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A rise in the number of bacterial strains resistant to both front-line treatment and "last resort" antibiotics means there is an urgent need for the development of new antimicrobial agents, or the modification of old antibiotic classes to extend their lifespans. The Trojan Horse approach offers a potential means of achieving both of these objectives by exploiting bacterial nutrient transport for antibiotic delivery. The primary aim of this research was the development of new biolabile linkers that can undergo cleavage inside the cytoplasm of bacteria, releasing an active antimicrobial species. A series of conjugates containing electrondeficient aromatic sulfonamide linkers were designed for this purpose; these can undergo release of sulfur dioxide (SO2) on reaction with biological thiols, and can be modified to undergo concurrent release of an antimicrobial unit. A conjugate bearing an SO2-releasing 2,4-dinitrobenzenesulfonamide group, and containing monocatecholate siderophore aminochelin was synthesised and characterised to determine whether siderophore units could potentiate the antimicrobial activity of SO2. While rapid release of SO2 is observed on reaction with thiols, the conjugate displayed poor antimicrobial activity against a range of bacteria, likely a combination of poor bacterial uptake, and SO2 release alone being insufficient for good antimicrobial activity. Two conjugates containing ciprofloxacin as an antimicrobial component were synthesised and characterised. Conjugates based on a 4-carboxyl-2- nitrobenzenesulfonamide linker displayed slow SO2 release on reaction with glutathione. Screening of a desferrioxamine conjugate of this type vs. a panel of bacteria showed reduced activity compared to the parent ciprofloxacin, with the slow release of ciprofloxacin potentially a limiting factor. In contrast, rapid SO2 release was observed for conjugates containing a novel pyrazine-sulfonamide linker, although these display instability in MHII broth, a common bacterial growth medium. An azotochelin conjugate containing this linker also displayed reduced activity compared to ciprofloxacin in bacterial assays.

Venous thromboembolism (VTE) is a global disease with high incidence, prevalence, morbidity and mortality. The risk of VTE is elevated during postpartum period. Vitamin K antagonists (VKAs) and low molecular weight heparins (LMWH) are the only anticoagulants currently available for the prophylaxis and treatment of VTE during the postpartum period. However, the parenteral nature of LMWH and frequent monitoring required with VKAs, act as barriers for many women. Direct oral anticoagulants (DOACs) have been demonstrated to be effective alternatives to LMWH and VKAs for VTE prophylaxis and treatment, but they are contraindicated in breastfeeding women due to limited clinical data. The aim of this thesis is to investigate how much DOACs transfer into human breastmilk and estimate infant exposure. The work initially involved completion of a qualitative study which aimed to investigate the factors that influence women's participation in a clinical trial involving DOACs whilst breastfeeding. Following the completion of 3 focus groups involving a total of 8 volunteer breastfeeding mothers, a qualitative data analysis was performed using Nvivo software. Several themes were identified using thematic framework approach, including: perceptions of what a clinical trial is, decision-making process, motivations for clinical trial participation, barriers to clinical trial participation, views on the proposed clinical trial whilst breastfeeding, and suggestions for the proposed clinical trial. Based on the findings, a revised protocol for the proposed DOACs trial was devised to include the provision of home trial visits, the follow-up procedure, prior training of using breast pumps, and actively recruiting mothers at the 'weaning' stage of lactation. To allow DOACs to be accurately quantified in human plasma and breastmilk, an ultra-highperformance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) assay was developed and validated for quantifying three DOACs - apixaban, edoxaban and rivaroxaban in human plasma and breastmilk. Protein precipitation with methanol was performed for sample preparation. Chromatographic analysis was performed using a C18 column. The MS detection was performed in MRM mode. The assay fulfilled the European Medicine Agency (EMA) bioanalytical method validation guideline and was shown to be simple, fast, and accurate. The assay was then utilised to measure samples collected for a phase IV clinical trial (pilot study) involving apixaban and rivaroxaban. In the pilot study, two breastfeeding women received a single dose of rivaroxaban 20mg, and one woman received two doses of apixaban 5mg (12 hours apart) and had their plasma and breastmilk DOAC concentration measured over a 24-hour period. The pharmacokinetic data were analysed using noncompartmental modelling in R. The results indicate a small amount of rivaroxaban transfers into breastmilk (milk to plasma ratio (M/P) = 0.27 (±0.02), estimated relative infant dose (RID) = 1.63% (±0.01)), in contrast to apixaban, which transfers into milk in significant concentrations (M/P = 2.61, RID = 12.78%). This work suggests that rivaroxaban holds promise to be an effective alternative to LMWH and VKAs for postpartum VTE prophylaxis and treatment, and should be built on, so that what is offered to breastfeeding women can be improved.

Multidrug-resistant pathogens are prevalent in chronic wounds. There is an urgent need to develop novel antimicrobials and formulation strategies to overcome antibiotic resistance and provide a safe alternative to traditional antibiotics. Chapter 2 aims to create a novel nanocarrier for two cationic antibiotics, tetracycline hydrochloride and lincomycin hydrochloride, overcoming antibiotic resistance. In this study, the use of surface-functionalised polyacrylic copolymer nanogels as carriers for cationic antibiotics is investigated. These nanogels can encapsulate small cationic antimicrobial molecules and act as a drug delivery system. They were further functionalised with a biocompatible cationic polyelectrolyte, bPEI, to increase their affinity towards the negatively charged bacterial cell walls. These bPEI-coated nanocarrier-encapsulated antibiotics were assessed against a range of wound isolated pathogens, which had been shown through antimicrobial susceptibility testing (AST) to be resistant to tetracycline and lincomycin. The data reveals that bPEI-coated nanogels with encapsulated tetracycline or lincomycin displayed increased antimicrobial performance against selected wound-derived bacteria, including strains resistant to the free antibiotic in solution. Next, after experimentation into the use of Carbopol nanogels against antibiotic-resistant wound-derived pathogens, in planktonic form, the work in chapter 3 investigated their use against biofilm-formed pathogens. Biofilms are prevalent in chronic wounds and once formed, are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Novel nanotechnology is applied to do this, based on protease-functionalised nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase, "digest" their way through the biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls, which overwhelms their defences. This thesis's work demonstrates that they are effective against six wound biofilm-forming bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis. Additionally, it is shown that co-treatments of ciprofloxacin and Alcalase-coated Carbopol nanogels led to a 3-log reduction in viable biofilm-forming cells when compared to ciprofloxacin treatments alone. Encapsulating an equivalent concentration of ciprofloxacin into the Alcalase-coated nanogel particles boosted their antibacterial effect much further, reducing the bacterial cell viability to below detectable amounts after 6 hours of treatment. Chapter 4 combines the work of chapter 2 (NPs against antibiotic-resistant pathogens) and chapter 3 (NPs against biofilm-forming pathogens). This concept is demonstrated by encapsulating Penicillin G and Oxacillin into shellac nanoparticles, subsequently coated Alcalase. It is shown for the first time that these active nanocarriers can destroy biofilms of S. aureus resistant to Penicillin G and are significantly more effective in killing the bacterial cells within compared to an equivalent concentration of free antibiotic. The approach of concentrating the antibiotic by encapsulating it into a nanocarrier allows a localised antibiotic delivery to the anionic cell wall, facilitated by coating the NPs with a cationic protease. This approach allowed the antibiotic to restore its effectiveness against S. aureus, characterised as resistant to the same antibiotic and cause rapid bacterial biofilm degradation. This approach could be potentially used to revive old antibiotics which have already limited clinical use due to developed resistance. Chapter 5 continued investigating the antimicrobial properties of antibiotic-loaded shellac NPs, with a cationic protease surface functionalisation, however this time on a pathogen fungal species, Candida albicans. These Amphotericin B (AmpB)‐loaded shellac NPs are fabricated by pH‐ induced nucleation of aqueous solutions of shellac and AmpB in the presence of Poloxamer 407 (P407) as a steric stabiliser (in the same fashion as penicillin G and oxacillin in chapter 4. The AmpB‐loaded shellac NPs are surface coated with the cationic protease Alcalase. The AmpB‐loaded shellac NPs show a remarkable boost of their antifungal action compared to free AmpB when applied to C. albicans in both planktonic and biofilm forms. The surface functionalisation with a cationic protease allows the NPs to adhere to the fungal cell walls, delivering AmpB directly to their membranes. Additionally, the hydrolysing activity of the protease coating degrades the biofilm matrix, thus increasing the effectiveness of the encapsulated AmpB compared to free AmpB at the same concentration. Additionally, these protease‐coated nanocarrier-based antibiotics showed no detectable cytotoxic effect against human keratinocytes. It is envisaged these antibiotic-loaded NPs. Subsequently, surface functionalised with the cationic protease could be potentially used to treat antibiotic-resistant biofilm infections in the clinic, for example, in recalcitrant chronic wounds. Chapter 6 outlines future work which could be performed using these NP formulations.

Clinical imaging including as PET, CT and MRI have revolutionised the detection and management of diseases such as cancer. When imaging agents are coupled onto nanosized carriers they provide information on the tumour location as well as provide an insight into their biodistribution. Therefore, these nanocarriers can be involved in integration of diagnosis and therapy in a single platform, which is called theranostics. Image Guided Focused Ultrasound drug delivery is a promising strategy for enabling both cancer diagnosis and treatment using the same nanocarrier delivery/diagnostic system. The aim of this project is to develop near infrared fluorescence (NIRF) and magnetic resonance imaging (MRI) labelled liposomes for targeted image guided drug delivery when combined with focused ultrasound (FUS). FUS can be applied for regional increase in temperature (hyperthermia) whereas MR guided FUS (MRgFUS) is a clinically used instrument that can provide this controlled hyperthermia. Previous studies have shown that incorporation of chelated Gd3+ lipids into liposomal formulations empowered these nanoparticles with MRI contrast enhancement. Here, various spacers between the chelating ligand (head group) and the lipid tail were introduced, to investigate their effects on liposomes' T1 relaxivities, a parameter used for measuring the contrast efficiency. Image guided thermosensitive PEGylated liposomes (iTSLs) were prepared with various Gd3+ chelated lipids that were made for the purpose of this study. In addition, a second, near infrared fluorescence (NIRF) label was also included in the liposomes for optical imaging. The prepared iTSLs were characterised by T1 relaxivities using a 400 MHz NMR and 9.4 T MRI. Gd3+ concentrations of liposome formulation were determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and Total Reflection X-Ray Fluorescence (TXRF). In addition, these two analytical methods were compared for reproducibility and accuracy for assessment of Gd3+ concentrations in liposomes. Moreover, the liposomal formulations incorporating with MRI and NIRF lipids were loaded with a chemotherapeutic agent (doxorubicin). In vivo, the tumours were monitored with doxorubicin when released via heat activation by FUS, which showed intrinsic drug fluorescence change. iTSLs accumulation in tumours at defined time points (post injection) in vivo were investigated with both imaging techniques (NIRF and MRI). Both NMR and MRI relaxometries studies showed potential for MR contrast enhancement. T1-weighted images showed positive enhancement for all iTSLs, with longer spacers apparently having a stronger effect. In mice, administration of iTSLs have shown a time-dependent tumour contrast enhancement and the change in T1 was quantified over time. The studies on xenograft mice models provided evidence that mild FUS-induced hyperthermia greatly improves the iTSLs uptake in tumours and trigger rapid drug release which improves the overall therapeutic index.

Calcium-activated chloride channels (CaCCs) are a class of ion channel involved in a multitude of cellular functions. The role of CaCCs is especially well recognised in vascular smooth muscle cells (VSMCs), where CaCCs are activated by agonist-induced Ca2+ release and their opening triggers depolarisation leading to vasoconstriction. CaCCs have been proposed as new potential therapeutic targets for diseases of altered blood vessel tone including systemic and pulmonary hypertension, as well hypotension and shock. Selective and potent CaCC modulators, however, are currently missing. Our lab has previously demonstrated that anthracene-9-carboxylic acid (A9C) has a biphasic effect on TMEM16A currents, producing both activation and inhibition depending on the concentration range and membrane potential. Defining the mode of action of this test compound can therefore help the design of both synthetic inhibitors and activations. Patch-clamp electrophysiology of cloned and native ion channels was used in conjunction with single point mutagenesis and wire myography to gain insights on the molecular pharmacology of TMEM16A-CaCCs. The main findings of this thesis are: 1. The selectivity of two of the most potent modulators of TMEM16A currently available 2-(4'-chloro-2'-methylphenoxy)-N-[(2''-methoxyphenyl)methylideneamino]-acetamide (Ani9) and N-((4'-methoxy)-2'-naphthyl)-5-nitroanthranilic acid (MONNA) was studied on isolated mouse VSMCs and isolated aortic rings. Ani9 potently blocked cloned and native TMEM16A channels, without interfering with other conductance's in mouse aortic SMCs. MONNA was found to inhibit cloned and native TMEM16A channels. However, MONNA also activated BKCa channels and inhibited CaV channels, significantly affecting the tone of aortic rings through a combined action of multiple conductance's. 2. The mode of action of A9C on the TMEM16A channel was elucidated. TMEM16A channels with the activation gate constitutively open were used to dissect the mechanism of A9C inhibition and activation. These channels were found to be entirely insensitive to A9C in the absence of intracellular Ca2+. However, when intracellular Ca2+ was presence, the mutant channels regained their sensitivity to extracellular A9C. Thus, intracellular Ca2+ is mandatory for the channel response for extracellular A9C. The underlying mechanism is an unanticipated conformational change in the outer pore, revealing a novel aspect of Ca2+-gating in the TMEM16A channel, and its implication for channel pharmacology. 3. A fluorescence-based screening cellular system was developed to enable a higher through-put evaluation of possible compounds that modulate TMEM16A function. This method was tested using known modulators of the TMEM16A channel. This cell system may help future drug screening exercises. To conclude, the research in this thesis has led to the determination of the binding site of A9C on the TMEM16A channel and revealed that the binding site become accessible to A9C only when intracellular Ca2+ is present; this indicates a possible site for therapeutic drug targets that can become accessible during stimulation of the cell by factors that trigger Ca2+ release (such as stimulation of VSMCs by vasoactive agonists). The pharmacology of TMEM16A was also systematically studied and the selectivity of two of the most commonly used tool compounds defined, which enables a more in-depth interpretation of published results involving these modulators. Collectively, these data advance our understanding of the pharmacology of the TMEM16A channel. The definition of a ligand binding site may form the basis for the rational design of small molecules acting at this site.

