The efficient encapsulation of active ingredients within formulated products and their controlled, targeted delivery to the sites of action, is very important to a range of industries such as pharmaceuticals, agrochemicals, home and personal care and cosmetics to name but a few. By successfully stopping the release of active chemicals and triggering their release where and when they are needed, product efficiency can be improved which reduces the required dose, lowering costs, environmental impact and/or side effects. Such active chemicals include pharmaceutical drugs, pesticides, fragrant and flavour oils, enzymes, vitamins. Encapsulation and full retention of small molecular weight actives is a particularly challenging task that remains unsolved by current technologies used in industry and academia. In particular, certain everyday product formulations provide difficult environments in which preventing active leakage through capsule walls is not feasible. For example, a continuous phase that can fully dissolve an encapsulated active will typically force full release over a fraction of the intended lifetime of a product. This is due to the inherent porosity of polymeric membranes typically used as capsule wall materials in current technologies. In this work, a method for preventing undesired loss of encapsulated actives under these extreme conditions using a simple three step process is developed. The developed methodology forms an impermeable metal film around polymer microcapsules, prevents loss of small, volatile oils within an ethanol continuous phase for at least 21 days while polymeric capsules lose their entire content in less than 30 min under the same conditions. Polymer shell−oil core microcapsules are produced using a well known cosolvent extraction method to precipitate a polymeric shell around the oil core. Subsequently, metallic catalytic nanoparticles are physically adsorbed onto the microcapsule polymeric shells. Finally, this nanoparticle coating is used to catalyse the growth of a secondary metallic film. Specifically, this work shows that it is possible to coat polymeric microcapsules containing a model oil system or typical fragrance oil with a continuous metal shell. It also shows that the coverage of nanoparticles on the capsule surface can be controlled, which is paramount for obtaining a continuous impermeable metal film. In addition, control over the metal shell thickness is demonstrated without altering the capability of the metal film to retain the encapsulated oils. In addition, a method to grow a continuous, non-porous metallic film directly onto nanoparticle stabilised Pickering emulsion droplets is demonstrated, negating the need for an underlying polymeric shell.

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Standardised [cAMP]_iand [Ca²⁺]_iassays (96-well plate) were used to directly compare hVPAC/PAC receptor pharmacology in CHO-K1 cells stably expressing the receptors, with additional studies evaluating cell lines with endogenous receptor expression. A range of peptide agonists were utilised in these studies, including non-selective (e.g. PACAP-27) and classical ligands (e.g. VIP), as well as recently described (e.g. R3P65) and highly selective compounds (e.g. maxadilan). The agonist rank order of potencies were identical between assays for each receptor in all of the cell lines examined, with EC₅₀ values consistently ~ 100 fold lower in the [Ca²⁺]_iassay. The pharmacology of the reportedly selective peptide antagonists PG7-269 (VPAC₁), PG99-465 (VPAC₂) and M65 (PAC₁) was also examined, with these studies identifying complexities in the pharmacology of PG99-465. The standardised assay conditions and the excellent correlation between the [cAMP]_iand [Ca²⁺]_idata, underpinned the establishment of an HT [Ca²⁺]_iassay (384-well plate) which was used to identify hVPAC receptor selective ligands from the compound libraries of the Fujisawa Pharmaceutical Company. Approximately 100,000 compounds were tested, with several non-peptides identified as potential ligands. The potency and selectivity of these compounds were subsequently fully characterised using the 96- and 384-well [Ca²⁺]_iassay formats, although none were as potent as the peptides examined in chapter one. In the final section of this thesis, putative roles for VPAC/PAC receptors in the brain were investigated. Firstly, as VIP and PACAP are thought to stimulate the release of cytokines from astrocytes in the brain, VPAC/PAC receptor pharmacology was examined in primary cultures of rat cortical astrocytes (RCA). Receptor characterisation studies ([cAMP]_iassays) established that the PAC₁ was the predominant VPAC/PAC receptor subtype in RCA, with preliminary studies also showing that VIP and PACAP-27 stimulated the release of the cytokines IL-1β and TNF-α from these cells. In addition, behavioural studies using mice further examined the role of the VPAC₂ receptor in the maintenance of circadian activity rhythms.

