Neuronal death is induced by a series of pathogeneses in different neurodegenerative diseases, and one of them, which has been widely accepted previously, is the overload of intracellular calcium (Ca2+) in neurones. This study has investigated ryanodine receptor (RyR) on the endoplasmic reticulum (ER) and several potassium (K+) channels which might be neuroprotective, such as the large conductance Ca2+-activated K+ channel (BK) and adenosine-5’-triphosphate (ATP)-sensitive K+ channel, in both neuronal and astrocytoma cell lines. The reverse transcription (RT)-polymerase chain react (PCR) results in this Thesis show that the messages of RyR, BK channel and ATP-sensitive K+ channel (KATP) exist at the messenger ribonucleic acid (RNA) level in those cell lines. And, the expression of RyRIII message was found being increased in SH-SY5Y cells but decreased in NTERA-2 cells after differentiation. The BK channel was confirmed as functional in SH-SY5Y cells with patch clamp recording. Cell insults which increase intracellular Ca2+ ([Ca2+]i), such as cobalt (II) chloride (CoCl2), hydrogen peroxide (H2O2), beta-amyloid (Aβ1-42) and glutamate, were found to reduce cell number in those cell lines in cell proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assays, and the cells with higher expression of RyRIII message were more sensitive to CoCl2 and H2O2 insults. In cell proliferation assays testing RyR or K+ channel modulators in the presence of insults, it has been found that generic blockade of RyR or K+ channel might be neuroprotective, and the activation of RyR or BK channel and the blockade of KATP channel may aggravate the insults. Selective blockade of RyRI and RyRII cannot protect the cells, which probably indicates that RyRIII is the key target in neuroprotection. Activators of the KATP channel cannot protect the cells at a low dose of CoCl2 but might be protective at a high dose, although cromakalim was an exception. Hence, blockade of RyRIII and the BK channel, and activation of the KATP channel, could be possible neuroprotective strategies. Future study should measure [Ca2+]i and cell apoptosis through Ca2+ imaging and fluorescence-activated cell sorting (FACS) respectively, and design selective a RyRIII blocker.

The genesis and outgrowth of pituitary adenomas is consequent to the combined contributions of genetic and epigenetic aberrations. Although the relative contribution of each is not known it is likely that particular aberration may be common or particular to an adenoma subtype(s). The impact of subtype specific changes presents significant challenges for clinical management options. In this thesis the potential impact of epigenetic aberrations, that target the Dopamine D2 Receptor (D2R) and the cytokine, Bone Morphogenic Protein 4 (BMP-4) were investigated. In these cases their expression patterns are frequently comprised in a subtype-specific context. This thesis reports that in pituitary cell lines, reduced expression of D2R and that of BMP4 are associated with CpG island methylation and histone modifications indicative of gene silencing. In these cases, incubation of cells with epidrugs, designed to reverse epigenetic silencing, restores their expression.

The reduction of bacteria on the skin results in prophylactic and therapeutic benefits by reducing the occurrence of skin infections. Currently, chlorhexidine digluconate, a conventional topical antiseptic, permeates the skin poorly leaving viable opportunistic pathogens below the superficial layers of the stratum corneum. The aim of this study was to use polyamidoamine (PAMAM) dendrimers to enhance the topical delivery of chlorhexidine digluconate to improve its antimicrobial efficacy.

This thesis is based on a critical re-appraisal of Richard Titmuss' classic formulation of gift relationships, which has long been a point of reference for thinking about blood donation in Britain. It argues that Titmuss' interest in the intersections of social systems and health care, together with his concern with mutuality, has been lost in the characterisation of blood donation as a uniquely altruistic activity. This argument is applied to some key assumptions about blood donation in Britain in the thesis, which considers their historical and political contours, and interrogates them in the light of the development of large biobanks which require blood samples for genetic research. In examining the revival of this 'mobilising metaphor' for genetic biobanks, interview data from UK National Blood Service donors and with others donating blood for a genetic research project is generated and analysed. This reveals that the notion of gifted blood has considerable acuity in summoning up social allegiances based on a sense of community. It is suggested however that mutuality (not one-dimensional altruism) is the model implied by these participants' stance to blood donation or participation in research. This resonates with the re-evaluation of Titmuss' work, in which debates about practical mutual provision and social insurance are more prominent than is generally acknowledged. Biobanks, as with blood banks of a traditional kind, are bound up with an assertion of common interests. The tacit use of notions of gifted blood and solidarity in the context of contemporary policies on biobanks are revealed as problematic. The thesis concludes by underlining the importance of having an explicit political debate about the UK Biobank, and of developing mechanisms to negotiate and protect the collective interests to which it refers.