The pharmaceutical heparin is a complex polysaccharide of the glycosaminoglycan family, usually derived from porcine mucosa, and is a mainstay of anticoagulant therapy worldwide. Its activity can be modified, most notably, through depolymerisation. Yet, owing largely to the chain complexity, the progressive effects of environmental conditions on the heparin structure have not been fully described. A systematic study of the progressive effects of acidic and alkaline hydrolysis on heparin chain length and sulfate substitution has therefore been conducted. The initial analysis concerned the changes of weight-average molecular weight of heparin, induced by applied conditions. In acidic environments, the relation between the molecular weight loss and pH was inversely correlated, whilst the rate of degradation increased with temperature. In alkaline environments, the molecular weight loss was proportional to pH and temperature, although less effective. Under milder acidic conditions, desulfation was the major factor affecting the molecular weight of the polysaccharide, whilst in alkaline conditions, the hydrolytic sulfate scission was not as prominent. Glycosidic scission was observed only after the prolonged hydrolytic processing at pH 1, at 80 C. Stability studies confirmed the chain stability between pH 2 and pH 12. To understand desulfation order and further investigate possible rearrangements, selected acid and alkaline hydrolysates were monitored via 2D (HSQC) NMR. This study revealed that in acidic environments all sulfate groups of heparin were altered. It was observed that the sulfate groups were hydrolytically removed in the following order: N-sulfate (NS-), then 2-O-sulfate (2OS-), and finally 6-O-sulfate (6OS-), although the selectivity of last two was strongly dependent upon applied conditions. On the other hand, the NMR analysis of alkali treated heparin demonstrated that applied environments catalysed only iduronate desulfation (2OS-), followed by its exclusive rearrangements to a galacturonic residue. The research discussed in this thesis investigated the behaviour of heparin in aqueous systems altered by temperature and pH over time. The selected stressing factors closely resembled conditions applied over the industrial and/or pharmacological processing. Therefore, the contribution of presented findings reaches beyond theoretical knowledge and extends to possible practical applications, i.e., optimisation of manufacturing, storage and administration of pharmacologically active heparin products.

In this project, the aim is to develop a new multipurpose prevention technology (MPT) platform to help address the global health burdens associated with sexually transmitted infections (e.g. HIV-1, HSV-2, HPV) and unmet need for contraception, which disproportionately affect young women in low and middle-income countries. The new MPT product concept is based around an innovative vaginal ring in which drug-containing cores are physically inserted into and retained within a ring-shaped frame using 'interference fits'. Compared to more conventional vaginal ring designs, this vaginal ring technology platform offers ease of fabrication of complex devices containing multiple drugs and multiple materials and can offer different kinetics and mechanisms of drug release within the same device. Various model drugs will be used to demonstrate the concept: MIV-150 is an experimental antiretroviral drug owned by Population Council, New York; Nestorone (NES) is a contraceptive progestin; metronidazole (MET) is an antibiotic commonly used to treat bacterial vaginosis; and lactic acid (LA) and its cyclic dimer form lactide (LT) can maintain protective vaginal pH and provide an anti-inflammatory effect. Cores containing these drugs will be prepared using a thermosetting silicone elastomer material and tested for in vitro drug release. By applying a modular construction approach based around a common architecture, this device may allow healthcare professionals to select the appropriate combination of therapies to suits women's circumstances and family planning aspirations.

Type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVDs) often coexist. Oral anticoagulants (OACs) are prescribed for the management of CVDs. However, the safety of OACs in T2DM patients remains unclear. This PhD research aimed to evaluate the use and safety of OACs in T2DM patients. The Health Improvement Network primary care database of the United Kingdom was used to achieve the project aims. Firstly, a systematic review and meta-analysis were conducted to review the literature. Secondly, a drug utilisation study was conducted to evaluate the prescribing of OACs in T2DM. Then, two studies were conducted to explore the epidemiology and treatment of AF in T2DM. Finally, an analytical cohort study using the propensity score and Cox regression models was conducted to investigate the safety of the use of OACs and oral hypoglycaemic agents (OHAs) in T2DM. The pooled average of the prevalence of hypoglycaemia in diabetes was 11.0% (95% confidence intervals, 7.0% - 17.0%). The prevalence of OACs prescribing increased by 50.8% from 4.4% (4.2% - 4.6%) in 2001 to 6.6% (6.5% - 6.7%) in 2015. The prevalence of AF increased from 2.7% (2.5% - 2.8%) in 2001 to 5.0% (4.9% - 5.1%) in 2016. T2DM patients with AF, aged 60-79, males, and BMI ≥25, were more likely to receive OAC. The cohort study results showed that compared with sulfonylurea only, concurrent use of warfarin and sulfonylureas, increased the risk of hypoglycaemia and bleeding, (HR 1.38, (1.10 - 1.75)), (HR 1.12, (1.01 - 1.24)), respectively. However, there was no association between the use of DOACs and sulfonylureas concurrently and the risk of hypoglycaemia, (HR 0.54, (0.27 - 1.10)). In conclusion, the prevalence of AF and the use of OACs in T2DM patients have increased over the last decade. T2DM patients are at a higher risk of developing serious adverse events when warfarin and sulfonylureas are used concurrently.

Continuous upstream processing is becoming increasingly popular for the industrial production of biopharmaceuticals. Despite this, cell line and process development remains reliant on batch-wise experimentation to acquire data; this approach is time consuming, costly and labour intensive as the demands put upon these processes increase. A shift to continuous experimentation, with perfusion utilised to keep cells sustained for longer periods with continual testing, may alleviate these demands. However, the lack of small-scale, high throughout perfusion systems makes this difficult to achieve. For the commercial systems that are available, the cell retention technology employed is frequently not compatible with familiar labware, instead requiring dedicated equipment for successful operation. This makes adoption with existing development protocols challenging. Microcarriers have been used previously to adhere CHO cells in suspension culture. Microcarriers are easily handled, scalable and compatible with many vessel formats. Their use as a potential cell retention device has been greatly overlooked. Commercial microcarriers are available, but they often require attachment proteins for cell adhesion, despite the discouragement of animal-derived products in biopharmaceutical processing. The work here has developed an in-house, microsphere-based cell retention device for a suspension-adapted, IgG-producing CHO cell line. An animal origin-free, inexpensive polymer, which has been neglected in the literature, has been shown to enable the attachment of these cells to vessel surfaces. Alongside this, acrylic-based polyHIPE microspheres with diameters from 270 to 1100 µm have been fabricated at negligible cost. A novel monomer has been included from 2 to 16 % to give the resulting materials increasing carboxyl functionality. These carboxyls have both increased the stability of microspheres in solution and enabled the rapid adsorption of this polymer. Cell loading, where high cell density suspensions are forced to interact with microspheres, has been introduced as a technique for guaranteed cell retainment. With this method, 30-100 million cells per mL of microspheres were retained within an hour, depending upon conditions used. To demonstrate the versatility of the cell retention device, a pseudo-continuous culture was performed for over 30 days using Erlenmeyer flasks, with continual IgG production demonstrated. A simple perfusion system, employing these microspheres within a modified spinner flask, was also tested. It has been shown here that sophisticated cell retention technology is not necessary for effective retainment of CHO cells. Because of this, continuous processing has been performed in the laboratory without costly equipment or prior experience. It is hoped the data obtained may inspire others to explore continuous operations for the pre-clinical development of biopharmaceuticals.

As the global threat of antibiotic resistance grows the approval rate of new antibiotics falls. Pharma companies are dropping their antibiotic research programmes at the time when new antibiotics are needed the most due to antibiotic resistance. Paradoxically antibiotic resistance is one of the many reasons that the discovery of new antibiotics is not lucrative enough for it to be a financially viable pursuit. Promising high-throughput technologies have been unsuccessfully employed to streamline drug discovery due to the lack of novel chemicals within libraries. Natural products have contributed massively to drug discovery in the past although its contribution to the declining number of antibiotics discovered recently has also diminished. To revive the drug discovery pipeline invasive weeds were targeted as a potential source of novel chemical compounds. Three species; Fallopia japonica, Impatiens glandulifera and Rhododendron ponticum were collected. Each species was extracted and fractioned to discover any potential antimicrobial compounds. A particularly active compound 2-methoxy-1,4naphthoquinone (MNQ) was discovered. This compound was found to have a broad range of activity against clinically relevant bacteria. The methoxy group was found to be crucial for the potent antimicrobial activity of this compound. It was also a potent inhibitor of Schistosoma mansoni. Cytotoxicity of this compound was found to be a potential issue with mixed results. High throughput metabolomic methodologies were developed to understand the mechanism by which MNQ inhibits the growth of MRSA. The metabolomic effect of MNQ was compared to other antibiotics, it was found that MNQ had a distinctive metabolomic effect. This unique effect was further investigated with in-depth metabolic pathway analysis using statistical methods twinned with KEGG metabolomics pathway database. Once a tentative mode of action was identified transmission electron microscopy and specific antimicrobial assays were used to support this theory. This research project has discovered a promising antimicrobial and anthelminthic compound and the developed metabolomic methodology yielded a large amount of useful data regarding the mode of action of MNQ and other antibiotics.

Tetrahydrobiopterin (BH4) is a cofactor in the conversion of amino acids such as phenylalanine, tryptophan and tyrosine to precursors of neurotransmitters and signalling molecules including dopamine, adrenaline, noradrenaline and nitric oxide. The involvement of BH4 is vital to a range of biochemical processes associated with the cardiovascular system, inflammation, mood, and neurotransmission. Growing evidence implicates high BH4 levels in chronic pain states. Therefore, enzymes that are involved upstream of BH4 biosynthesis within the de novo pathway offer an exciting pharmacological target for small molecule inhibitors to modulate BH4 production and possibly relieve chronic pain. Pharmacological targeting of both GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the pathway, and sepiapterin reductase (SPR), the terminal enzyme in BH4 production, were both investigated. A combinational approach of various in silico and in vitro techniques was used to generate compound libraries and demonstrate that inhibition of either GCH1 or SPR activity reduces BH4, revealing novel inhibitory interactions for a number of compounds. In particular, tranilast, licensed for use since 1982 in bronchial asthma treatment, inhibited SPR activity (IC50 3.85 ± 1.9 μM), reducing sepiapterin reduction, BH4 production, and downstream reactive oxygen species (ROS) and nitrite production in vitro. Computational ligand docking and molecular dynamics (MD) simulations suggest that tranilast interacts with human SPR at the same spatial position in the active site as sepiapterin, including one of the catalytic triad residues of SPR, S157. This thesis demonstrates that pharmacological targeting of both GCH1 and SPR in the BH4 pathway are attractive options for chronic pain treatment, and reports novel inhibitory properties of the approved drug tranilast on SPR, a viable prospect for drug repurposing in chronic pain treatment.

Protein drug development is a risky, lengthy and expensive process. Accordingly, there are significant time and cost implications for stakeholders regardless of the stage at which a prospective candidate is discontinued. Mitigating this risk depends critically on making informed decisions as early as possible during the process. Some of the most indispensable decision-making aids are analytical techniques used in the characterisation of protein stability. Protein stability is a multifaceted and crucial determinant for progressing a candidate through development to commercialisation. The inherent complexity of proteins, the evolving pipeline of engineered protein constructs, as well as limitations of current techniques place a demand for the expansion and development of more analytical probes for protein stability. This thesis focuses on the development of Raman spectroscopy and 2D-correlation analysis (2DCA) for monitoring conformational stability during early protein drug development. Although Raman spectroscopy has been used for many decades to study protein conformational changes in proteins; it is highly underutilized during protein drug development, owing partly to the complexity of Raman protein spectra. 2DCA is a mathematical technique that can significantly improve the visualization of and clarify the interpretation of complicated spectral features arising from an applied perturbation. The specificity of 2DCA in extracting such spectral variations directly linked to a perturbation is apt for protein stability testing, where the identification of a protein's response to stress is pivotal. In this work, we demonstrate this combined technique as a sensitive means of distinguishing the conformational stability profiles of proteins under an applied perturbation on the basis of post-translation modification, metal-ligand and surface charge mutation. In all cases, we show that the technique is capable of informing the choice of the most stable candidate and or conditions, even where subtle differences exist.

This thesis describes the synthesis and evaluation of cysteine-derived tetramate (pyrrolidine-2,4-dione) derivatives for the development of novel antibacterial drugs. Chapter 1 gives a brief overview of the history of antibiotics and clinically available antibacterial drugs. It addresses the need for novel antibacterial drugs due to the emergence of bacterial resistance. While exploring different chemical approaches to tackle the problem of drug resistance, it highlights the re-emergence of natural products as a crucial source for drug development. In particular, it focuses on tetramic acid containing natural products and discusses their value in antibacterial drug discovery, their properties and synthesis. In the end, it exposes the need for optimisation of cysteine-derived tetramates due to their apparent reduction of antibacterial activity when tested in vitro in the presence of blood, hence establishing the hypotheses and aims of this project. Chapter 2 describes the synthesis, structural and brief antibacterial evaluation of a library of cysteine-derived tetramates with increased hydrophilicity by incorporation of pendant heterocyclic rings. An effective synthetic protocol for accessing polar tetramate derivates is established and a computational study has developed detailed understanding of the controlling factors of the key Dieckmann cyclisation step. Direct evidence of strong metal chelation in these tetramate systems is also demonstrated. Chapter 3 and 4 discuss chemical modifications on cysteine-derived tetramates to modulate their metal chelation property. Structural dependency of the metal binding ability is established, with a library of cysteine-derived tetramates exhibiting evidence for reduced metal chelation. The antibacterial activity of these tetramates is evaluated in relation with their metal binding ability. Chapter 5 provides a detailed biological evaluation of the synthesised cysteine-derived tetramates with regard to their antibacterial potency, physiochemical properties and metal chelation ability. The potential for multi-targeting mechanisms coupled with a low cytotoxicity is also briefly addressed. It demonstrates the types of structural modifications possible to assess hydrophilic cysteine-derived tetramates with reduced metal chelation, while preserving their selective Gram-positive activity with MIC against MRSA < 8 μg/ml.

