Hydrogen sulfide (H2S) has relatively recently been added to a list of endogenously produced gaseous signalling molecules. Our understanding of the science of H2S has advanced rapidly in recent years as exemplified by the fact that within a mere 10 years a range of H2S releasing drugs have already been discovered and some indeed are entering clinical trials. However, the precise biological roles of endogenous H2S are not fully understood. In this respect, slow releasing H2S donors, such as GYY4137, have played a part in elucidating the complex roles of this gas in the body and are also beginning to show promise as possible therapeutics in inflammation - an area in which the function of H2S remains ambiguous. This thesis attempts to provide some additional clarity to the biological significance endogenous H2S. The first part of this work examines the release of endothelial cell derived H2S in vitro and the consequences of knocking out nitric oxide synthase on tissue H2S biosynthesis in mice. As part of this study, I show that the methods currently utilised to measure H2S are insufficiently sensitive/reliable to demonstrate the release of H2S synthesis from endothelial cells in vitro. In addition, data reported herein has demonstrated that knocking out endothelial cell nitric oxide synthase (eNOS) results in a presumably compensatory increase in tissue H2S synthesising activity associated with increased protein levels of the H2S synthesising enzyme, cystathionine-γ-lyase (CSE). The second part of this thesis examines the role of H2S in inflammation and provides further evidence for its anti-inflammatory activity both in vitro and in vivo. In addition, this thesis has shown the ‘added benefit’ of slow-releasing H2S donors (c.f. 3 conventional sulpide salt based donors) in that the H2S released from slow-releasing donors is sustained and does not instantaneously expose cells to potentially cytotoxic amounts of H2S. Identifying a need for additional slow releasing H2S donors attempts were made to examine the H2S releasing ability and antioxidant capacity of a library of additional compounds. As a result of this work, a novel compound, ZJ802 was shown to exhibit more potent antioxidant ability than the currently commercially available H2S donors and was further shown to exhibit anti-inflammatory activity both in vitro and in vivo. Overall, the roles of H2S in physiology are not clear. Current methods to detect H2S are flawed. Thus, the necessity for pharmacological tools, such as slow releasing H2S donors and selective H2S synthesising enzyme inhibitors, cannot be overemphasised. Whilst the possible use of H2S donors in the clinic has been raised there is still a need for more detailed preclinical, pharmacokinetic and long term drug safety and toxicological studies.

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitises activated G protein-coupled receptors (GPCRs). In this thesis, I identify ezrin, a member of the ezrin-radixin-moesin (ERM) family of actin/plasma membrane cross- linkers, as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates ezrin within a 53 amino acid region that encompasses the phosphorylation site required for ezrin activation, threonine-567. Two lines of evidence indicate that GRK2 acts as an effector of activated GPCRs through phosphorylation of ERM proteins. Firstly, in Hep2 cells muscarinic Ml receptor (MlMR) activation promotes cytoskeletal reorganisation and membrane ruffling. This ruffling response is ERM-dependent and is accompanied by ERM protein phosphorylation. Inhibition of GRK2, but not Rho kinase, protein kinase C or cAMP-dependent protein kinase, prevents ERM protein phosphorylation and membrane ruffling. Secondly, agonist-induced internalisation of the (-adrenergic receptor (P2AR) and Ml MR is accompanied by ERM protein phosphorylation and localisation of phosphorylated ERM proteins to receptor-containing endosomal vesicles. The Na+/H+ exchanger regulatory factor, however, is not required for co-localisation of phosphorylated ERM proteins to the p2AR. Inhibition of ezrin function impedes P2AR internalisation, further linking GPCR activation, GRK activity and ezrin function. However, ERM protein phosphorylation does not occur following M2 muscarinic receptor (M2MR) activation and ERM proteins do not colocalise with internalised M2MR, suggesting that ERM proteins may be required for the internalisation of a subset of GPCRs. Overall, my results indicate that GRK2 serves not only to attenuate but also to transduce some GPCR-mediated signals.