P-glycoprotein is an ATPase binding cassette (ABC) drug-transporter protein represented by MDR1 in humans and mdr1a and mdr1b in mice. The substrate specificity of this protein is unusually large due to the polyspecific nature of its binding pocket and as a result a wide range of endogenous and exogenous compounds are effluxed by this transporter. There is a large body of literature about the role of P-glycoprotein in limiting the absorption of drug substrates at a number of barriers but currently data in the lung is limited. P-gp expression and localisation studies have confirmed its presence in lung tissue but there is conflicting data with regards to the functional relevance on lung absorption. The aims of this project were to establish a knockout, mdr1a/b (-/-), isolated perfused mouse lung model (IPML) to investigate the role of pulmonary P-gp upon lung delivery. Utilising the lungs of knockout mice in the IPML allowed the investigation of P-gp function with the effects of chemical inhibition which may have resulted in the discordance observed in its functional significance in the literature. IPML experiments were conducted with a panel of 18 P-gp substrates which were chosen for diversity in their physicochemical characteristics. The discordance in P-gp functionality in the lung observed in the literature was replicated in the IPML model. Ten of the substrates showed significant efflux from the lung by pulmonary P-gp whereas the absorption of the remaining eight was unaffected. Further studies into the mechanism behind the differential effects of pulmonary P-gp showed that it was a barrier specific effect as it was not observed in the intestine. The effect was also not explained by differences in the P-gp binding kinetics off the substrates. Investigations in to the membrane affinity of the panel revealed a potential mechanism for the selectivity of P-gp in the lung with regards to the passive permeability of the molecules and specifically the paracellular pathway.

Chronic pain {CP} is a major health problem that affects about 20% of adults worldwide. Unlike acute, physiological pain, which resolves promptly once the painful stimulus is removed, CP can last for months, years or even a lifetime. CP, which includes inflammatory pain {IP} that is associated with both tissue injury and the accompanying inflammation and peripheral neuropathic pain {NP} that is a direct consequence of a lesion or disease affecting the peripheral nervous system {PNS}, often is a constant burden, degrades peoples’ quality of life, and costs billions of pounds. Patients with CP usually complain of either: a) continuous or intermittent spontaneous, un-provoked pain; or b) hypersensitivity, due to either increased pain from a stimulus that normally provokes pain or pain due to a stimulus that does not normally provoke pain. Successful therapy for CP, particularly NP, remains a challenge because the currently available drugs are largely ineffective and many result in adverse side effects. Therefore, there is a pressing need to understand the pathophysiology of CP, in order to devise appropriate palliative and curative strategies. CP is believed to be due, at least partly, to increased excitability of normally quiescent dorsal root ganglion {DRG} neurons, which convey sensory information from the periphery to the central nervous system {CNS}. However, the underlying ionic and molecular mechanisms of this neuronal hyperexcitability and spontaneous activity {SA} are poorly understood. The aim of this research project was to examine the hypothesis that during peripheral CP states, hyperexcitability in DRG neurons could be due to increased expression of hyperpolarisation-activated cyclic nucleotide-gated {HCN} channels, possibly in combination with a change in their activation properties. This is because these channels, which are composed of 4 subunits {HCN1-4}, produce an excitatory inward current, termed the hyperpolarisation-activated {Ih} current in neurons, that depolarizes the membrane potential toward the threshold of action potential {AP} generation. To test this hypothesis, several integrated approaches, including behavioural pharmacology, in vivo electrophysiology, and immunofluorescent staining, were used in two rat models of CP that were compared to appropriate controls. The rat models of CP were: a) chronic inflammatory pain {CIP} model, which involved induction of hindlimb inflammation with complete Freund’s adjuvant {CFA}; and b) chronic neuropathic pain {CNP} model that involved L5 spinal nerve {SN} axotomy, in addition to loose ligation of the L4 SN with neuro-inflammation inducing chromic gut, referred to as modified SN Axotomy {mSNA}. The objectives of the current project were to: (i) evaluate, using behavioural pharmacology, the influence of modulating the HCN channels with the Ih-specific blocker, ZD7288, on pain hypersensitivity in both CIP and CNP. (ii) determine, using in vivo intracellular voltage and current recordings, the difference in AP parameters between normal and mSNA-treated L4 DRG neurons and the effect of ZD7288 on SA in the mSNA-treated L4 DRG neurons. (iii) determine, using immunofluoresence, the types of DRG neuron that express HCN1-HCN3 subunits in normal rats and whether expression of these subunits is altered in both CIP and CNP. The results showed: 1. In both CIP and CNP, peripheral administration of ZD7288 resulted in significant attenuation of mechanical hypersensitivity and a non-significant absence of spontaneous pain {SP}. 2. In the L4 DRG neurons of mSNA-treated animals with CNP, ZD7288 had no effect on the frequency of SA from low-threshold mechanoreceptors {LTM} and induced changes in hyperpolarisation-associated AP parameters in Aα/β-fibre DRG neurons. 3. In both CIP and CNP, an increased proportion of small and medium sized DRG neurons express HCN2, but not HCN1 or HCN3, channel protein. Taken together, the findings suggest that HCN channels, particularly HCN2, in specific sub-populations of DRG neurons contribute to the development!of CIP and CNP.