The group 1 metabotropic glutamate receptors (mGluR1/5) are of key interest in the synaptic plasticity that underlies learning and memory and have been implicated as a cause or target in many disease models such as Parkinson's, Huntington's, Alzheimer's, depression, schizophrenia and fragile X syndrome. Alongside this, group 1 mGluRs are linked, via the production endocannabinoid 2-archidonyl glycerol (2-AG), to cannabinoid receptor 1 (CB1). CB1 is one of the most highly expressed G-protein coupled receptors (GPCRs) in the central nervous system (CNS) and its function has not been confirmed in Xenopus laevis tadpoles. Debate and research are still ongoing as to how CB1 can be targeted to improve disease outcomes and the extent of its effect in vivo. The pattern of neuronal signalling in the X. laevis central pattern generator (CPG) has been well categorised using electrophysiological recording in the immobilised tadpole at stages 37-42. We sought to build on these previous experiments to assess the swimming behaviour of the X. laevis tadpole in vivo using slow-motion high frame rate video (400 frames per second) to determine the frequency of tail swim cycles and the angle of tail flexion achieved during these swim cycles. Using this well-characterised model the effects of the two GPCRs can be determined in the unadulterated swimming behaviour. X. laevis tadpoles are also advantageous because of low cost in maintenance, a simple primary culture that can be set up at room temperature and an ectothermic development meaning large batches can be staggered in age. We looked to assess well-known plasticity inducing modulatory receptors mGluR1/5 and corroborate electrophysiological recordings previously showing mGluR1/5 activation via Dihydroxyphenylglycine (DHPG) increased motoneuron output frequency. We tested if, as in other models such as lamprey, this increase is partly mediated by retrograde cannabinoid signalling. Our results show group 1 metabotropic glutamate receptors cause increases in the frequency of X. laevis swim-cycles at stage 40-42 evidenced through the application of DHPG, a group 1 mGluR agonist. This increase appears to be mainly through mGluR1 as inhibition of this receptor via LY367385 caused significant decreases in swim cycle frequency whereas inhibition of mGluR5 with 2-Methyl-6-(phenylethynyl)pyridine (MPEP) caused no significant decreases, indicating an intrinsic role for mGluR1 over mGluR5 in the maintenance of normal swim cycle frequency. Antagonism of CB1 with AM-251 caused significant decreases at 50µM and 10μM. However, a significant increase was observed at 2μM indicating a biphasic effect dependent on concentration. Inhibition of CB1 with AM-251 (10μM) followed by application of DHPG (50μM) had no significant effect on the frequency of swim cycles when compared with vehicle control, indicating that the increase seen with DHPG application may be blocked by CB1 inhibition. Application of endogenous CB1 agonist N-arachidonoylethanolamide (AEA) decreased frequency of swim cycles at lower concentrations (0.1-10μM), but no significant change was observed at the highest concentration (50μM). This may be some form of partial antagonism due to a higher affinity of AEA than other endogenous ligand 2-AG, but lower efficacy, effectively occupying the CB1 receptor without activation. Our data suggest that mGluR5 and CB1 may be involved in the normal muscle flexion during swimming with the application of MPEP alone or with AM-251 causing significant decreases in the angle of tail flexion. We wanted to see if these changes in behaviour reflect in the morphology of neurons, particularly the dendritic spines dimension and membrane viscoelasticity. To do this, a primary X. laevis neuron-muscle co-culture was developed from the literature. The cultures were treated with the same pharmacological treatments and fixed in glutaraldehyde 5%. The dendritic spines of the neurons were scanned with an Atomic Force Microscope (AFM). From these scans the dendritic spine dimensions measured were; radius, volume, cross-sectional area, and membrane roughness. Using the phase contrast images, the loss tangent was calculated to give a unitless ratio of membrane stiffness. This measure of the stiffness/viscosity of the sample was used to determine if the dendritic spine head changed significantly in stiffness after group 1 mGluR or CB1 targeted drug treatment. With AFM analysis of dendritic spine morphology and membrane stiffness, we found that group 1 mGluR activation elongated dendritic spines corroborating previous evidence in immature developing spines and in mature hippocampal cultures. Interestingly inhibition of mGluR5 also elongated the spines alongside increasing the volume of the spines. Both mGluR5 inhibition with MPEP and group 1 mGluR activation with DHPG caused significant increases in dendritic spine membrane stiffness compared with vehicle controls. We aimed to investigate if the 20-minute application of treatments induced changes in protein expression, particularly postsynaptic density protein-95 (PSD-95) a postsynaptic structural protein present in glutamate synapses linked to NMDAR function, a key receptor in the glutamate excitation of descending interneurons (dINs). After the behavioural assessment, the tadpoles were fixed and embedded in paraffin wax for microtome sections, which enabled observation of the spinal cord, which was investigated for changes in PSD-95 density using immunohistochemical means. After scanning with AFM, the cultures were stained with anti-PSD-95 antibodies and changes in fluorescence were measured. The results of this investigation were inconclusive due to large autofluorescence meaning a clear positive signal could not be identified. The in vivo swimming analysis gives almost completely unadulterated tadpoles for analysis of swim-cycle output with a very well understood neuronal network that serves as an excellent model for the interplay between excitatory and inhibitory signalling of CPG networks. In future AFM scanning can be used alongside fluorescent confocal microscopy to build a detailed picture of morphology and changes in membrane dynamics that may aid our understanding of synapse formation in normal development and in genetic disease such as fragile X.

Adverse drug effects are unintended and undesirable effects of medicines, causing attrition of molecules in drug development and harm to patients. To anticipate potential adverse effects early, drug candidates are commonly screened for pharmacological activity against a panel of protein targets. However, there is a lack of large-scale, quantitative information on the links between routinely screened proteins and the reporting of adverse events (AEs). This work describes a systematic analysis of associations between AEs observed in humans and bioactivities of drugs while taking into account drug plasma concentrations. In the first chapter, post-marketing drug-AE associations are derived from the United States Food and Drug Administration Adverse Event Reporting System using disproportionality methods, while applying Propensity Score Matching to reduce confounding factors. The resulting drug-AE associations are compared to those from the Side Effect Resource, which are primarily derived from clinical trials. The analysis reveals that the datasets generally share less than 10% of reported AEs for the same drug and have different distributions of AEs across System Organ Classes (SOCs). Using the drugs from the two AE datasets described in the first chapter, the second chapter integrates corresponding bioactivities, i.e. measured potencies and affinities from the ChEMBL database and ligand-based target predictions obtained with the tool PIDGIN, with drug plasma concentrations compiled from literature, such as Cmax. Compared to a constant bioactivity cut-off of 1 uM, using the ratio of the unbound drug plasma concentration over the drug potency, i.e. Cmax/XC50, results in different binary activity calls for protein targets. Whether deriving activity calls in this way results in the selection of targets with greater relevance to human AEs is investigated in the third chapter, which computes relationships between targets and AEs using different measures of statistical association. Using the Cmax/XC50 ratio results in higher Likelihood Ratios and Positive Predictive Values (PPVs) for target-AE associations that were previously reported in the context of secondary pharmacology screening, at the cost of a lower recall, possibly due to the smaller size of the dataset with available plasma concentrations. Furthermore, a large-scale quantitative assessment of bioactivities as indicators of AEs reveals a trade-off between the PPV and how many AE-associated drugs can potentially be detected from in vitro screening, although using combinations of targets can improve the detection rate in ~40% of cases at limited cost to the PPV. The work highlights AEs most strongly related to bioactivities and their SOC distribution. Overall, this thesis contributes to knowledge of the relationships between in vitro bioactivities and empirical evidence of AEs in humans. The results can inform the selection of proteins for secondary pharmacology screening and the development of computational models to predict AEs.

Antimicrobial resistance is a global public health issue which significantly threatens human life. There are approximately 50,000 deaths in the USA and Europe per year owing to antimicrobial resistance infections. This burden of resistance has increased resulting in an increase in morbidity and mortality in clinical and community setting. Thus, global collaborative action is needed for developing effective strategies to combat antimicrobial resistance. International and local approaches including antimicrobial surveillance, guidelines for treatment of bacterial infections, regulation of the availability of antibiotics, improving hand hygiene, understanding the mechanism of bacterial resistance and development of new antimicrobial agents have been advised. Aminoacyl tRNA synthetases are valuable targets for antibiotic development as they have a fundamental role at a cellular level during the translation process of the genetic code. Mupirocin (Bactroban ®) is an approved isoleucine tRNA synthetase inhibitor which is used for the treatment of methicillin resistant Staphylococcus aureus (MRSA). High and low level of mupirocin resistance has been demonstrated in most S. aureus isolates due to acquired plasmid-mediated mupA, which encodes a novel IleRS and mutation, respectively. Thus, the design of multitarget aminoacyl tRNA synthetases inhibitors could be an effective way to make significant reductions in the biological fitness of bacteria leading to a reduction in drug resistant microorganisms. Class IIb aminoacyl tRNA synthetases of which AspRS and AsnRS belong is a target of the project in Staphylococcus aureus and Enterococcus faecalis. A computational study of both enzymes in both microorganisms including homology modelling, validation techniques, molecular dynamics and docking of the natural substrates (aa-AMP) were used to be a platform for the design of dual site inhibitors containing a sulphamoyl linkage which mimic aa-AMP in both enzymes. Different series of AspRS/AsnRS inhibitors were designed to occupy both pockets of the target enzymes then synthesised after optimisation their synthetic routes. The minimum inhibitory concentration of compounds against a panel of microorganisms were evaluated compared with ciprofloxacin as the standard and one compound showed good inhibitory activity against Enterococcus faecalis (MIC = 2 μg/mL).

Background and Aim: Since ketamine was first trialed as an anti-depressant in 2000, it has been growing in popularity due to its fast-onset anti-depressant effects. The cognitive effects of ketamine when self-administered recreationally or administered acutely to healthy participants have been examined in many studies (Morgan & Curran, 2006). However, it is unclear how ketamine may impact cognitively on people with treatment-resistant depression, as mood and cognitive functioning are closely linked. The aim of this paper is to review current understanding of the cognitive effects of ketamine when used to treat individuals with treatment-resistant depression. Method: A systematic review of PsycINFO, Embase and OVID MEDLINE was conducted to find studies that utilised cognitive assessments during ketamine infusions in patients with treatment-resistant depression. Seventeen articles were identified and met the inclusion criteria for the review. There were three types of articles included that explored the cognitive effects of ketamine. Firstly those that used sub-anaesthetic ketamine infusions, secondly those used sub-anaesthetic ketamine infusions plus an anaesthetic agent, and thirdly those that used anaesthetic ketamine infusions. Included studies were quality assessed using the Cochrane Risk of Bias 2 and Risk of Bias in Non-Randomised Studies – of Interventions. Results: Of the 17 articles included, 10 found that when changes in depression symptomology were controlled for, both acute and repeated ketamine infusions were not significantly associated with cognitive performance. Three of the papers found that baseline cognitive functioning could predict individual’s improvements in mood following ketamine administration. Methodological variations meant a wide range of cognitive domains were explored, using various ketamine dosages and assessment intervals. There was considerable variability in the quality of the research. Conclusions: Methodological heterogeneity rendered findings inconclusive as to whether ketamine affects cognitive functioning within a treatment-resistant depression population. The relationship between ketamine’s impact on mood and subsequent cognitive changes are important to assess. Further high quality research needs to be done to fully document the cognitive side-effects of ketamine infusions so that patients and their doctors can make fully informed decisions.

Designer benzodiazepine is the term used when referring to benzodiazepines, which have been available for recreational use since the late 2000s. Some designer benzodiazepines are prescribed in other countries and became popular in the UK as a drug of abuse, others were investigated as medicines in the 1960s and 1970s but were never brought to market. A few designer benzodiazepines are novel drugs created solely for the recreational market. Originally sold as “research chemicals” or “legal highs” they circumvented the law by having small structural differences to the traditional benzodiazepines and sold in packages containing the disclaimer “Not for Human Consumption.” The introduction of the Psychoactive Substances Act 2016 (PSA) put new UK legislation in place to control the distribution and manufacture of any compound that is “capable of producing a psychoactive effect,” this captured the designer benzodiazepines as well as other “legal highs”. This legislation works in conjunction with the current Misuse of Drugs Act 1971 (MoDA), thereby legislating against a distinct list of drugs in the MoDA and any drug producing a psychoactive effect in the PSA. While there is now clarity of the legal status of these drugs, the scale of use in different sub-populations in Scotland, before and after, this legislation is unknown. There is little literature exploring how commonly designer benzodiazepines are detected in post-mortem cases from both drug related and non-drug related deaths. It has been demonstrated that etizolam is a common finding in drug-related deaths in Scotland but there is a lack of data regarding the designer benzodiazepines that emerged after etizolam. This makes toxicological interpretation and the decision to include the drug in the cause of death very difficult for toxicologists and pathologists, respectively, as there is a lack reference ranges to consult. Similarly the scale of use in living populations who are required to abstain from drugs for reason such as treatment or incarceration is unknown. The initial legality and the belief they may evade detection by simple screening tests make the designer benzodiazepines an attractive option. Two Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) methods were developed in order to test the different sub-populations. The urine method developed was a qualitative screen and was validated for use. The blood method developed was used to quantify the designer benzodiazepines and was validated for use. A total of 2,582 samples were analysed from the different sub-populations. Of these, 893 were urine samples from living participants and 1691 were blood samples from deceased individuals. All blood samples were from the post-mortem (PM) cohort and 369 (22%) of the cases were positive for the designer benzodiazepines tested. Diclazepam was detected in 212 cases and gave a median concentration of 0.017 mg/L (n=157, 0.005 – 0.211 mg/L), Delorazepam was detected in 339 cases and gave a median concentration of 0.043 mg/L (n=311, 0.005 – 1.50 mg/L), Lormetazepam was detected in 144 cases and gave a median concentration of 0.010 mg/L (n=85, 0.005 – 0.18 mg/L), Flubromazepam was detected in 18 cases and gave a median concentration of 0.66 mg/L (n=15, 0.01 – 2.30 mg/L), Pyrazolam was detected in 9 cases and gave a median concentration of 0.033 mg/L (n=6, 0.008 – 1.10 mg/L). These concentrations can assist in the toxicological interpretation of these drugs. The urine samples, which were screened for a wider range of benzodiazepines, were from three different cohorts. These were made up of individuals being admitted to or liberated from one of the seven Scottish Prison Service (SPS) facilities included in this study, individuals under the supervision of a Drug Treatment and Testing Order (DTTO) through the Scottish Drug Court (SDC) system in Glasgow and patients undergoing psychiatric treatment from NHS Greater Glasgow and Clyde Forensic Directorate (NHS GGC FD). The analysis found that 55% of the 73 urine samples from the SDC were positive, 41% of the 725 SPS urine samples were positive and there were no positive samples found in the 95 NHS GGC FD urine samples. The results of the studies show that benzodiazepines and designer benzodiazepines are widely used in the Scottish population. The individuals from the SDC and NHS GGC FD were able to refuse to take part in the study or able to abstain before their known drug test date. The SPS samples only gives a snapshot of those being admitted to or liberated from prison, which is not a reflection of the general inmate population and not every post-mortem case was tested for designer benzodiazepines. However despite these limitations, key information about the scale, nature and blood concentrations of the designer benzodiazepines being abused in Scotland was gained.