The human cytochrome P450 3A subfamily metabolises endogenous substances and approximately half of all currently available drugs. There is marked inter-individual variation in hepatic expression of the major adult isoform, CYP3A4; the genetic component of this variability is estimated at 60-90% and, as yet, remains largely uncharacterised. Elucidation of genetic factors determining CYP3A4 activity would permit personalised dose-adjustment in therapies with CYP3A4 drug substrates. CYP3A4 genomic variation within the 5’ regulatory region, 3’ UTR, exons and regions of flanking adjacent introns was characterised by sequencing 752 chromosomes from five Ethiopian ethnic groups supplemented by sequencing data from The 1000 Genomes Project. Considerable novel variation was found within Ethiopia. CYP3A4 variants were identified that will form the foundations for future in vitro and in vivo work to determine the functional impact of variation on CYP3A4 expression/activity in vivo. Evidence of purifying selection was observed and genomic variation was consistent with the hypothesis that, within humans, the expression of CYP3A4 is essential to the maintenance of life. Conversely, regulatory region variation revealed the potential for considerable ethnic variation in the amount of protein expressed. Adult expression of the foetal isoform, CYP3A7, is associated with significantly altered levels of steroid hormones and may impact drug metabolism. A promoter variant, CYP3A7*1C, associated with adult expression of CYP3A7 was found at high frequency among populations of North Africa. Significant linkage disequilibrium was observed between CYP3A5*3 and CYP3A7*1C indicating that, within the populations studied, adult expression of CYP3A7 is likely to be accompanied by reduced/null expression of CYP3A5.

Caffeine is commonly used as a model hydrophilic compound in skin research, and it also has pharmaceutical and cosmetic applications. Several topical products containing caffeine are currently being marketed, including anti-cellulite creams and gels, moisturizers, serums and shampoos. As the skin permeation of this compound is expected to be low, the aim of this work is to investigate how the topical delivery of caffeine may be optimized using chemical penetration enhancers. A range of penetration enhancers for caffeine were selected, namely ethanol (EtOH), propylene glycol (PG), isopropyl myristate (IPM), propylene glycol dipelargonate (DPPG), oleic acid (OA), dimethyl isosorbide (DMI), 1,2-pentanediol (1,2-PENT), propylene glycol monocaprylate (PGMC), propylene glycol monolaurate (PGML), Transcutol-P (TRANS), isostearylisostearate (ISIS), isopropyl isostearate (IPIS), geraniol (GER), d-limonene (LIM) and 1,8-cineole (CIN). Solubility and miscibility studies were conducted using these solvents. Based on the miscibility and solubility data, caffeine permeation using infinite dose studies in human skin was evaluated. The studies were performed for caffeine in simple single solvents namely, PG, DMI, 1,2-PENT and IPM. The effects of using more complex binary solvent systems and ternary solvent systems of these solvents were also studied. The highest flux values were observed from the ternary solvent system PG:1,2-PENT:IPM (50:45:5 v/v), which enhanced caffeine permeation up to 24 times, 43 times and 7 times compared with the single solvents PG, 1,2-PENT and IPM, respectively. Following infinite dose studies in human skin, caffeine permeation was evaluated in finite dose studies. Because of the high number of experiments required, this section of the work was conducted with porcine skin, which was easier to source compared to human skin. Porcine skin was chosen over other skin models because from the literature it has been shown that this skin model is the closest animal model to human skin. The enhancement effects on caffeine permeation by binary and ternary solvent systems were also evaluated in this study. Following the finite dose studies, mass balance studies were conducted in order to understand the distribution of the actives in the skin. After 24 hours of finite dose studies, the permeation profiles did not reach a plateau. However, the studies could not be prolonged to 48 hours with porcine skin since the solvents damaged the skin barrier, as shown by a sudden increase in permeation. Therefore, the two best ternary solvent systems were selected. It was found that the same trend of caffeine permeation was observed (PG:DMI:IPM> PG:1,2-PENT:IPM) from the finite dose studies using porcine skin and that obtained from human skin. These formulations were further evaluated in in vivo studies. The formulations were applied on human volunteers in order to investigate the effect of the formulations on the skin barrier in vivo. These studies were carried out by analysing the trans-epidermal water loss (TEWL), Attenuated Total Reflectance Fourier Transform Infrared(ATR-FTIR) scans and protein content. The TEWL measurements from the PG:1,2-PENT:IPM treated site showedsignificantly higher TEWL values up to the removal of 15 tape strips (p<0.05). It was speculated that this may be due to irritation caused by the formulation, which results in damage to the skin barrier as shown by the high TEWL values. However, no further macroscopic changes in the skin barrier were observed using the ATR-FTIR. Further evaluations of the effects of the formulations on caffeine permeation were assessed by using the tape-stripping technique. A general trend of higher protein content was observedin the stratum corneum removed from the PG:1,2-PENT:IPM treated site, compared with the PG:DMI:IPM treated site and the control site. It was noted from this study that a higher caffeine amount was detected from the PG:1,2-PENT:IPM treated site compared tothe PG:DMI:IPM treated site. In conclusion, this thesis presents evidence that caffeine permeation can be enhanced by using combinations of chemical permeation enhancers in increasingly complex combinations. The effects of the formulation on the skin barrier in vitro were successfully elucidated by modelling the permeationdata. Furthermore, in vivo data provides further insight into the effects of the formulations on caffeine permeation and on the skin barrier, which will be useful in taking forward these formulations for use in cosmetic or pharmaceutical preparations.