NR678, a small thiourea-based rodenticide candidate, is lethal to rats when administered orally at relatively small doses (5 and 10 mg/kg) and causes rapid life-threatening respiratory impairment, characterised by severe hydrothorax and pulmonary oedema. However, rats are protected from a normally lethal dose of NR678, after prior exposure to a low dose (0.5 mg/kg). Similar to other molecules containing the thiourea moiety, NR678 is mainly metabolised by flavin containing monooxygenases (FMOs), a class of enzymes involved in the detoxification, or more rarely, bioactivation of N- or S-containing molecules during phase I metabolism. Rat FMO2, the main FMO expressed in the lungs, like the correspondent human isoform, exhibits a genetic polymorphism which may influence the responses of this organ to NR678 and other drugs metabolised by FMOs. The aims of this thesis were to assess the morphological and functional aspects of NR678-induced acute lung injury (ALI) and to investigate the role of the adaptive pulmonary defence response involved in the prevention of the oxidative injury. In addition, the wild rat, which, in contrast to the laboratory rat, possesses a functional pulmonary FMO2, was explored as a possible animal model to evaluate the metabolic and toxicologic consequences of FMO2 polymorphism in humans. It was shown that pulmonary endothelial cells are the main target of acute NR678 oxidative injury and exhibit ultrastructural alterations, which, coupled with a marked depletion of glutathione (GSH) levels, are likely responsible for the perturbation of pulmonary vascular permeability. The lungs of tolerant animals showed mild and transient changes of the vascular permeability and mildly increased cellularity in the lungs, which was characterised by increased numbers of alveolar macrophages and immature pneumocytes within 24 h after dosing, followed by a rise in the number of mature type II pneumocytes one week later. The microscopic changes resolved by the end of the second week, when rats were found to be again susceptible to a lethal dose of NR678. It was speculated that the adaptive response of the lungs to NR678 could be related to irreversible inhibition of FMOs, rather than increased clearance of the oedema fluid by macrophages and type II pneumocytes, down-regulation of FMOs, evaluated by in situ hybridisation and qPCR, or decreased GSH levels. In addition, it was found that the presence of an active FMO2 isoform enhances pulmonary FMO catalytic activity and leads to increased susceptibility to the toxic effects of NR678. In conclusion, the work presented here contributes evidence that FMO2 polymorphism may be relevant to humans and provides an animal model which can be used to study its implications. NR678 represents an interesting and unique approach to investigate the pathogenesis of ALI and, at the same time, understanding the defence mechanisms involved in adaptation may bring new insight into potential therapeutic strategies of lung diseases.