This thesis aimed to establish the potential of extremely halophilic microorganisms, isolated from Kilroot Salt Mine, as a reservoir of unexplored natural product chemistry with antimicrobial activity against the clinically relevant ESKAPE pathogens. The capacity of haloarchaea to modulate bacterial quorum sensing via the production of cross-domain induction and inhibitory compounds were also investigated. Both conventional and enhanced culturing methods including the iChip device were employed to obtain a collection of morphological/genetically distinct halophilic bacteria and archaea from brine samples and PFA, resulting in a collection of 51 Haloarchaea and 10 halotolerant Actinobacteria isolates. Crude extracts from these haloarchaea and bacteria isolates, alongside 16 extremophilic fungi were investigated for antimicrobial activity against the ESAKPE pathogens and QSI production. The screening revealed extracts from Brevibacterium sp., NAT-30 and NAT-27, exhibited antimicrobial against MRSA at 2 mg/mL and 0.5 mg/mL respectively. The isolate Halorubrum sp. iNAT-14 exhibited antimicrobial activity against P. aeruginosa and A. baumannii, with a MIC of 4 mg/mL. The most potent haloarchaea extract was from Haloferax volcanii DS2 with a MIC of 0.5 mg/mL against E. faecium. The yeast-like fungus, A. pullulans EXF-9635, displayed the most potent antimicrobial activity at 0.125 mg/mL against MRSA. Furthermore, this extract exhibited anti-biofilm activity with a MBIC of 2 mg/mL, and 1 mg/mL against MRSA and Staphylococcus aureus biofilms, respectively. In vitro studies revealed no toxicity at concentrations ≤ 2 mg/mL using NCTC 929 cell line and in vivo analysis with G. mellonella larvae revealed 80% survival in concentrations ≤1 mg/larvae. The highest percentage survival of infected larvae was at 0.125 mg/larvae. The extract of Halorubrum sp. CSM52 was demonstrated to induce bacterial QS using the bioreporters A. tumefaciens NTL4(pZLR4), E. coli pSB401, pyoverdine production in PAO-MW1, and pyocyanin inhibition in PAO1. Initial characterisation of the bioactive(s) using TLC-overlays, biochemical tests, and LC-MS/MS analysis excluded structural similarity to known AHL molecules, suggesting that it may be a chemically modified AHL or a DKP. Fractionation of the crude extract revealed that fraction nine induced pyoverdine production, while fraction 4 and 6 were capable of inhibiting pyocyanin production in PAO1. Initial results suggest the role of this AI may be involved in haloarchaeal biofilm formation. Metagenomic analysis of Kilroot Salt Mine using Kaiju and Kraken2 classifiers, revealed a reduction in the relative abundance of Salinobacter spp. and Haloquatrum spp. compared to hypersaline lakes. Metagenome mining revealed both bacteria and archaea harbour AMR genes in a relatively undisturbed environment. Genomic mining of brine and salticle metagenomes for halocin AMPs revealed that several halocin-S8 homologs existed. A putative AMP termed, Halocin-KSM was developed from a consensus sequence of known halocin homologs and was predicted to possess antimicrobial activity using in silico analysis. This putative activity was verified using disc diffusion and MIC assays, revealing that the C-terminal amide species of Halocin-KSM had a MIC of 0.06125 mg/mL against S. aureus. Both culture-dependent and culture-independent approaches have provided a detailed insight into the microbiome of Kilroot Salt Mine and provide an excellent framework for optimising future isolation procedures. Structural elucidation of bioactives from Halorubrum sp. CSM52, A. pullulans EXF-9635, and Haloferax volcanii DS2 will provide an invaluable insight mechanism by which halophilic microbes exert their activity. In conclusion, these results have established that halophilic microorganism from all three domains of life are capable of producing antimicrobial compounds against clinically relevant pathogens.

Conventional antidepressants have a relatively slow onset of action, requiring a number of weeks before clinical improvement in depressive symptoms is apparent. The delay in onset of action of antidepressants may result in patients suffering increased morbidity due to the persistence of depressed mood and associated symptomatology. More recently, animal and human studies suggest that N-methyl-D-aspartate (NMDA) receptors may participate in the pathophysiology of depression and play an important role in effective pharmacological treatment. Indeed, several NMDA receptors antagonists, such as ketamine, lanicemine and nitrous oxide (N2O) have been shown to produce an antidepressant effect within hours in some clinical trials. Another group of drugs, acting at the glycine site of the NMDA receptor, have also shown potential as antidepressant agents. The cognitive neuropsychological model of antidepressant drug action proposes that in the early stage of conventional antidepressant treatments the primary effect of antidepressants is to modify negative biases in the processing of emotional information. However, there have been no systematic studies to identify how NMDA-targeting antidepressants may change emotional processing in humans. Thus, this thesis aimed to investigate the effects of different NMDA receptor ligands on emotional processing in both depressed patients and healthy participants using a combination of behavioral and neuroimaging approaches. The first study assessed the effects of single doses of lanicemine and ketamine on neural and behavioural measures of emotional processing in depressed patients, 24 hours post infusion. It was found that ketamine did not show the broad range of effects on emotional processing seen with conventional antidepressants but instead significantly increased the error rate for recognizing facial expression as positive, and also produced higher false positive memory intrusions in the emotional recall task. Lanicemine only showed a trend for better recognition of facial expression across all emotions and did not produce positive shifts in any emotional processing tasks (Chapter 2). Furthermore, a single dose of ketamine increased brain activation in the right dorsolateral prefrontal cortex region during negative self-referential processing (Chapter 3). The second study assessed the effects of a single dose of N2O 24 hours post infusion using behavioural measures of emotional processing in treatment-resistant depressed (TRD) patients. It was found that N2O did not change the processing of emotions in TRD patients compared to those in the placebo group (Chapter 4). The third study was designed to assess the effects of an acute dose of an NMDA receptor glycine site, partial agonist D-cycloserine (DCS), on autobiographical memory, emotional processing and stress response in healthy volunteers. Relative to placebo, DCS enhanced autobiographical memory specificity at 3 and at 24 hours post treatment. DCS also increased positive bias in emotional word categorisation and subsequent free recall at 3 hours; however, this effect did not extend to other emotional processing tasks (chapter 5). In addition, further investigation on the effects of DCS on stress responses (chapter 6) found no effect of DCS on psychophysiological changes and self-reported mood and anxiety in an acute psychosocial stress paradigm. These findings suggested that NMDA receptor targeting antidepressants showed early effects on emotional processing in the absence of subjective improvement in mood symptoms, though the changes seen differ from those observed with conventional antidepressants. These findings are important for understanding the therapeutic action of NMDA receptor targeting antidepressants in the treatment of depression and stress-related disorders and also for the ability of DCS to augment psychological treatment in patients with anxiety and depression. This thesis also outlines the challenges and future research directions for better understanding the cognitive neuropsychological mechanisms underlying the effects of NMDA receptor drugs in depression. This could have implications for the development of antidepressants with an early onset of clinical action.

Thalidomide is a highly teratogenic medicine and an effective frontline treatment for multiple myeloma when used in combination with other therapeutic agents. The global burden of multiple myeloma has been steadily increasing over the last decade, with the most significant rise being in middle and low sociodemographic index countries. The increasing burden of multiple myeloma poses particular concerns regarding the safe use of thalidomide and the need for risk management programmes to prevent foetal exposure. Evidence suggests that the effectiveness of risk management programmes for thalidomide has not been extensively investigated in spite of the serious risks of the medicine. Such lack of research on the risk management of thalidomide is of particular significance in countries where the incidence of multiple myeloma is increasing. Consequently, the programme of research presented in this thesis focuses on understanding the risk management of thalidomide in Jordan, a middle income country in which the burden of multiple myeloma has shown a recent increase. Two empirical studies were carried out in this programme of research to address the aim of understanding the risk management of thalidomide in Jordan and identify affecting factors within the Jordanian health system. The first study was informed by findings from a systematic review of the literature on teratogenic risk management that was carried out as the initial step in this programme of research. The systematic review found a lack of research on risk management of thalidomide, particularly in countries of the Middle East. It also found variation in the reported use of teratogenic risk management measures which included reporting low levels of adherence to these measures. While this might constitute a significant medicine safety concern, it may be the result of using data sources that might have been unable to generate complete data. Therefore, the review concluded by highlighting the need for future research to investigate multiple sources to confirm consistency of the obtained findings on the use of teratogenic risk management. Therefore, the first study of this programme of research utilised primary data reported by patients through filling a questionnaire and secondary data extracted from medical records to investigate risk management of thalidomide in Jordan. The cross-sectional study aimed to profile the characteristics of patients using thalidomide in Jordan and describe patients' use of teratogenic risk management measures using quantitative methods. Subsequently, the second study sought to contextualise findings by exploring those factors within the health system that have an effect on risk management using qualitative methods. This was achieved by applying the World Health Organization's (WHO) health systems framework to explore the experiences and views of patients, industry, drug regulators, and health care professionals of risk management of thalidomide in Jordan. Findings of this programme of research show that that implementing the risk management programme of thalidomide in Jordan is failing to consider the sociodemographic characteristics of patients using the medicine. Although few exceptions exist, the programme generally does not fit the profile of patients using thalidomide due to their old age. In addition, the programme overlooks the cultural dimension of family members' involvement in handling thalidomide which might constitute a significant safety concern among those caregivers. Moreover, by applying the WHO health systems framework, this research identifies which of the health system components have the most significant impact on the risk management of thalidomide and therefore, need to be improved. Consequently, it can be concluded that there is a real need for the patient voice in preventing foetal exposure to thalidomide, and this has to be at the centre of a well-functioning health system.

Antimicrobial resistance has become one of the major hurdles to overcome when developing novel antibiotics. The number of deaths related to resistant bacteria is expected to increase dramatically even overtaking cancer. One of the main drivers of this resistance is the formation of biofilms. Biofilms are collectives of one or more bacteria that adhere to biotic or abiotic surfaces. This structure confers on the bacteria a biological advantage over planktonic cells. The biofilm structure amongst many other benefits confers antimicrobial resistance to the organisms. One of the main contributors to this resistance is the extracellular DNA found in the biofilm matrix. Targeting the biofilm structure and its components has become an important way to tackle biofilm derived resistance. A family of compounds found to possess activity against Staphylococcus aureus biofilms are pyrrolomycins. Pyrrolomycins are highly substituted natural products biosynthesised by Streptomyces vitaminophilum. Despite their simple core structure, the different members of the family display a high degree of variation in substitution pattern: from highly halogenation compounds to fairly uncommon β-nitropyrrole moieties. Further investigation on their biosynthesis, synthesis, complexation abilities and mode of interaction with DNA has been carried out. In this work, the synthesis of naturally occurring pyrrolomycins and their derivatives has been investigated. Several previously unreported compounds have been synthesised as well as developing a novel coupling strategy for the synthesis of the core structure that presents advantages over previously reported chemistry used for them. Limitations on reduction and nitration have been encountered and alternative enzymatic synthesis has been explored. Synthetic investigation has led to a novel synthesis of pyrrolomycin derivatives.

Functional psychosis is a severe mental illness with often devastating consequences for individuals and a high cost to society. One leading aetiopathological hypothesis is the ‘aberrant salience theory’ whereby abnormal salience is attributed to stimuli leading to psychotic symptoms mediated by striatal dopamine. Reward processing is also thought to be mediated by striatal dopamine and is disrupted in psychosis. One aspect of reward processing is motivational salience, whereby perception of an incentivising stimulus leads to approach behaviour in an organism. Accumulating data points to a role for the endocannabinoid system in both psychosis and reward processing. In particular, the phytocannabinoid cannabidiol (CBD) is a promising candidate as a novel antipsychotic. At the core of this thesis are two published articles using functional magnetic resonance imaging (fMRI) to investigate the neural substrate of motivational salience in health, how it is affected in psychosis and the neurocognitive effects of CBD. The first article is an international meta-analysis of fifteen original fMRI group maps sourced from healthy adult participants (n=346) undertaking the Monetary Incentive Delay Task (MIDT). We analysed the anticipation of monetary reward and loss contrasted with the neutral condition. In both contrasts there was consistent activation of the striatum and salience network, with more complex patterns of activation and deactivation in the central executive network, default network and cerebellum. We subsequently compared the anticipation of reward with loss and found significantly greater relative deactivation in the left inferior frontal gyrus. The second publication reports a randomised double-blind placebo-controlled study using the MIDT in which thirty-three participants at clinical high risk for psychosis (CHR) were administered either 600mg oral CBD or placebo and compared with nineteen healthy controls (HC). CHR subjects represent a population of help-seeking individuals who have experienced psychotic-like symptoms or have significant genetic risk of developing a psychotic illness, but they remain antipsychotic-naïve. Reward and loss anticipation conditions were combined to create a condition of motivational salience, subsequently contrasted with neutral. Region-of-interest analysis was confined to two masks consisting of striatum/midbrain/hippocampus and the core salience network (anterior cingulate/insula). Differences were detected between CHR-placebo and HC groups and CHR-placebo and CHR-CBD groups within the core salience network but not within the mask containing the striatum. There was increased activation in a region of posterior left insula/parietal operculum in CHRplacebo compared to HC. Activation in this area in the CHR-placebo group positively correlated with psychotic symptoms and negatively correlated with salience perception, as indexed by reaction time difference between salient and neutral stimuli. Thus, psychopathology and aberrant salience were 8 linked to increased activation in the same region of brain in CHR. Increased activation in the left insula/parietal operculum in CHR was attenuated by CBD. In conclusion, this thesis establishes that the salience network is activated in motivational salience, and that the neurocognitive dysfunction associated with reward processing in psychosis may be based in the salience network, in particular insula/operculum regions. Moreover, the effect of CBD in attenuating activity in the insula/operculum suggests a potential antipsychotic mechanism-of-action by normalising aberrant motivational salience.