Background: The management of pain in children is a common but difficult symptom to treat and the use of strong analgesics is often limited, for a variety of reasons, including fear of inducing side effects. Morphine is normally considered to be a centrally acting analgesic but recently, the beneficial effects of peripheral opioids have been demonstrated in adults for a number of painful inflammatory conditions. When administered in this non invasive way, opioids provide analgesia without achieving significant plasma concentrations and therefore it can be assumed that many of the adverse effects associated with oral or systemic opioids are avoided. Methods: A) The first part of the thesis describes the mechanism of action of peripheral opioids through development and particularly their effect during inflammation. This work was conducted using different ages of Sprague Dawley rat pups and skin inflammation was induced using carageenan. B) The second section is a description of a double-blinded, randomised-controlled, placebo-controlled trial with crossover design, to assess the efficacy of peripheral opioids in paediatric inflammatory pain. This was conducted in children with a diagnosis of Epidermolysis Bullosa (EB), as a model for acute and chronic inflammatory pain Results:. The laboratory study demonstrates that mu opioid receptor (MOR) expression is up regulated in neonatal plantar skin and significantly up regulated in neonatal lumbar dorsal root ganglion (DRG) four hours post hind paw inflammation, MOR protein levels in the rat hind paw plantar skin are significantly up regulated post-natally, and MOR protein levels are significantly up regulated in both neonatal and young adult plantar skin four hours post hind paw inflammation. Clinically, pain reduction was most significant with background pain Conclusion: The developmental regulation of peripheral MOR both in naive and inflamed cutaneous tissue may have implications for the use of topically / peripherally applied opioids in infants and children. "I, Gillian Watterson confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis".

Sensitivity to aspirin, non-steroidal anti-inflammatory drugs (NSAIDs) and also dietary salicylate is an increasingly important and observed phenomenon. The reliable diagnosis of individuals with these disorders is of great importance, given the use of these drugs as a prophylactic medication. Many of the symptoms seen in patients associated with this sensitivity to these drugs include dermatitis, nasal polyps, persistent cough, rhinitis, skin problems, stomach irritation, swelling of face and urticaria are also seen in salicylate intolerance. These symptoms are similar to those seen in the allergic patient, however, the reaction to salicylate is not an allergy. A major clinical problem is that no current method of food intolerance or allergy in vitro testing and diagnosis has currently been established to predict those people with this condition. This study was designed to search for new markers, which could be used to predict with high specificity and sensitivity, subjects with aspirin induced asthma (AIA). This was done by investigating patients with known aspirin sensitivity and comparing them with healthy individuals and atopic non-aspirin sensitive patients. All subjects used (male/females- age range 21-68 years old) were divided into 4 groups (atopic aspirin sensitive, non-atopic aspirin sensitive, atopic non-aspirin sensitive, and healthy volunteers) according to the clinical history and confirmed positive skin test to common allergens. Flow cytometer was used to assess the role of activated basophils in AIA and detect the expression of CD63 in their blood after stimulation of the cells with L-ASA. A posterior rhinomanometer was used to assess a challenge technique that was sensitive to local stimulation by L-ASA. Cytokine (IL-4) expression in lymphocytes was determined using ELISA and IL-4 messenger RNA levels investigated using RT-PCR. The release of PGF2α from plasma protein on those patients was detected by ELISA. The results obtained in this study, suggest that up regulation of CD63 expression by aspirin and Nasal airways resistance test (NART) following aspirin challenge, may prove to be useful diagnosis tool in these patients. The study also indicates that lymphocyte responses as evidenced by IL-4 release are not affected by salicylate sensitivity. The significance of these results is also discussed.