We studied the expression of genes regulating steroid sensitivity in the healthy Wistar rat and found genes were differentially expressed along the length of the colon. Notably, P-gp expression was increased in proximal compared to distal colon. A reverse gradient was noted for GR. Systemic dexamethasone treatment as well as endogenous corticosterone were shown to regulate these genes, and thereby these data support a role for steroids regulating tissue sensitivity to steroids. To further clarify the role of bacteria and disease on expression of P-gp and GR, HLA-B27 transgenic and non-transgenic rats were studied. Colonic inflammation in HLA-B27 transgenic rats decreased P-gp and GR. Bacteria were also shown to regulate expression of these genes in a site-specific manner along the colon. These data emphasise the complex gene-bacterial interactions within the colon in health and disease. Dexamethasone administered to rats with/out inflammation differentially regulated GR expression in a site-specific manner along the colon. Steroid treatment did not alter P-gp expression in the inflamed colons. These data show the complexity of the intestinal regulation of these genes in the inflamed and non-inflamed colon; corticosteroid treatment may have selected efficacy in different regions of the colon. Collectively these data suggest a role for dexamethasone treatment as well as bacteria in the regulation of genes determining steroid sensitivity in the healthy and diseased rodent intestinal epithelium. The complex interaction between P-gp and GR expression in response to bacteria has implications for potential mechanisms by which inflammation is induced in the colon.

Hypoxia sensitivity of STREX has previously been shown to be dependent upon a cysteine motif (-CSC-) in the STREX insert that is also within a putative phosphorylation motif. This thesis thus had three main aims: 1) to develop a high throughput assay to allow discrimination of different BK channel splice variants and their modulation 2) to characterise the role of cysteine residues within the STREX exon that may determine hypoxia sensitivity of the channel, and finally 3) to examine the modulation of the hypoxia response by intracellular ATP. During the course of these experiments the monovalent ion lithium (Li⁺) was observed to robustly block the STREX response to hypoxia. Li⁺ shares physiochemical properties with the divalent magnesium (Mg²⁺) ion hence the role of Mg²⁺ in the STREX hypoxia response was also investigated. Mg²⁺ modulated the response in a similar fashion to ATP. In addition it was seen that Li⁺ and Mg²⁺ decreased the single channel conductance of STREX and ZERO variants, Mg²⁺ also increased STREX channel open probability. However, while Li⁺ and Mg²⁺ may share similar mechanisms/sites to modulate channel conductance the differential effect of Li⁺ and Mg²⁺ on the hypoxia response suggest Li⁺ acts via a site/mechanism distinct from that of Mg²⁺. Taken together, these data suggest that alternative pre-mRNA splicing is an important determinant of BK channel pharmacology and that cysteine and other residues within the STREX insert play a key role in function of the channel and its response to hypoxia.

The aim of this thesis is to investigate the use of NMDAR antagonists in preventing naturopathic sensitisation. The chronic constriction injury (CCI) model of neuropathic pain is used to study the role of NMDARs in the development of neuropathic pain and subsequent modulation. Behavioural effects are assessed in association with changes in NMDAR subtypes. Memantine and ketamine (NMDAR antagonists) are shown to attenuate typical behavioural responses (thermal hyperalgesia and cold allodynia) to nerve injury. In addition, NMDAR antagonist pre-treatment is shown to effect the subsequent NMDAR subunit expression, with improved susceptibility to subsequent NMDAR antagonist treatment. The clinical use of epidural ketamine as a preventative drug prior to lower limb amputation is investigated in a double blind randomised placebo controlled study. No significant effects on the incidence of post-amputation pain were found, although the overall incidence of pain was lower than in comparable studies. Ketamine is shown to improve peri-operative analgesia and have long lasting effects (up to one week) on sensory processing in the remaining stump. In summary, NMDAR antagonists seem to be effective in attenuating neuropathic pain in animal models. The promise shown in these studies has not translated into a reduction in post-amputation pain in a clinical study. Ketamine remains a clinically useful drug in peri-operative pain management but the role of ketamine and other clinically available NMDAR antagonists in the prevention of neutropathic sensitisation is still not clearly defined.