Introduction: Epidemiological and biological evidence show no boundaries between diagnostic categories of non-affective and affective psychoses, thus challenging the current nosological model developed from Kraepelin’s paradigm. My thesis aimed to address this limitation by 1) examining the transdiagnostic dimensional structure of i) psychotic symptoms in first episode psychosis (FEP) patients and ii) psychotic experiences in the general population; 2) investigating the relationship between these dimensions and a set of external factors, such as the use of cannabis and genetic common variant liability for psychotic disorders. Overall, I expected that differences in symptom profiles at FEP reflected gradients of neurodevelopmental impairment in psychosis. Methods: This thesis uses data from a multisite incidence and case-control study, which I worked on, conducted across six countries [i.e. the ‘European Network of National Schizophrenia Networks studying Gene-Environment Interactions’ (EU-GEI) study]. To examine the latent structure of psychopathology, I analysed ratings of psychotic symptoms and experiences using multidimensional item response modelling in Mplus, and I estimated different theory-based models of psychosis, including unidimensional, multidimensional, bifactor, and hierarchical solutions. To examine the common variant liability to psychosis, I examined the population structure in the EU-GEI sample and computed ancestry-specific schizophrenia (SZ), bipolar disorder 7 (BP), and combined schizophrenia-bipolar disorder (SZ-BP) Polygenic Risk Scores (PRSs) in PRSice. To examine the relationship between the latent structure of psychopathology and demographic and context determinants, detailed patterns of cannabis consumption, and PRSs, I used multiple linear regression models fitted in STATA14. Results: The associations among ratings of both psychotic symptoms in FEP patients and psychotic experiences in population-based controls were best represented by a bifactor model, composed of one general psychosis factor and multiple specific dimensions. In FEP patients, the examination of general and specific dimensions with external factors showed that 1) higher scores on the negative symptom dimension were associated with being a male, and having never used cannabis; 2) higher scores on the positive symptom dimension were associated with exposure to socioenvironmental risk factors in psychosis, such as being part of an ethnic minority, and having had exposure to cannabis in a dose-response fashion, with those having used high potency varieties on a daily basis having the highest score; 3) both higher scores on the positive and negative symptom dimensions were associated with a higher SZ-PRS. In population-based controls, the examination of general and specific dimensions with external factors showed that 1) higher scores on the positive psychotic experience dimension were associated with current use of cannabis but not with the extent of lifetime exposure to cannabis; 2) higher scores on the general and all the specific psychotic experience dimensions were associated with a high SZ-PRS. Conclusions: My thesis shows that symptom dimensions are useful psychosis phenotypes, that are validated by psychometric data and socioenvironmental and genetic factors. Specifically, the bifactor model of psychopathology holds across diagnostic categories of non-affective and affective psychosis at FEP and in the general population. Furthermore, my findings indicate that use of cannabis is associated with more positive and less negative symptoms at FEP, consistently with the hypothesis that cannabis users who develop psychosis have less early neurodevelopmental impairment than their non-user counterparts. Overall, these findings indicate that it is appropriate to conduct research using enhanced phenotypes, and they have translational relevance, they are important for developing secondary prevention strategies in psychosis. Currently, symptom dimensions at FEP could be used for formulating clinical impressions regardless of diagnostic categories according to a developmental-symptom approach, and for guiding tailored treatments.

Mephedrone is a synthetic cathinone known for its psychostimulant properties. Despite its ban in the United Kingdom in 2010, mephedrone use in London remains popular and there are reports describing an increasing problem of the drug being injected. Nevertheless, there is little known about the distribution of mephedrone and its metabolites in humans as only two controlled mephedrone administration studies and one dose-finding pilot study have been previously reported. In recent years, there has been a growing interest in the use of alternative biological matrices for determining drug abuse. The collection of these samples is usually non-invasive, fast and cost effective which allows for drug testing in the workplace, by the roadside and in addiction treatment centres. A single dose administration study of 100 mg mephedrone hydrochloride via nasal insufflation to six healthy male volunteers was performed to determine the distribution and pharmacokinetics of mephedrone and its metabolites in conventional (whole blood, plasma, urine) and alternative (oral fluid, fingerprint sweat, dried blood spots, head hair) biological matrices. Samples were collected at different timepoints after mephedrone administration and were analysed for the presence of mephedrone, dihydro-mephedrone (DHM), nor-mephedrone (NOR), hydroxytolyl-mephedrone (HYDROXY), 4-carboxy-mephedrone (4-CARBOXY) and dihydro-nor-mephedrone (DHNM) by validated liquid chromatography-tandem mass spectrometry methods. All analytes were detected in whole blood and plasma, where 4-CARBOXY reached the highest concentration. The mean Tmax for mephedrone (55.0 ± 18.2 min in whole blood and 52.5 ± 20.7 min in plasma) correlated well between both matrices, indicating rapid absorption of the drug after nasal insufflation. Other analytes had a more delayed Tmax but were all detected up to 6 h in both matrices, with mephedrone also being detectable on Day 2 in one participant in whole blood. Mephedrone had a mean half-life of 2.12 ± 0.33 h and 1.98 ± 0.30 h in whole blood and plasma, respectively. In addition, statistical analysis showed that median whole blood to plasma distribution ratios, reported here for the first time, were statistically different from 1 (unity) for mephedrone (median: 1.11), DHM (median: 1.30) and NOR (median: 0.765). Chiral analysis revealed that R-mephedrone reached higher concentrations than S-mephedrone in whole blood and had comparable pharmacokinetic parameters to total mephedrone. It has been shown that the two enantiomers of mephedrone exhibit different pharmacokinetic profiles in humans, but the clinical significance of this finding is not yet fully understood. In urine, 4-CARBOXY and DHNM were the only metabolites detectable on Day 3, making them promising markers of mephedrone use. In the alternative biological matrices, mephedrone metabolites were detected for the first-time in head hair one month after mephedrone administration. Calculated NOR:mephedrone and DHNM:mephedrone ratios were 0.19 (n=1) and 0.21 (n=1), respectively. However, sample size was too small to suggest robust metabolite to mephedrone ratios that would differentiate external drug contamination from drug consumption. In fingerprint sweat, mephedrone and NOR were detected above the limit of detection in 62% and 3.8% of all post administration samples, respectively. Inter- and intra-subject variability was observed which can be attributed to the differences in pressure applied during fingerprint deposition, the angle and duration of contact with the deposition surface coupled with an inability to control the ‘amount’ of collected sweat. Given these limitations fingerprint sweat may not be ideal for use in quantitative analysis until practical solutions to these problems are found. In dried blood spots, mephedrone, NOR and 4-CARBOXY were the only analytes detected in the majority of samples. In oral fluid, mephedrone and NOR were detected but their concentrations peaked earlier than in whole blood and plasma which may be due to the contamination of the oral cavity with mephedrone after nasal insufflation. It is hoped that this work will help with interpreting results and reporting findings from the analysis of conventional and alternative biological matrices following mephedrone use in forensic (drug-related deaths and crime) and clinical (acute drug toxicity and drug dependence) toxicology as well as in the workplace and roadside drug testing.

Resistance to certain antimicrobial agents is poorly understood, with knowledge gaps regarding both the prevalence and genetic basis of resistance. In this thesis, these aspects were examined for three antimicrobial agents (silver, bacitracin, and nitrofurantoin) that are used in the prevention and treatment of bacterial infections. Overt silver resistance was not prevalent in a cross-section of clinical Gramnegative isolates. A large proportion of Klebsiella spp. and Enterobacter spp. isolates have the potential to readily acquire silver resistance, with resistance arising as a consequence of single point mutations in silS that led to the activation of the Sil system. The development of silver-resistant strains is likely in clinical settings, and this could affect the clinical utility of silver. Resistance to bacitracin was identified in ~3% of Staphylococcus aureus isolates tested, and an epidemiological cut-off (ECOFF) point was proposed for 'true' bacitracin resistance of < 512 mg/L. All such strains carried the bcrABRS/bacA locus that encodes bacitracin resistance determinants. This locus is on a plasmid which suggests that bacitracin usage could induce the dissemination of this resistance in the future. After examining the nitrofurantoin susceptibility profile in a group of S. aureus isolates; it was apparent that MICs for nitrofurantoin naturally span a broad range. No nitrofurantoin resistance was detected, and a suggested ECOFF point for nitrofurantoin resistance in S. aureus was therefore 64 µg/mL. No evidence for nitrofurantoin resistance determinants was obtained among the isolates exhibiting reduced susceptibility to nitrofurantoin. Nitrofurantoin seems to be of value in the treatment of S. aureus, and it could be the drug of choice for urinary tract infections caused by S. aureus strains. In conclusion, this study provides insights that could help to inform the clinical application of these antimicrobial agents and can underpin future work to further dissect the biochemical and structural basis for resistance.

Recent attempts to reconcile neuropharmacological and cognitive accounts of depression have led to the development of a cognitive neuropsychological model of antidepressant treatment action. This model suggests that antidepressant drugs exert their clinical effects by acutely inducing positive biases in “hot” (i.e. emotionrelated) cognitive processes which can counteract the negative biases in affective information processing that are inherent to depression. The validity of this model is well supported by a multitude of empirical studies investigating the effects of different established antidepressant drugs which, taken together, suggest that the induction of positive biases is a common and crucial causal mechanism of antidepressant action. The aim of this thesis is to investigate whether potential novel antidepressant treatments could have similar effects. Taking an experimental medicine approach, three different interventions were studied in healthy volunteers: bright light treatment, practising positive psychology, and administration of the dopamine D2/D3 receptor-preferring agonist pramipexole. In addition to their effects on hot cognitive processes, for each of these interventions, potential effects on subjective state and, where of interest, also effects on other relevant measures (e.g. HPA axis activity for practising positive psychology, gustatory processing for pramipexole treatment) were tested. Contrary to our predictions, we did not find conclusive evidence for an antidepressant-like effect on emotional information processing for any of the three treatments we studied. However, several interesting patterns in other measures were observed: First, an early memory practice that has previously been used as a supposedly inert control intervention in positive psychology studies showed a blunting effect on cortisol awakening responses and potentially also a general improvement of hot cognitive processes. Second, subacute pramipexole treatment showed a potential effect on exploration-exploitation strategies in an information bias learning task, maintaining random exploration after repeated-testing as compared to placebo. Furthermore, pramipexole also showed a potential effect on gustatory processing, enhancing taste recognition but blunting subjective evaluation of appetitive and aversive taste samples. In conclusion, previously reported antidepressant effects of the putative novel treatments that were studied in this thesis seem not to be explainable by the induction of positive biases in emotion-related information processing. Further studies on the mechanisms behind their efficacy are warranted. The observed effects of the mental practice and of subacute pramipexole treatment deserve further attention and should be interrogated in closer detail in future research.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause harmful and potentially deadly infections, including neonatal sepsis. Vancomycin, a glycopeptide antibiotic, remains the most effective antibiotic to treat this condition. In the case of systemic diseases, vancomycin must be administered via intravenous injection. However, administration of vancomycin via this route has some associated drawbacks, such as the fact that the patient can experience pain and discomfort upon administration, as well as the possibility of transmission of blood borne infections as a result of needle-stick injuries. Vancomycin displays low absorption when administrated orally, due to the large molecular weight and low permeability in the gastrointestinal tract. As a consequence, utilising a novel transdermal drug delivery system to deliver vancomycin could prove a promising alternative. One approach that can be used to enhance transdermal drug delivery is microneedle (MN) technology. MN is micron-sized needles on a solid backing or support that can easily penetrate the skin’s stratum corneum barrier, accessing the viable epidermis and dermis. This thesis aimed to investigate the development of dissolving and hydrogel-forming MN for the facilitated delivery of vancomycin hydrochloride (VCL). VCL was formulated into dissolving MN (DMN), in combination with a variety of different excipients. In an alternate approach, VCL reservoirs, namely film dosage forms, lyophilised wafers and compressed tablets, were integrated with hydrogel-forming MN (HFMN). The physicochemical properties of the VCL-containing DMN, including mechanical strength, insertion ability and drug content, were evaluated. In vitro permeation studies were then carried out using the selected VCL-containing DMN and VCL reservoirs integrated with HFMN. Based on the outcomes of those studies, candidate VCL vehicles/MN were used in pharmacokinetic and biodistribution studies in an appropriate rat model. For the first time, VCL was successfully delivered transdermally into the systemic circulation using DMN and HFMN. The findings of this thesis explore the potential of appropriately formulated and characterised MN to enhance VCL delivery and bioavailability, with the potential to ameliorate approaches to the treatment of neonatal sepsis. Before these novel delivery systems can be employed in clinical settings however, pharmacodynamic assessments of VCL efficacy in models of infection must be determined, as well as considering patient compliance and industrial regulatory standards.

Biologically-based medicines such as vaccines and therapeutic proteins are routinely used in the clinic. While recombinant technologies are firmly entrenched and biotherapeutics will continue to be developed, maximising clinical efficacy requires optimal pharmacokinetics and duration of action. Treatment failures have also been known to occur due to poor adherence, compliance and access to healthcare. Generally, it is advantageous to have sustained protein delivery systems to reduce dosing frequency, to have fewer side effects, and to increase patient compliance. Controlled release protein-loaded particles or depots are options for less frequent dose administration. Similarly, with vaccines, there is often a need to administer boosting dose, so long acting or pulsatile vaccine formulations could provide clinical benefit. This thesis focuses on using electrohydrodynamic processes to fabricate polymeric fibres and particles that could form the basis for controlled release of biotherapeutics. Chapters 1 and 2 describe the relevant background and the materials and methods that were used. Chapter 3 reports the processes essential for the fabrication of alkaline phosphatase (ALP) encapsulated by polyethylene oxide (PEO) fibres and particles, to help understand the effect of electrohydrodynamic atomisation (EHD) processing techniques on the enzyme activity. There was evidence of change to the activity of the enzyme, which can be minimised by reduction of voltage used in processes. Chapter 4 describes the production of albumin-loaded from electrosprayed PLGA and Eudragit particles. Biphasic release systems could be generated by either mixing the two particle sets, or by forming core-shell structures. Albumin was used as a model antigen. Physicochemical and in vitro cytokine secretion assays were conducted to correlate particle properties with processing parameters. It was possible to vary the albumin release profiles and the poly(lactic-co-glycolic acid) (PLGA) particles significantly increase the production of tumour necrosis factor (TNF- α) but no other cytokines from macrophage-like cells. Chapter 5 describes optimisation of the processing parameters that affect particle shape and examines the effect of PLGA particle morphology on macrophage-like cells. Rod-like particles had more of an immunostimulatory effect than spherical particles and might hence be better suited to delivery of vaccines. In conclusion, materials fabricated using EHD can be tailored to achieve desired structures, shapes and release profiles.