The transporter hypothesis has been postulated to explain pharmacoresistance in epilepsy. Despite over a decade of research surrounding the drug transporter hypothesis, the role that solute carrier (SLC) transporters might play in this theory remains largely unaddressed. Hence, the major focus of this thesis was to investigate and identify SLC transporter systems of interest that are expressed at the blood-brain barrier (BBB) and to determine which, if any, of commonly prescribed antiepileptic drugs (AEDs) are substrates for such transporter systems. Characterisation of AED transport was undertaken using widely reported model systems such as Xenopus laevis oocytes and the human cerebral microvascular endothelial cell line (hCMEC/D3), together with novel stably-transfected MDCK II cell lines. Organic anion transporter 1A2 (OATP1A2), the monocarboxylate transporter (MCT) family and the organic anion transporter (OAT) family were specifically selected for investigation. Valproic acid and gabapentin showed the greatest evidence for SLC-mediated transport by OAT1/OAT3 and MCT1 respectively, while other compounds were largely unremarkable in this respect. Valproic acid transport increased OAT1 overexpressing cells compared to control but decreased in OAT3 overexpressing cells. Gabapentin uptake increased in MCT1 transfected Xenopus laevis oocytes and was shown to decrease in hCMEC/D3 cells in the presence of a panel of MCT inhibitors. The induction/suppression of expression of SLC transporters by AEDs was explored in the hCMEC/D3 cell line, in an attempt to understand how AEDs might influence the functionality of endogenous transport pathways. A number of AEDs were observed to induce/suppress expression of transporter genes involved in transport and detoxification. A further study explored the fundamental physiochemical properties of AEDs, which is relevant to their penetration into the brain. A number of AEDs, including lamotrigine, gabapentin and topiramate, observe adequate uptake in the hCMEC/D3 model of the BBB despite having physiochemical properties, such as a high polar surface area and negative log D value which may limit passive entry into the brain. This would suggest that a carrier mediated system may be involved in the uptake of these drugs into the brain. The work described in this thesis has shown that a number of AEDs may be subject to carrier mediated uptake into the brain. Individual differences in transporter expression at the BBB may be responsible for variability in brain concentrations of AEDs. However, at present, this does not provide us with an adequate explanation for why some people with epilepsy experience pharmacoresistant seizures.

Malaria remains a major public health concern for billions of people worldwide. Achieving the ambitious goal of malaria eradication requires co-ordination of control strategies dealing with a range of parasite, vector, human, social and environmental factors. Availability of effective antimalarial treatment is a key component in malaria control. However the number of drugs available is limited and drug resistance, particularly in Plasmodium falciparum, has now been reported for all currently available antimalarials. Mathematical models provide the opportunity to explore key features underlying antimalarial drug action, effectiveness and resistance. They further allow investigation into questions that cannot otherwise be easily addressed, either because they are too expensive, unethical or logistically too complex. This thesis aims to develop pharmacological models to investigate antimalarial drug treatment. In Chapter 2 we develop a pharmacokinetic-pharmacodynamic (PK/PD) model of antimalarial drug treatment (calibrated using published data) and use it to investigate the efficacy of artemisinin combination therapies (ACTs). Chapter 3 addresses two assumptions built into the methodology that limit the models future application. The model now allows for (i) time lags and drug concentration profiles for drugs absorbed across the gut wall and, if necessary, converted to another active form (ii) multiple drugs within a treatment regimen (iii) differing modes of drug action in combinations (iv) modelling drugs converted to an active metabolite with similar modes of action. In Chapter 4 we extend the methodology to allow for i) the presence of more than one clone when treatment begins (ii) the acquisition of new clones during treatment follow-up (iii) the tracking of individual clones using molecular markers. We then use these extensions to simulate clinical trial data to determine the best methods of analysis. Chapter 5 details how the drug action components of the extended PK/PD model were incorporated into OpenMalaria; a mathematical model of malaria epidemiology allowing investigation of the effects of various intervention strategies including malaria vaccines, vector control strategies and antimalarial drug treatment. In Chapter 6 we investigate the ability of clinical trials to accurately estimate (WoS) using the extended PK/PD model. Windows of selection (WoS) are often used to quantify the genetic process whereby parasites evolve increasing tolerance to antimalarial drugs. We noted a conspicuous lack of comprehensive, good-quality PK datasets currently available in the literature. Despite this, the models produced results highly consistent with field data. They were applied to investigate the potential implications of drug resistance and to make predications about the future effectiveness of antimalarials. We emphasise the value of mathematical models by simulating ‘field data’ to assess the best methods of analysing clinical trials and to investigate the predictive ability of WoS. While we do not suggest models can replace the information gained in clinical trials, this work does demonstrate the importance of mathematical models capable of generating results consistent with field data.