In obese versus lean Zucker rats, hepatic 5α-reductase type 1 mRNA expression and protein levels were increased. They also had increased activity of hepatic 5β-reductase activity. By contrast, 3α-hydroxysteroid dehydrogenase mRNA expression was unchanged in obesity. Greater inactivation of B by A-ring reductases in liver may decrease local corticosterone (B) concentrations in these sites, and increase the metabolic clearance rate of glucocorticoids, thus increasing drive to the hypothalamic-pituitary-adrenal axis (HPA). To investigate whether 5α-reduced metabolites of corticosterone are glucocorticoid receptor agonists, competition binding studies were carried out. In displacing tritiated dexamethasone from binding sites in hepatocytes from male lean Zucker rats, B and 5α-tetrahydrocorticosterone (5αTHB) had similar affinities which were greater than 5α-dihydrocorticosterone (5αDHB) and 5β-reduced metabolites. Binding of B and 5αDHB binding was impaired in obesity whereas 5αTHB binding was unaltered suggesting that 5αTHB may modulate GR activation in obesity. Activation of flucocorticoid receptors was assessed following transient transfection into HeLa cells with an MMTV-luciferase reporter. By comparison with corticosterone, 5αTHB was active and additive. 5β-Reduced metabolites did not activate glucocorticoid receptors. In addition, in H4IIe cells which express endogenous glucocorticoid receptors, 5αTHB induced tyrosine aminotransferase mRNA expression albeit to a lesser extent than corticosterone. 5αTHB was also found to possess glucocorticoid activity <i>in vivo</i> as suppression of plasma ACTH was demonstrated in adrenalectomised lean Zucker rats following i.p. administration of B or 5αTHB. We conclude that hepatic A-ring reduction is enhanced in the obese Zucker rat producing increased concentrations of 5αTHB. Transcription of glucocorticoid regulated genes in tissues which express 5α-reductases will thus be influenced by intracellular levels of both corticosterone and its 5α-reduced metabolites. Manipulation of this enzyme may prove to be a useful therapeutic target in obesity.

The prognosis for patients with lung cancer remains extremely poor. Newer, more effective treatment regimens are required. In this thesis, the effectiveness of combining human recombinant interferon alpha with various estalished cytotoxic agents was assessed in laboratory models of human lung cancer and a clinical study. The majority of the experimental work was performed on a series of human bronchial carcinoma xenografts established in immune deprived CBA mice. Interferon alone had no cytotoxic effect but appeared to potentiate the activity of various anti-cancer agents in non-small cell tumours. No such effect was seen in two small cell xenografts. Further studies suggested that the dosing schedules of each agent in the combination was important in producing positive effects. The exact mechanisms by which interferon may interact with cytotoxic drugs remains unexplained. It was shown that the results obtained in the xenograft model were not mediated through increased toxicity to the host or by modulation of cell cycle distribution within the tumour cells. Assessment of interferon/drug combinations was also performed using in-vitro models of lung cancer. Significant synergistic interactions were not demonstrated. A pilot clinical study investigating the potential of combined treatment with interferon and Cisplatinum was performed in a group of patients with non-small cell lung cancer. The toxicity of the treatment was predictable and an encouraging response rate was seen.