The burden of antimicrobial resistance is a daily challenge in clinical settings, especially in intensive care units. The escalating trends of multidrug-resistant (MDR) bacteria are reported on a global scale today, and the loss of effective antibiotics and disinfectants undermines the ability to fight infectious diseases. The development of novel antibacterial agents is urgently needed in healthcare settings to reduce bacterial resistance and potential nosocomial infections. This study aimed at assessing the antimicrobial effect of metal ions and graphene-based compounds against multidrug resistant (MDR) isolates of Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. It also aimed at incorporating the successful combinations of metal ions and graphene composites into novel formulations of skin cleansers and nasal sprays/ointments that are effective against MDR chlorhexidine-adapted K. pneumoniae and mupirocin-resistant S. aureus in clinical settings. The antimicrobial efficacy of eighteen metal ion solutions, graphene and graphene oxide were tested, individually and in combination, against ten clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae using minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The antibiofilm activity of the compounds was tested using crystal violet biofilm assays (CVBA) and XTT assays. The synergistic effect between metal ions or combinations showing the best inhibitory activity against the three bacterial species and the excipients of the skin cleanser or nasal spray/ointment was assessed using fractional inhibitory concentration (FIC). Compounds that did not show any antagonistic effect were tested for their cytotoxicity using MTS assay against human skin fibroblast cell line, and were incorporated into different formulations of skin disinfectant, and nasal spray/ointment. The antimicrobial efficacy of the formulations was tested using the agar well diffusion method and CVBA assay. The organoleptic characteristics, uniformity, spreadability, thermal stability and other physical characteristics of each formulation were also evaluated. Morphological changes in bacterial xvi cells treated with the tested formulations were visualized using scanning electron microscopy (SEM). Results showed that platinum, palladium, gold, tin and molybdenum ions exhibited the best inhibitory effect against the planktonic cells of P. aeruginosa, S. aureus and K. pneumoniae. Graphene oxide demonstrated no antimicrobial effect (>500 mg/L) against the 10 isolates, whilst graphene showed an excellent against S. aureus and K. pneumoniae only. It was evident, however, that the addition of graphene or graphene oxide enhanced the antimicrobial effect of metal ions against the three bacterial species. Cytotoxicity readings revealed that gallium, indium, platinum, palladium, gold, graphene and graphene oxide were not toxic to the tested skin fibroblasts. Various formulations of skin disinfectants (14), topical ointments (9) and nasal sprays (9) containing metal ions and graphene composites were tested. The best inhibitory and antibiofilm activities were revealed by formulations containing gold/graphene, gold, platinum, platinum/graphene and palladium. The antibacterial effect of the different tested formulations was further confirmed by the clear morphological damages shown in bacterial cells characterized by the formation of deep grooves, pores, or cuts in their cell walls. In the light of the inefficacy of the currently used antibiotics and disinfectants, this study highlighted specific metal ions, used alone or in combination with graphene composites, owing their antibacterial activity, as promising potential novel antibacterial agents in clinical settings.

Background - Under-reporting of adverse drug reactions (ADRs) is a challenging medication safety problem globally. Even though ADRs are associated with significant morbidity and mortality, poor reporting among healthcare professionals (HCPs) persists, particularly in resource-limited settings. This study aimed to explore HCP experiences and factors influencing ADR reporting in the Ghanaian hospital setting. Methods - A concurrent mixed methods design was undertaken using face-to-face semi-structured qualitative interviews, focus groups and a survey. Nursing, pharmacy and medical staff were sampled using a stratified random sample from five hospitals in Tamale, Ghana coupled with purposive sampling for interviews. Survey data were analysed descriptively using SPSS and in-depth interviews and focus group discussions analysed using a six-stage thematic analysis using NVivo. Findings - 386 HCPs (86% response rate) participated in the survey. Pharmacovigilance (PV) knowledge was low (19%) with the majority being unaware of the national PV centre (68%) and basic information on reporting forms (65%). Pharmacy staff were however more knowledgeable compared to nursing and medical staff. Only 13% of HCPs reported to have observed an ADR at least once in a year and another 14% had completed a form. The majority (92%) of HCPs agreed that patient safety could improve if they reported ADRs and disagreed that litigation (82%) and lethargy (81%) were a hindrance. Pharmacists were perceived to have a key ADR reporting role. Use of verbal reporting was perceived to reduce ADR reporting formally along with complex interrelated system and human factors, such as lack of forms, inadequate infrastructure, stakeholder issues, uncertainty about reporting responsibilities, poor interpersonal relations, perceive patient attitudes, bureaucracies, fear of wrongdoing and blame. Conclusions – This study suggests that ADR reporting is low and often informal in the Ghanaian hospital setting but enhancing the role of pharmacists may be important in improving ADR reporting, as well as increasing HCP awareness through training – particularly for non-pharmacy staff - and logistical changes such as electronic ADR reporting.

Endocannabinoids, such as Anandamide (AEA), are lipid compounds which, together with the receptors they bind to, form the endocannabinoid system (ECS) which, in animals, modulates mood, cognition, appetite etc. The ECS can be activated by phytocannabinoids such as cannabidiol (CBD) and as such, interest in its therapeutic use has grown substantially over recent years. However, not all of CBD’s effects can be explained by its binding to cannabinoid receptors and many other targets have been proposed. A better understanding of these alternative targets would increase the therapeutic potential of this phytocannabinoid in the future. Single-celled protists, such as amoebae, do not possess cannabinoid receptors yet they respond negatively to endo- and phyto-cannabinoids, suggesting they possess alternative targets only. This study therefore examined the involvement of three alternative targets, i.e., the Peroxisome-Proliferator Activated Receptor (PPAR), Dopamine Receptor and Serotonin Receptor, in the action of AEA and CBD on amoebae. Of the 20-amoeba species tested, only 6 showed a reduction in population growth in the presence of CBD (IC50, 0.98-7.31μM), i.e., Hartmannella cantabrigiensis, Naegleria gruberi, Vahlkampfia avara, Vermamoeba vermiformis, Acanthamoeba castellanii and Flamella arnhemensis. All but the latter two species also showed reduced population growth in the presence of AEA (IC50, 0.96-9.89μM). The negative effect of AEA could not be alleviated by blocking receptors with antagonists against the three PPAR isoforms (PPARs α, β and γ), the dopamine receptor or the serotonin receptor, suggesting that none were involved in the mode of action of AEA. However, the negative effect of CBD was alleviated with the antagonist for the PPARα receptor in V. vermiformis and that for the serotonin receptor (specifically 5-HT1A) in N. gruberi. Interestingly, CBD significantly affected the feeding behaviour of these two amoebae (but AEA did not), by stopping amoebic feeding completely (and causing a lag in the ingestion of prey), which was then followed by a reduced ingestion rate; both of which were dose-dependent. Further work with V. vermiformis showed that this CBD-induced cessation in feeding was not due to the halting of phagosome processing and defecation however, there was an interaction between CBD and C-type lectins recognising mannose, N-Acetyl-D-glucosamine (GlcNAc) and 2 N-Acetyl-D-galactosamine (GalNAc). Receptor-mediated phagocytosis in V. vermiformis involved all three C-type lectins and although CBD did not appear to directly interfere (bind) with any of them, its presence (together with sugars to block these receptors) led to a synergistic reduction in ingestion rate with mannose and GalNac (but no effect on lag) and an extension of the lag with GlcNAc (but no effect on ingestion rate). The ‘CBD-receptor’ (putative PPARα) and GlcNAc receptor were therefore considered to be involved with phagosome formation and not prey capture and phagosome filling. It is therefore hypothesised that, because only the three PPAR isoforms exist in vertebrates, V. vermiformis possesses a promiscuous PPAR-like molecule that can bind CBD at the same site it binds the PPARα antagonist. The instantaneous nature of the feeding lag suggests the presence of a non-genomic PPAR response whereby in its ligated state PPAR cannot bind with a necessary protein (possibly Syk, LAT or PKC) to initiate a downstream signaling cascade, which would normally culminate in actin polymerisation and phagocytic cup formation. In conclusion, amoebae provide an ideal model organism to evaluate the significance of alternative targets in the functioning of the ECS. And, as is the case with animals, multiple alternative targets appear to be present in amoebae; PPARα (for CBD) in one species, Serotonin Receptor (for CBD) in another species, with the other alternative targets for CBD and indeed AEA currently unknown. And, considering multicellularity originated from free-living single-celled protists, amoebae have also provided an opportunity to investigate the historical functioning of the ECS which appears to be in the main, involved with the feeding response.

The human body is a complex piece of organic machinery, and as such, can be prone to faults and deterioration. There are many medical conditions that can be attributed to bone or cartilage deterioration and damage, like osteoarthritis and osteoporosis. Regeneration of these tissues is an on-going challenge, which have been targeted in many different ways. MicroRNAs are short sequenced RNAs that are involved in the regulation of nearly 60 percent of the genes in the body. This project aims to exploit their function to promote differentiation into bone, fat or cartilage cells. This is achieved using nanoparticles as the delivery platform. The use of nanoparticles in medicine has picked up at a rapid rate, and its small size, non-toxicity and multi-valancy allows for a safer and customizable method of cargo delivery. A novel form of inducing osteogenesis is the using of nanovibrations, known as nanokicking. This project aims to use antagomirs or anti-microRNAs as the primary cargo, which is a nucleotide sequence that can bind to the microRNA, causing a loss of function. A combination of nanokicking and functionalised GNP treatment will be used to see if there is an improvement in the efficacy and time taken for differentiation.

Background: In the late 1950s/early 1960s Thalidomide was given to thousands of pregnant women across the world to relieve morning sickness. The drug caused severe birth defects including missing/short limbs and sensory impairments, collectively referred to as Thalidomide Embryopathy (TE). However, Thalidomide is not just a historical tragedy, it is a contemporary disability issue. Across the world more than 5000 Thalidomide survivors are still living with TE. Aim and Objectives: We know relatively little about the long-term effects of Thalidomide damage nor about the survivors’ present day experiences. The aim of this study was to address this knowledge gap by exploring the changing nature of health and independence amongst UK Thalidomide survivors as they age. Methods: A mixed methods grounded theory study, comprised of four stages: a scoping literature review; primary content analysis of semi-structured interviews with a sample of 38 Thalidomide survivors; a cross-sectional survey of all UK Thalidomide survivors (to which 375/75% responded); and secondary grounded theory analysis of the semi-structured interviews. Findings: Whilst TE is regarded as a non-progressive condition, it is not static. As they age Thalidomide survivors are experiencing new Thalidomide-related health problems and deterioration in their original impairments alongside the accumulated disabling consequences of a life lived with a rare condition. Shifting impairment was leading to efforts to preserve function and the need to rethink independence, which in turn had implications for mental wellbeing. Discussion: Thalidomide survivors are at a stage in their lives where disability and ageing are beginning to intersect. They are ageing differently to their peers in the general population but there are both differences and similarities to other people with early acquired disabilities. Importantly, what is happening to Thalidomide survivors, needs to be seen in the particular historical, social and economic context of their lives.

Heparin has been extensively explored for cancer metastasis treatment owing to its ability to competitively inhibit metastatic cancer cells adhesion to P-selectin which is overexpressed on vascular endothelium and its inhibitory activity of platelet-tumour cells interaction. Moreover, heparin plays a key role in inhibiting the heparanase that is overexpressed during the early stage of metastasis process. However, heparin polymer is rapidly excreted through renal filtration. Detection of metastasis is of a paramount importance as it allows an early clinical intervention which could increase the survival rate of cancer patients. In the first part of this thesis, heparin was conjugated to silica NPs surfaces in order to investigate its P-selectin blocking activity simultaneously exploiting the prolonged circulation of the NPs. Moreover, two structurally related polymers; chondroitin sulphate and sodium alginate were grafted onto the surface of silica NPs in order to be used as controls. Heparin, chondroitin sulphate and alginate were attached to NPs’ surface by coupling of the EDC-activated polymers to amine functionalised NPs. TGA, azure A and DMMB assays confirmed the attachment of these polymers to the NPs surfaces. Polymer grafted NPs had a particle diameter circa 200 nm and negative zeta potential values (~ -56 mV). Heparin, chondroitin sulphate and alginate conjugated NPs were fluorescently labelled with different fluorophores; TAMRA, FAM and Alexa 633 respectively, in order to monitor their cell surface binding under flow conditions that mimic the in vivo circulation. The study revealed that heparin and chondroitin sulphate conjugated NPs were more bound to the surface of human umbilical vein endothelial cells (HUVECs) compared to alginate conjugated NPs as indicated by fluorescence microscopy. Furthermore, silica microparticles with mean size of ~ 1.4 μm were also synthesised and cellular uptake study was conducted which has shown that heparin conjugated microparticles exhibited less cellular internalisation compared to heparin conjugated NPs. However, the latter exhibited greater P-selectin dependent binding to HUVECs’ surface under flow conditions. To conclude, heparin conjugated NPs can interfere/bind with P-selectin receptors as the competitive assay showed that these NPs were blocked by anti-P-selectin antibodies. Consequently, this could suggest that heparin conjugated NPs could be further investigated and observed in the future as potential tools to reduce the dissemination of circulating tumour cells. In the second part of this thesis, heparin was chemically modified to produce desulphated heparin and glycol split heparin for the inhibition of heparanase as they have been reported to have less side effects and greater heparanase inhibitory activity compared to unmodified heparin. Successful synthesis of the modified heparin derivatives was confirmed by 1H-NMR. An anticoagulant assay (Anti Xa), which measures heparin activity by indirectly measuring its ability to activate antithrombin III, had indicated that glycol split heparin polymer exhibited lower anticoagulant activity compared with native heparin and thus it would have lower adverse reactions e.g. haemorrhage. Desulphated heparin is expected to have an additional clinical advantage compared to heparin such as higher antiangiogenic activity. Both heparin derivatives were attached to the surface of silica NPs using EDC coupling reaction. Heparin analogues conjugated NPs had a particle diameter around 200 nm so the EPR effect, which is a phenomenon that hypothesises a preferential accumulation of the NPs in the tumour interstitial fluid, could be exploited to accumulate the modified heparins in the extracellular space of the tumour where heparanase is overexpressed. Anti Xa assay also showed that glycol split heparin conjugated NPs exhibited lower anticoagulant activity compared with heparin conjugated NPs. Desulphated heparin conjugated NPs showed both significantly less blue shift effect on azure A λmax and lower zeta potential value compared to heparin conjugated NPs. The data together indicate successful conjugation of the modified heparin polymers to the surface of silica NPs that could be further employed to inhibit heparanase. In the final part of this thesis, different FRET based models were designed to detect heparinase which is the bacterial form of mammalian heparanase overexpressed during metastasis, in order to enable the early diagnosis of cancer. In model I, both FRET pair were attached to heparin chain to attain fluorescence quenching using EDC/NHS coupling reaction. The fluorophore was either attached to the end or within the chain of heparin; however, the probe prepared by intrachain conjugation of the fluorophore showed significantly higher fluorescence quenching. This model showed promising results with respect to quenching efficiency (QE), unfortunately the restoration of the fluorescence signal after heparinase treatment was minimal compared with the chemical depolymerisation. Model II was then developed which adopts the use of core-shell NPs composed of 12 nm thick shell containing a fluorophore (TAMRA) and a black hole quencher conjugated heparin (BHQ2-Heparin) probe attached to NPs surfaces. Where the thinnest possible shell was used in order to maintain the FRET pair close to each other to achieve an optimum FRET energy transfer and maximal QE. The QE of this system was measured with either blank core or with a core containing 7-methoxy coumarin, however a discrepancy between the QE measurements of the core-shell nanosensors was observed with dramatically lower value obtained by coumarin based system. Due to the inconsistency of QE measurements of model II, model III was developed which comprises BHQ1 incorporated silica NPs and fluorescently labelled heparin appended to the NPs surfaces. In summation, Model III showed the best potential for bacterial heparinase detection and thus it could be further improved and utilised for detection of mammalian heparanase sampled from the blood of cancer patients for metastasis diagnosis. In conclusion, the results highlight that heparin conjugated nanoparticles could be manipulated for versatile nanomedicine applications, thus they could be further investigated as novel potential therapeutics and diagnostics for cancer metastasis.