Protein therapeutics or biologics represent 30 % of current licensed pharmaceutical products. In general, biologics offer superior safety profiles compared to small molecules. However, significant clinical concerns have emerged in terms of development of anti-drug antibodies (ADAs), a phenomenon that is covered under the term, immunogenicity. Anti-drug antibodies can alter pharmacokinetics, reduce efficacy of the therapeutic and also can in some cases induce allergic reactions. Human recombinant interferon beta (IFN-β) is a biologic used for the treatment of multiple sclerosis (MS) – a chronic, inflammatory and demyelinating disease of the central nervous system. Long-term treatment with IFN-β has been shown to lead to the development of anti-IFN-β antibodies that can cause total loss or reduced efficacy. Anti-drug antibodies can be non-neutralising (N-NAb) and neutralising (NAb) depending on the site to which they bind. This study aimed to conduct a systematic review to determine factors affecting the formation of neutralising antibodies against three different formulations of IFN-β Avonex™, Rebif™ and Betaseron/Betaferon™. Findings from the systematic review highlight the relative differences in immunogenicity risk of different IFN-β formulations Avonex™, Rebif ™ and Betaseron/Betaferon™ with Avonex™ having the lowest risk, Rebif™ has moderate risk and Betaseron/Betaferon™ has high risk. Characterising the immunoglobulin profile of the IFN-β ADAs from the plasma of ADA positive MS patients revealed that IFN-β ADAs are predominantly of the IgG1 and IgG4 subclass. We also characterised the neutralising potential of the major ADA IgG4 subclass using a IFN-β bioactivity assay and show that IgG4 antibodies may likely contribute to the neutralisation activity. The potential of the neutralising ADAs to cross-react with endogenous IFN-β was investigated using an in vitro bioactivity assay. Findings from this set of experiments revealed varying degrees of neutralisation of endogenous IFN-β. We next explored the potential immunological consequence of ADA with regards to formation of immune complexes and activation of complement. The interaction of ADAs with the biologic can result in formation of immune complex. Immune complexes can activate the complement system. The data revealed IFN-β ADAs can form immune complexes with IFN-β and therefore activate complement. We also attempted to identify IFN-β linear epitopes that the ADAs had the ability to bind. However, a combination of multipin peptide technology and in vitro peptide competitive binding assay failed to reveal a definitive linear epitope although there was some evidence for the existence of potential linear epitopes. We also examined the involvement of T helper cells and T regulatory cells (Tregs) in ADA development. The data revealed no significant differences in the frequency of Tregs among IFN-β ADAs positive, negative and healthy donors. Attempts were also made to identify T helper epitopes within IFN-β that could potentially drive immunogenicity. Using T-cell epitope prediction tools (IEDB-AR and ProPred) and T-cell functional assays we identified an immunogenic sequence of 36 amino acids within IFN-β (position 130-166). Our data revealed that one IFN-β peptide within this sequence is a potential T-cell immunogenic epitope. In addition we identified a possible association of one IFN-β derived peptide with the DRB1*1501HLA haplotype. In summary, the results presented in this thesis have provided essential information on subclass profile of IFN-β ADAs, the possible involvement of T helper cells and potential antibody epitopes within IFN-β. Future studies should be aimed at providing greater detail on the evolution of the ADA response and test strategies to remove immunogenic determinants from IFN-β.