The blood-CNS barriers form a highly selective biological firewall; regulating the passage of solutes between the blood and cerebral microenvironment. This thesis modelled the blood-CNS barriers in vivo to determine the transport of nutrients, metabolic products and drugs across these barriers, and was split into two main parts: 1. L-arginine and ADMA transport - Circulating levels of the cationic amino acid L-arginine and its endogenously produced homologue asymmetric dimethylarginine (ADMA) are in delicately poised equilibrium. Dysregulation of this balance has well-documented implications in cardiovascular disease and also more recently conditions of the brain, as ADMA is a potent inhibitor of nitric oxide synthase (NOS) enzymes, including the endothelial (eNOS) and neuronal (nNOS) isoforms. Until now it has been postulated that ADMA competes for transport across membranes via the same cationic transport system as L-arginine (system y+), however this hypothesis has surprisingly never been tested. The major contributors to transport of both amino acids were investigated to determine how their respective intracellular and extracellular concentrations affect transport across the blood-CNS barriers. These data also have strong relevance to other research areas due to the widespread influence of NO in physiological pathways in health and disease. At the time of writing, this study represents the first true characterisation of ADMA transport across any physiological membrane in vivo and reveals a likely mechanism for explaining ‘the L-arginine paradox’ – the clinical observation that NO-deficient patients respond well to oral supplementation with L-arginine even though [arginine]plasma is easily sufficient to saturate eNOS. 2. ‘NanoHAT’ - Efflux transporters expressed at the blood-CNS barriers remain one of the biggest hurdles for the efficient delivery of therapeutic agents to the brain. The anti-trypanosomal drug pentamidine was shown previously by our group to be a substrate for efflux from the blood-CNS barriers and does not ordinarily accumulate to pharmacologically relevant levels in the brain. Pluronic® copolymers are known biological response modifiers that are approved for use in humans and have generated a great deal of interest due to their ability to inhibit efflux transporters and spontaneously form micelles in solution. In this study, a coformulation of pentamidine and Pluronic® P85 was evaluated as a potential future treatment for CNS-stage Human African Trypanosomiasis, using a combination of in silico, in vitro, and in vivo methodologies. Combining our group’s knowledge and expertise in blood-brain barrier research with colleagues in Molecular Biophysics and Materials & Molecular Modelling has resulted in the creation of a ‘mini formulation-development pathway’ that can be utilised to optimise and develop formulations in the future.

Little is known about the actions of the cannabinoid CB, receptor antagonist SR141716A on food intake and body weight, or the behavioural mechanisms that underlie these effects. The aim of the experiments presented in this thesis is to explore the influence of SR141716A on some of the signals that modulate feeding behaviour and help determine meal characteristics. These include: reward and motivational processes, hedonic processes and the development of satiety. Further experiments analysed interactions with serotonin, a neurotransmitter system important in appetite regulation, and effects on the adiposity signal adiponectin, which is involved in homeostatic control of energy balance. Using a novel operant schedule I studied the effects of SR141716A on reward processes initiated prior to and during feeding. These data are presented in chapter 2. Treatment with SR141716A suppressed both appetitive and consummatory components of feeding behaviour. This argues strongly for an involvement of SR141716A in the motivational aspects of consumption. Microstructural data presented in chapter 3 support this argument and suggest a role of the cannabinoid system in motivational processes associated with meal initiation änd satiation. In the experiments presented in chapter 4 differences between the effects of SR141716A on carbohydrate and fat diets are highlighted suggesting specific effects on fat consumption. Using both pharmacological tools and genetically modified animals, the experiments of chapter 5 investigated interactions between cannabinoids and serotonin, specifically, the CB1,5-HT, B and 5- HT2C receptors. The results revealed no functional interactions between the two systems with regards to feeding. The effects of chronic drug administration on body weight and food intake in an animal model of obesity, and the extent to which these effects were attributable to hypophagia, were determined and the data presented in chapter 6. Molecular biology techniques were employed to establish whether SR141716A acts on adipose tissue to modulate the adiposity signal Acrp30.