Each year, a large number of people develop scars all over the world as a result of abnormal wound healing. However, the current treatment strategies available have demonstrated insufficient outcomes and limitations. Even though studies have shown that angiotensin converting enzyme inhibitors (ACEIs) can be a cutting edge scar treatment, licensed topical formulations of ACEIs have not been yet available for use. Therefore, an attempt has been made in the present work to design stable topical formulations of ACEIs and evaluate the percutaneous absorption of the drugs across porcine ear skin as a surrogate model of human skin. Since the quality of any formulation relies on its stability and efficacy, three different ACEIs, moexipril (MOX), ramipril (RAM), and cilazapril (CILA), were underwent stability studies at 32 ̊C in a wide range of dermal vehicles. Among these three compounds, CILA exhibited good stability while the others were degraded under the experimental conditions resulting in diketopiperazine formation. Based on the findings of the stability studies, simple and complex topical formulations of CILA were developed. The permeation data demonstrated the feasibility of dermal delivery of CILA across porcine skin. Furthermore, the results indicated that the complexity of the topical formulation had a pronounced effect on the permeation rate. The hydrolysed form of CILA, cilazaprilat, was also found in the receptor phase, indicating that CILA may undergo metabolism during the permeation. This was confirmed by evaluating the susceptibility of CILA to metabolism by porcine skin homogenate. Overall, the findings indicated that CILA was extensively metabolized by skin esterases leading to metabolite formation, specifically, cilazaprilat. We reported for the first time the possibility of the topical delivery of cilazapril. Ultimately, the work described here will lead to the design and development of new and better topical formulations for the management of wounds.

One explanation as to why atypical antipsychotic drugs (APDs e.g. clozapine) show reduced extrapyramidal side effects (EPS) involves their rapid dissociation from the dopamine D2 receptor (D2R). Their selective regional action in the brain permits an antipsychotic effect through inhibition of mesolimbic dopamine signalling but spares physiological dopamine neurotransmission in the striatum. This hypothesis is based largely on direct kinetic measurements using radiolabelled APDs and achieved under varied conditions and over many years. The alternative, ‘serotonin-dopamine hypothesis’ of atypicality suggests that antagonism at the 5-HT2A receptor and agonism at the 5-HT1A receptor may account for reduced EPS, due to increased nigrostriatal dopamine release. The strength of this hypothesis is currently limited to a consideration of the relative receptor affinities of APDs for the 5-HT2A and dopamine D2R, and limited data exploring their 5-HT1A agonist effects. To further explore these hypotheses, I developed a series of novel higher throughput time-resolved fluorescence resonance energy transfer (TR-FRET) binding assays to measure the kinetic properties of a series of clinically relevant typical and atypical APDs at the dopamine D2R, the serotonin 5-HT2A and 5-HT1A receptors. Relative 5-HT1AR APD efficacy was estimated using a [35S]-GTPS assay and through the application of the operational model of agonism. These studies establish a novel mechanism of atypical APD action based on a restricted drug diffusion model, suggesting that for the majority of clinically used APDs association rate and not dissociation rate, better predicts the overall rate of dopamine D2R reversal in the striatum. For compounds with negligible affinity for 5-HT2A receptors, EPS is robustly predicted by a D2R specific rebinding model that integrates D2R association and dissociation rates, to calculate the net rate of reversal of receptor blockade (kr). However, I show that for certain compounds (eg sertindole) a combination of high relative 5-HT2A occupancy and rapid dopamine D2R dissociation (koff), equates to a reduced propensity to cause EPS. Thus, optimizing the binding kinetics of compounds at both the serotonin 5-HT2A and the dopamine D2R may result in the development of APDs with an improved on-target side-effect profile.

Cannabinoid receptors (CB1 and CB2) are signalling proteins which belong to the largest family of transmembrane proteins called G protein-coupled receptors (GPCRs). Since their discovery, they have been widely studied for the notable pharmacological influences exerted by their interaction with cannabinoids and are hence viewed as druggable targets for a number of diseases. Along with their endogenous ligands and the metabolic enzymes that affect the bioavailability of endogenous cannabinoids, they form the endocannabinoid system. The discovery of crystal structures of CB1 and CB2 in recent years has relayed critical information regarding the conformation of the receptors in active and inactive states, and their binding pocket interactions. Though being invaluable sources of information, rigid crystal structures cannot completely rationalise structure-activity relationship for all classes of ligands that interact with the receptors. The work reported herein describes the exploration of the structures of CB1 and CB2 receptors via computational tools such as molecular modelling, docking, and molecular dynamics simulation. Accordingly, significant variations in the conformations of CB1 and CB2 in different states of activation were studied. It was observed that transmembrane helices 1,3,6 and 7 influence the structural features of both receptors at different states. In recent years, many ligands that are not classified as cannabinoids have been shown to influence the endocannabinoid system. In this regard, the work presented here also analyses the interaction of non-cannabinoid ligands at CB1 and CB2. In this regard, a select group of commonly used drugs were tested against CB1 and CB2 using [35S]GTPγS binding assay for agonist activity. Furthermore, non-cannabinoids that have been reported to show activity at CB1 and CB2 were docked to models of the receptors to decipher their binding mode. It was found that the binding mode depends on the binding pocket surface area and is stereoisomer specific. In general, the work documented herein provides an insight into the structural complexities of these receptors for the cannabinoid research community.

Ascorbic acid (vitamin C) is a popular topically applied cosmeceutical due to its antioxidative, photoprotective, antiaging and antipigmentary effects. Despite these beneficial effects to the skin, instability of ascorbic acid in aqueous solution has directed formulation scientists to focus on stable ascorbic acid derivatives that can be used in topical preparations. However, these derivatives are required to be metabolised by the skin to the active, free-acid form to observe efficacy. Accordingly, current knowledge regarding skin permeation of ascorbic acid is limited, with in vivo human skin permeation data unavailable. The aim of this work was to use imaging techniques, specifically time of flight-secondary ion mass spectrometry (ToF-SIMS), to visualise the spatial distribution of topically applied ascorbic acid in the skin without the requirement for chemical labelling. This was performed in conjunction with established analytical techniques, such as high performance liquid chromatography (HPLC), that are traditionally used for skin permeation studies to obtain absolute quantitative information but with no spatial component. This dual approach provides the opportunity to reveal new insights into ascorbic acid skin permeation that would not be possible with one analytical technique alone. A comprehensive study comparing ascorbic acid and caffeine permeation, believed to be the first of its kind, shows how a gel formulation can retard the permeation of ascorbic acid but enhance the permeation of caffeine through ex vivo porcine skin tissue. The gel formulations were rubbed into the skin, to mimic in use conditions. When examining the spatial distribution, ascorbic acid and caffeine were found to be non-uniformly distributed and primarily localised to the epidermis. Contrary to previous reports that the follicular route contributes approximately 50% of the total in vitro skin permeation, no localisation of caffeine was observed in the hair follicle. Nanostructured lipid carrier (NLC) formulations have been intensively investigated as topical formulation vehicles. Herein an NLC formulation, and a comparator cream formulation, were developed and tested on human skin in vivo for permeation. Analysis of tape strip samples by HPLC analysis showed the cream formulation to deliver the most ascorbic acid into the stratum corneum. However, analysis of tape strip samples from cream-treated skin, by ToF-SIMS, showed the ascorbic acid was mainly localised in the furrows of the skin. A direct correlation between ToF-SIMS and HPLC, performed for the first time, showed that for the NLC formulation, ascorbic acid was more laterally distributed over the corneocytes at all concentrations, and not just localised to skin furrows, than the cream formulation. An ideal topical preparation should deliver the active ingredient into the corneocytes. Uncovering the spatial distribution, and localisation, of the permeant in the skin barrier has facilitated identification of the NLC formulation to be the most suitable topical preparation for ascorbic acid; and therefore, have the potential to increase the therapeutic effects observed in the skin. Dependence on the established analytical techniques, used for skin permeation studies would have resulted in an altogether different conclusion being reached. The standard approach of using HPLC, as advised by regulatory bodies, provides limited information about skin permeation due to a lack of imaging capability. The application of imaging techniques, such as ToF-SIMS, in this work has shown unequivocally permeation behaviour different from current literature understanding and a permeation profile different from conventional chromatography based methodology. It was therefore demonstrated that information regarding spatial distribution of the permeant in the skin barrier is essential to gaining a more holistic understanding of the mechanisms and routes of skin permeation when evaluating the effectiveness of topically applied formulations.

Fungal infections now contribute significantly to microbe-related morbidity and mortality due to there being a limited number of available antifungal agents and limited modes of delivery. Over the past two decades, many pathogenic fungi have developed various modes of resistance to commonly used antifungals. Thus there is a need for novel antifungal agents with novel mechanisms of action. Inhibition of the essential plasma membrane (PM) H+-ATPase of fungi is a potentially effective therapeutic approach in antifungal drug discovery. In order to investigate this, three series (A-C) consisting of a total of thirty-three symmetrical 1,4-diene-3-one (22a-v, 23a-h and 24a-c) compounds have been synthesized. In vitro macro-broth susceptibility testing of 1,4-diene-3-ones showed wide range of inhibition against Saccharomyces cerevisiae (0.2 - 99%) and Candida albicans (0 - 99%). Compounds 22f, 22m, 22n and 22s exhibited highest potency then other compounds from the library against S. cerevisiae (IC50 = 1.21, 2.22, 0.62 and 1.87 μM), however these compounds demonstrated limited activity against C. albicans (IC50 = 114, 525, 474 and 666 μM). In contrast, compounds 23f, 23g and 23h exhibited a higher degree of antifungal activity against C. albicans (IC50 = 68.5, 57.6 and 50.7 μM) and these compounds also showed good potency against S. cerevisiae (IC50 = 8.46, 5.52 and 6.25 μM). To gain an understanding about the mechanism of action of 1,4-diene-3-ones, the H+-ATPase mediated proton pumping by S. cerevisiae was investigated by measuring the pH of the glucose-induced acidification of the external medium. The bis-pyridylidene derivatives of N-methylpiperidin-4-one (23a-c, 60 μM) were determined to be the most potent inhibitors of H+ efflux from S. cerevisiae and the steady state of proton flux from S. cerevisiae was achieved within 10 minutes of medium acidification. A similar result was observed with N-ethylmaleimide (NEM, 60 μM, positive control). Additionally, 23a, 23b and 23c have shown good potency in the macro broth susceptibility assay of S. cerevisiae (IC50 = 12.6, 8.84 and 9.45 μM). Moreover, the most potent compounds 22n and 23h in macro broth susceptibility assay against S. cerevisiae and C. albicans exhibited limited activity to inhibit the proton efflux from S. cerevisiae. To further elucidate the mechanism of action, preliminary structure-activity relationship (SAR) studies was performed. SAR of bis-benzylidene derivatives of N-methylpiperidin-4-one displayed reasonable correlation coefficient (R2 = 0.6746) between the inhibitory activity expressed as log (1/IC50) and an electronic parameter, the 1H-NMR δ-values of proton on the β-carbon. Conversely, SAR of log (1/IC50) and a lipophilic parameter, calculated logP (clogP) of bis-benzylidene derivatives of N-methylpiperidin-4-one showed R2 of 0.4138. This indicates that the inhibitory activity of compounds is due to the influence of electronic property rather than the lipophilic property. In conclusion, the inhibitory action of 1,4-diene-3-ones on yeast suggests a membrane-bound enzyme target for its action. It is hypothesized that these compounds form a covalent C-S thio-ether bond with cysteine residues of proteins of the plasma membrane and eventually inhibit the H+-ATPase.