Although the beneficial role of other anti-resorptive agents such as the Selective Estrogen Receptor Modulators (SERMs) in preventing bone loss in postmenopausal osteoporosis are well established, the effects of SERMs on the maintenance of the osteocytic population in vivo are less clear. Work in this thesis has investigated whether the SERM LY 117018, which is a Raloxifene analogue, can mimic the osteocyte-sparing effect of 17β-estradiol in a rat model of ovariectomy. Abrupt estrogen withdrawal was shown to increase osteocyte apoptosis in rat bone, while the ovariectomy-induced stimulation of osteocyte apoptosis in rats was shown to be reversed following administration of the LY 117018 SERM in a similar way to the known effect of 17β-estradiol replacement. These data point to the potential benefits of SERMs on bone viability in estrogen-depleted rats. However, the molecular mechanism by which 17β-estradiol and the LY 117018 SERM maintain the osteocytic population has not been well characterised. In vitro studies in this thesis indicated that 17β-estradiol; raloxifene and LY 117018 suppressed the pro-apoptotic stimuli induced in response to H₂O₂ in osteocyte cultures possibly by exerting direct anti-oxidant properties related to their chemical structure. These data introduced a novel mechanism of action for 17β-estradiol, raloxifene and LY117018 as antioxidants, in the protection of the osteocytic population increasing the knowledge available on their activity in bone. In addition, these data suggested that the effects of SERMs on preventing osteocyte apoptosis will in future help to determine their effectiveness and interest for the clinical development of estrogen replacement compounds with activities consistent with the maintenance of both bone mass and bone quality. Finally, the effects of 17β-estradiol on the viability of osteocytes were investigated in human cancellous bone ex vivo in the presence or absence of mechanical loading. Results pointed to important anabolic effects induced by either 17b-estradiol or mechanical stimulation and have suggested that the osteogenic response of loading was not enhanced by the presence of 17β-estradiol in this patient. In this study, identification of anti-apoptotic effects of SERMs on osteocytes in vivo and in vitro highlighted an important bioactivity that may explain part of the action of SERMs in vivo. Further characterisation of the anti-apoptotic effects of 17β-estradiol and SERMs in vitro indicated direct antioxidant capabilities pointing to the possibility the SERMs mimic not only the bone sparing activity of 17β-estradiol but its anti-oxidant nature as well.

In this study, the rat hypothalamic brain areas; the arcuate nucleus and the ventromedial hypothalamic nucleus, were selected for study of GHRP-6 and L-144,446 effects <i>in vitro</i>. Extracellular recordings showed that electrical activity of neuronal population in the arcuate nucleus was significantly excited by GHRP-6 and L-144,446. Patterns of excitation by either GHRP-6 or L-144,446 were closely similar. The increase in firing rate was observed with latency period of approximately 2 to 5 min. The excitation was sustained for more than 1 h after the secretagogue washout. Interestingly, GHRF-6 was also observed to increase the firing activity of neurones in the ventromedial hypothalamic nucleus. Patterns of increase in firing activity of ventromedial hypothalamic neurones were very similar to those observed in arcuate neurones. This is surprising, because in <i>in vivo</i> studies, Fos protein induction by systemic administration of secretagogues was consistently observed in the arcuate nucleus but not in the ventromedial hypothalamic nucleus. According to our evidence that ventromedial hypothalamic neurones were significantly excited to GHRP-6 <i>in vitro</i>, it was hypothesised that, <i>in vivo</i>, GHRP-6 stimulates neurones in the arcuate nucleus or other nuclei nearby which project their nerve endings to inhibit ventromedial hypothalamic neurones. The majority of neurones in the arcuate nucleus and the ventromedial hypothalamic nucleus that were excited by either L-144,446 or GHRP-6, were significantly inhibited by neuropeptide Y (NPY). Especially in the ventromedial hypothalamic nucleus, 68~% of GHRP-6-excited neurones, compared to 40% of those in the arcuate nucleus, were clearly inhibited by NPY, NPY is a potent orexigenic agent that was previously found to be produced in many neurones in the ventromedial arcuate nucleus. In addition, the existence of a NPY pathway that originates in the arcuate nucleus and projects to the ventromedial hypothalamic nucleus has previously been demonstrated. These results suggest that, <i>in vivo, </i>secretagogues may stimulate NPY neurones in the arcuate nucleus which project their nerve endings to inhibit a sub-population of the ventromedial hypothalamic nucleus. To investigate the mechanism of GHRP-6 action, immunohistochemistry technique was used to detect the induction of Fos, a protein product of the immediate early gene c-<i>fos</i> a protein product of the immediate early gene c-<i>fos</i>, in the accurate nucleus. The results showed that intravenous injection of GHRP-6 significantly increased immunostaining of Fos in the arcuate nucleus.  Pretreatment with intravenous administration of NPY caused 61% reduction of number of Fos-positive neurones in the arcuate nucleus induced by GHRP-6. This findings confirm the effects of GHRP-6 on the electrical activity of the arcuate nucleus and support the idea that this brain area is a central site of action of GHRP-6. This thesis describes site of action of secretagogues in the hypothalamus with <i>in vitro </i>and <i>in vivo</i> data. It also shows the functional involvement of NPY in effects of secretagogues on firing activity and induction of Fos protein in this area.