This study describes the organisation and characterisation of the genes involved in the polyketide antibiotic pseudomonic acid (mupirocin) biosynthesis by Pseudomonas fluorescens NCIB10586. Suicide vector and PCR strategies were used to isolate and clone chromosomal DNA segments flanking a 17.1 kb fragment which was previously cloned. The sequence analysis of the 83.993 kb from the whole DNA fragments obtained by these strategies revealed the involvement of approximately 74.5 kb genomic DNA in the mupirocin biosynthesis. The study predicted that this region encodes the whole biosynthetic pathway, since the disturbance of the upstream and downstream region of the 74.5 kb does not affect mupirocin production where many suicide plasmid insertions within the 74.5 kb region disturbed the biosynthesis. The sequence analysis revealed two sets of tRNA genes immediately upstream of the mup operon indicating that the mupirocin gene cluster might be integrated within the P. fluorescens genome like a pathogenicity island. The translated amino acid sequence revealed a unique order of biosynthetic genes comprising a mixture of (PKS) typel and typell systems. Four main large open reading frames designated orfl, orflI, orflII and orflV encoded multifunctional enzyme complexes resembling type I systems of polyketide synthases (PKS) and fatty acid synthases (FAS). Additional individual genes were identified within the mup cluster as type 11 systems. Some other genes were also identified as tailoring genes probably involved in modification functions such as oxidation, reduction, hydration and ligation. Also a putative mupirocin resistance gene which encodes isoleucyltRNA synthetase activity was identified within the cluster. In an attempt to accumulate intermediate products during mupirocin biosynthesis, in-frame deletions within some domains and genes involved in mupirocin biosynthesis were performed using the suicide vector strategy. This would help in studying the catalytic activity of these genes and subsequently understanding how mupirocin could be assembled. The regulation of mupirocin biosynthesis was first described in this study by the discovery of the mupirocin regulatory genes mupR and mupI. The regulation of mupirocin is controlled via a quorum-sensing system similar to LasR/LasI and LuxR/LuxI systems. A reporter gene fusion and deletion mutation studies were performed in order to determine the effect of mupR and mupI on the putative promoter region of mup operon and consequently on mupirocin productivity. According to information extracted from the putative activities of the identified domains and genes, the predicted pathway of mupirocin assembly was postulated.

Neurosteroids are important endogenous modulators of the major inhibitory neurotransmitter receptor in the brain, the γ-amino-butyric acid type A (GABAA) receptor. They are involved in numerous physiological processes, and are linked to several central nervous system disorders, including depression and anxiety. The neurosteroids allopregnanolone and allo-tetrahydro-deoxy-corticosterone (THDOC) have many effects in animal models (anxiolysis, analgesia, sedation, anticonvulsion, antidepressive), suggesting they could be useful therapeutic agents, for example in anxiety, stress and mood disorders. Neurosteroids potentiate GABA-activated currents by binding to a conserved site within α subunits. Potentiation can be eliminated by hydrophobic substitution of the α1Q241 residue (or equivalent in other α isoforms). Previous studies suggest that α2 subunits are key components in neural circuits affecting anxiety and depression, and that neurosteroids are endogenous anxiolytics. It is therefore possible that this anxiolysis occurs via potentiation at α2 subunit-containing receptors. To examine this hypothesis, α2Q241M knock-in mice were generated, and used to define the roles of α2 subunits in mediating effects of endogenous and injected neurosteroids. Biochemical and imaging analyses indicated that relative expression levels and localization of GABAA receptor α1-α5 subunits were unaffected, suggesting the knock-in had not caused any compensatory effects. Electrophysiological characterization of cells in hippocampal and nucleus accumbens brain slices revealed faster-decaying inhibitory synaptic transmission in α2Q241M mice. Furthermore, the response to applied THDOC was markedly reduced compared to wild-type cells. α2 subunits therefore formed a major component of synaptic GABAA receptors in these areas. The α2Q241M knock-ins showed greater anxiety levels in two classical rodent anxiety paradigms (light-dark box and elevated plus maze), consistent with endogenous neurosteroids mediating anxiolysis via α2-type GABAA receptors. In addition, the anxiolytic response to injected THDOC is impaired by the α2Q241M mutation, which would identify the α2 isoform as an appropriate target for generating receptor subtype-selective neurosteroid therapeutics for anxiety disorders.