Antibiotic resistance is considered a major public health concerns in the 21st century. Microbes that were once susceptible to antibiotics are becoming more and more difficult to treat as they acquire rapidly a wide range of resistance mechanisms including enzymes of the β-lactamase family. These proteins are typically plasmid-borne and inactivate beta-lactam ring containing antibiotics that are worldwide used to treat infections. This enzyme family with more than 2800 proteins is rapidly evolving through the spontaneous acquisition of missense mutations that enhance protein stability, increase catalytic turn-over and/or improve substrate specificity. This study is an epidemiological snapshot of the antibiotic resistance conferred by CTX-M-type extended-spectrum β-lactamases (CTX-M type ESBL) in patients based in North Wales who were diagnosed with urinary tract infection (UTI). Multiplex PCR amplification of β-lactamase genes of the blaCTX-M groups 1, 2, 9, and 8/25 followed by DNA sequencing of ESBL-producing isolates was performed with samples from three referral hospital in North Wales; Ysbyty Gwynedd, Glan Clwyd Hospital, and Wrexham Maelor Hospital In line with previous studies, the β-lactamase CTX-M-15 was found to be the dominant CTX-M variant in the study region. The analysis of samples from Ysbyty Gwynedd hospital at Bangor revealed however an as yet unknown CTX-M variant that differed from CTX-M-14 by only three missense mutations. The amino acid substitutions A55T, A273P and R277C reside in the beta-strand domain opposite of the active site of the enzyme. CTX-M-15, CTX-M-14 and the novel variant were expressed in the periplasm of recombinant E.coli using the inducible vector pASK-IBA2C. Determination of the Minimum Inhibitory Concentration (MIC) of a series of clinical relevant antibiotics using the induced recombinant strains showed a significant increase in Nitrofurantoin resistance and a partial increase in Cefoxitin resistance that were both specifically linked with the three mutations. In vitro Kinetic studies using protein from periplasmic extracts of induced strains showed however that neither the affinity for Nitrofurantoin nor for Cefoxitin was increased by these substitutions. Taken together, the three missense mutations in CTX-M-14 may render UTI patients isolates more resistant to Nitrofurantoin because the three amino acid changes increase enzyme stability, its expression level in the resistant coliform bacteria or result in a higher turn-over rate of the mutated β-lactamase by facilitating conformational changes.

This study focused on isolation, synthesis, development and evaluation of novel antimicrobial and anticancer agents from natural product. Natural products have found lead applications in the pharmaceutical industry as sources of chemotherapeutics against cancers and infectious diseases. This investigation focussed on isolating sesquiterpene lactones from natural products, and synthesis of flavonoid based anticancer and antimicrobial agents from the flavonoid – chrysin because sesquiterpene lactones and flavonoids are potent anticancer and antimicrobial agents. In addition to the sesquiterpene lactones – alantolactone (D1) and isoalantolactone (D2), this is the first time costunolide (D3) has been isolated from Inula helenium. A series of 8-novel and 10 known Chrysin-derivatives were also synthesised via microwave-assisted O-alkylation of chrysin and other synthetic methods, and their antimicrobial and anticancer activities investigated. Twenty compounds were screened against eight bacteria and one fungus - Candida albicans using Gentamycin and Fluconazole as standard drugs for antibacterial and antifungal studies. 4-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)butyl acetate (C1), 7-((3,5-dimethylbenzyl)oxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (C2) and 7-(2,4-Dinitrophenoxy)-5-hydroxy-3-phenyl-4H-chromen-4-one (C3) indicated bactericidal inhibition against a broad spectrum of bacteria and Candida albicans; Staphylococcus aureus, Escherichia coli, Bacillus cereus and Enterococcus faecalis were the most susceptible bacteria to inhibition by these compounds. MIC of C1 against these microorganisms was 25, 50, 50 and 50 μg / mL, C2: 50, 100, 100 and 100 μg / mL, C3: 12.5, 12.5, 25.0 and 50 μg / mL. MIC values indicated by these compounds were better than those indicated by chrysin and were comparable to those showed by the standard drug – Gentamycin. C1 and C3 also inhibited the growth of the drug-resistant bacteria MRSA at doses of 65 and 100 μg / mL respectively. Chrysin derivatives also inhibited the growth of extended spectrum beta lactamase (ESBL) bacteria – E. coli, K. pneumoniae and P. aeruginosa. C1 and C3 also showed superior antifungal activity against the pathogenic fungus – Candida albicans at MIC values of 50 μg / mL respectively compared to the standard antifungal drug – Fluconazole (MIC 125 μg / mL). 7-O-bromochrysin and 7-O-alkylchrysin derivatives of chrysin showed weak antimicrobial activities (MIC ≥ 125 μg / mL) against all bacteria and fungus tested. D1, D2 and D3 also inhibited the growth of S. aureus and B. cereus at MIC values ≤ 125 μg / mL. The best antimicrobial agent synthesised were C1 and C3. The antiproliferative activities of D1, D2 and D3 were also investigated in hypoxia and normoxia (under hypoglycaemic and hyperglycaemic conditions). In hypoxia D1, D2 and D3 elicited a 10 to 55-fold significant antiproliferative activity against leukaemia cell line (K562) in hypoglycaemic conditions with cytotoxicity (IC50 values) 0.58, 0.66 and 3.07 μM compared to the antiproliferative activity indicated in hyperglycaemic condition (IC50 values – 0.80, 1.18 and 4.77 μM) respectively. In normoxia (normal oxygen concentration), the IC50 values in hypoglycaemia were 1.80, 1.90 and 6.72 μM for D1, D2 and D3. The IC50 values in hyperglycaemia were 2.60, 3.29 and 7.15 μM for D1, D2 and D3 respectively. The antiproliferative activities indicated by D1, D2 and D3 under the conditions stated above were better than those indicated by the positive control – Chlorpromazine hydrochloride (CPZ) with IC50 values 32.88, 27.05, 9.36 and 16.21 μM respectively. D1, D2 and D3 were selectively cytotoxic against cancer cell lines but not the normal cell line – BEAS-2B. Anticancer activities against colorectal cancer cell lines (HCT 116 and Caco-2) indicated by C1, C2 and C3 (IC50 ≤ 2.0 μM for HCT 116, and IC50 ≤ 6.0 μM for Caco-2 cell line), were better than those indicated by chrysin and the positive control drug – (CPZ) – IC50 4.07 and 5.9 μM respectively, and IC50 4.84 μM (chrysin) and 5.17 μM (CPZ) for Caco-2 cell line. C1, C2 and C3 also elicited better anticancer activities against leukaemia (K562) at concentrations of 7.27 μM, 5.58 μM and 8.69 μM; and mesothelioma (Mero-14) cell lines at concentrations of 7.65 μM, 26.31 μM and 8.71 μM respectively, compared to chrysin (IC50 13.13 μM for K562 cell line and 45.99 μM for Mero-14 cell line) and CPZ (IC50 7.38 μM for K562 cell line and 13.42 μM for Mero-14 cell line). Similarly, C1 and C2 also elicited significant cytotoxic inhibition against the triple-negative breast cancer cell line – MDA-MB 468 at IC50 values 7.49 and 16.59 μM compared to chrysin. Anticancer activities elicited by long-chain 7-O-alkylchrysin derivatives (C10, C11 and C12) against HCT 116 cancer cells (IC50 16.4, 17.5 and 18.9 μM) was comparable to those indicated by chrysin and CPZ (IC50 5.9 μM). C16 and C17 also elicited good to moderate cytotoxic inhibition against a broad spectrum of the ten cancer cell lines tested. Interestingly, C1, C2 and C3 were selectively cytotoxic against cancer cell lines but not the normal cell line – BEAS-2B. Chrysin was selectively cytotoxic but CPZ killed both cancer and normal cell lines. D1, D2 and D3 also indicated anticancer inhibition against K562 cell lines during hypoxia and normoxia, and during hyperglycaemia and normoglycaemia. Anticancer inhibition during hypoxia and low glucose for D1 and D2 (IC50 ≤ 0.66 μM) was better than the anticancer inhibition elicited in normoxia and low glucose concentrations (IC50 ≤ 1.8 and 1.9 μM respectively). In this study, novel anticancer and antimicrobial agents which elicited better bioactivity and solubilities compared to standard antimicrobial agents (Gentamycin and Fluconazole), anticancer positive control agent (Doxorubicin) and chrysin (the parent compound) have been synthesised. Furthermore, hyperglycaemia in normoxic condition was associated with rapid cell proliferation in contrast to hypoxia in hypoglycaemic condition. Compounds isolated in this study were more cytotoxic under hypoxic conditions. Costunolide was isolated for the first time from Inula helenium. In-vivo cytotoxicity studies of costunolide against leukaemia cell line – K562 in mammalian models is necessary. Some modified chrysin-derivatives also indicated significant cytotoxicity in-vitro. In-vivo cytotoxicity studies using mammalian model is also required. Synthesised chrysin derivatives were selectively cytotoxic against cancer cell lines but were not toxic against normal cell line at IC50 ≤ 100 μM. From the structure–activity point of view, nature and position of the electron withdrawing and electron donating functional groups on the chrysin core might have contributed to the observed antibacterial and anticancer action. The observed anticancer activity of alantolactone, isoalantolactone, costunolide, C1, C2, C3, C6, C8 and C10 and the antimicrobial activity of C1, C2 and C3 makes them an attractive drug candidate than chrysin and shows that substitution at the 7-OH position of chrysin could enhance the bioactivity of chrysin.

The two main phytocannabinoids, ∆9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) are agents already licensed for clinical use (i.e. Sativex®and Epidiolex®). THC and CBD have numerous cardiovascular effects in vitro; however, their effects on haemodynamics in vivoin humans remain unclear. Two systematic reviews and meta-analysis were established to evaluate the effects of THC and CBD on the haemodynamics in vivoin animals and humans. Our analysis showed that THC acts differently according to species and experimental conditions, causing bradycardia, hypotension and increased blood flow (BF) in animals, and increased heart rate (HR) in humans. However, CBD has no effect on blood pressure (BP) or HR under control conditions, but reduces BP and HR in stressful conditions, and increases cerebral BF in mouse models of stroke. Both systematic reviews and meta-analysis highlighted the limited data available in humans and further studies should be considered. Our group’s recent study in healthy men showed that acute oral administration of CBD (600 mg) causes a reduction in BP at rest and in response to stress. Because of this, three human studies were conducted to see if the findings from our previous study are reproducible, as well as if tolerance develops and if other vascular endpoints were affected post-CBD. Continuous beat-to-beat haemodynamics was assessed at rest and in response to stress (isometric exercise) using Finometer®. Arterial stiffness and aortic blood pressure were assessed using Vicorder®.Duplex ultrasound was used to assess changes in cerebral blood flow, extra-cranial arterial diameter, and brachial artery endothelial function after CBD administration. In the first human study, 26 healthy men were given 600 mg of CBD or placebo orally for seven days in a randomised, placebo-controlled, double-blind, parallel trial (n=13 per group). Cardiovascular parameters were assessed after single (acute) and repeated (chronic) dosing for seven days. CBD reduced mean arterial pressure after acute dosing at rest and reduced systolic BP after acute and chronic dosing in response to stress. Repeated CBD dosing reduced arterial stiffness, induced vasodilation and augmented BF volume in the internal carotid artery and improved endothelial function compared to single CBD dosing. Based on these findings, the second study was conducted to investigate the effect of a single dose of CBD in healthy men on arterial stiffness and cerebral BF to determine whether the effects seen after repeated dosing of CBD can also be seen after a single dose compared to baseline. Ten healthy men were given a single dose of placebo and CBD (600 mg) orally with a washout period of at least a week between the two treatments in a randomised, placebo-controlled, double-blind, crossover trial. The cardiovascular system was assessed before and after receiving the treatment. No effect was seen on arterial stiffness or cerebral BF following acute CBD administration. Due to the lack of studies assessing the effects of CBD on the cardiovascular system in women, a third study was conducted to establish the acute effect of CBD on the haemodynamics in women. Thirteen healthy females were given a single dose of placebo and CBD (600 mg) orally with a washout period of at least a week between the two treatments in a randomised, placebo-controlled, double-blind, crossover trial. The cardiovascular system was assessed after receiving the treatment. No effects wereseen following acute CBD administration. The findings of this thesis indicate that CBD may induce positive effects on human haemodynamics and on vascular function following chronic administration, and that sex-difference could impact its effects and should be considered in future studies investigating the effect of CBD on the human cardiovascular system. Further research is needed with larger sample sizes in dose-escalation trials and patient populations with vascular diseases.

The 5-hydroxytryptamine (5-HT; serotonin) 5-HT3 receptor is an excitatory ligand-gated ion channel expressed in for example the brain and the gastrointestinal tract. Two major subtypes of the receptor have been studied in the most detail; the homomeric 5-HT3A receptor and the heteromeric 5-HT3AB receptor. 5-HT3 receptor antagonists are used clinically to treat chemotherapy induced and post-operative nausea and vomiting, and demonstrate symptomatic relief in diarrhoea-predominant irritable bowel syndrome (IBS-d); but unfortunately, these medications cause adverse effects such as constipation or rarely ischemic colitis in the latter condition. This study has characterised the pharmacology of two structurally distinct 5-HT3 receptor partial agonists (vortioxetine and CSTI-300); and identified the unique binding properties of the cryptic orthosteric modulator 5-chloroindole (Cl-indole) for the human (h) 5-HT3 receptor. Vortioxetine is a multi-modal antidepressant, which displays affinity for the 5-HT transporter as well as a multitude of 5-HT receptors, including the 5-HT3 receptor. The first part of this study has identified vortioxetine as a relatively high affinity, competitive 5-HT3 receptor partial agonist with an intrinsic efficacy of approximately 40-50% that of 5-HT. Given the safety of vortioxetine in patients, it has the potential to be trialled in other conditions for which 5-HT3 receptor antagonists demonstrate efficacy but cause adverse effects, such as IBS-d or even schizophrenia. The second section of this study has identified CSTI-300 as a selective, high affinity 5-HT3 receptor partial agonist (intrinsic activity 30-40% that of 5-HT). Moreover, in a rodent model of IBS-d, CSTI-300 demonstrated comparable efficacy to alosetron, a 5-HT3 receptor antagonist with established efficacy in treating IBS-d. CSTI-300 is predicted to be able to relieve symptoms of IBS-d in patients without eliciting the side effect profile associated with 5-HT3 receptor antagonism. The final part of this study has demonstrated that the binding mechanism of Cl-indole for the 5-HT3A receptor is modulated by 5-HT3 receptor agonists, but not 5-HT3 receptor antagonists. This could have implications in designing 5-HT3 receptor allosteric ligands for potentially treating conditions such as IBS-d.