ro65-6570 displayed partial agonist activity at MOP and full agonist activity at MOP. A second MOP/NOP bivalent pharmacophore, (DeNO), based on the peptides Dermorphin (MOP) and N/OFQ demonstrated full agonist activity at both receptors. A tetrabranched ligand formed from N/OFQ, displayed increased potency at the NOP receptor compared to N/OFQ. DeNO was investigated in human embryo kidney (HEK) cells which co-expressed MOP and NOP. The results of functional assays demonstrated a loss of MOP activity caused by the presence of NOP. Further studies with the opioids, Dermorphin and N/OFQ, and antagonists naloxone (MOP) and UFP-101(NOP), have demonstrated a structural interaction between MOP and NOP in this cell line. The work in this thesis demonstrates how modification of peptide structures was more successful in the development of multitarget ligands. The findings from this thesis provide a significant contribution to theory of receptor heterodimerisation between MOP and NOP, as demonstrated by the loss of potency of MOP agonists in the co-expression system.

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