Impaired nitric oxide (NO)-mediated vasodilatation has been implicated in the increased peripheral vascular resistance (PVR) associated with chronic heart failure (CHF). However, there is evidence that basal synthesis of NO may be preserved or even enhanced in CHF, perhaps due to the expression of the inducible NO synthase (iNOS). Increased superoxide production has been demonstrated in CHF and, some superoxide destroys NO, a reduction in NO bioavailability may be responsible for impaired NO-mediated relaxations and may also explain why PVR remains elevated despite increased NO production. Therefore, the aims of this thesis were to investigate the expression of iNOS in the cardiovascular system of rats with CHF following coronary artery ligation, to determine the functional significance of this potential source of NO on responsiveness of the peripheral vasculature, and to investigate the role that superoxide plays in modulating vascular function. Immunohistochemical studies revealed that iNOS was expressed in all cell types of small mesenteric arteries and in thoracic aortae from CHF rats. iNOS was also identified in coronary vascular, endocardial and myo-endothelial cells in hearts from CHF rats. Immunoreactive iNOS was also found throughout the viable left ventricle of hearts for CHF rats. No staining was found in arteries or hearts from sham-operated rats. The salient finding of this thesis is that iNOS is expressed in the peripheral vasculature and in the heart in this model of CHF. However, in small mesenteric arteries, which play a pivotal role in determining PVR, substrate deficient iNOS-derived superoxide is responsible for increased responsiveness to constriction agents. In thoracic aortae, however, iNOS appears to play no role in modulating vascular function. In conclusion, the findings of this thesis may represent an important mechanism for the raised PVR and endothelial dysfunction associated with CHF.

The aims of this thesis was (i) to assess the effect of cirrhosis on the responses of human hepatic artery to three vasoconstrictor agonists, arginine vasopressin (AVP), 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), which may contribute to the development of the hyperdynamic circulation and (ii) to clarify the mechanism(s) of any abnormally detected. The endothelium was shown to be damaged considerably in isolated human and porcine hepatic arteries, hence, all subsequent studies used denuded vessels. The protocol developed using porcine hepatic arteries demonstrated that preservative solutions had no effect on contractile function but did alter endothelium-independent relaxation. Responses to vasoconstrictors were altered in arteries from patients with hepatic cirrhosis although the nature of this alteration was agonist-dependent: the maximum contraction to AVP was impaired whilst that to 5-HT was augmented. In contrast, the sensitivity of response to ET-1 was increased, whereas KCl-mediated contraction was unchanged. The contractile responses to AVP, 5-HT and KCl were unaffected by NOS inhibition. There was no immunoreactivity for iNOS in donor hepatic arteries nor in those from most patients: low level of iNOS were only detected in arteries from patients with alcoholic liver disease. Use of antagonists demonstrated that AVP and ET-1 contracted human hepatic arteries via the V₁ and ET_A receptors, respectively, whilst 5-HT acted predominantly via 5-HT₁-like receptors with a small contribution from the 5-HT_2A subtype. These studies demonstrated that cirrhosis of the liver is associated with a agonist-selective alteration of receptor-mediated contraction in denuded human hepatic arteries. Inducible nitric oxide synthase activity did not contribute to these alterations and unaltered responses to KCl suggest that these abnormalities are not due to structural changes in the vessel wall. These results indicate, therefore, that altered contractile function in hepatic arteries from patients with cirrhosis are due to changes in receptor activity and/or post-receptor signal transduction pathways.

ACTH is the principal factor stimulating the formation and release of cortisol from the adrenal cortex and is controlled, in part, by the negative feedback inhibition of cortisol. Investigation into the kinetics of cortisol production by cultured bovine adrenocortical cells in response to ACTH-treatment and the relationship between the cortisol secretion and the expression of the steroidogenic acute regulatory (StAR) mRNA was undertaken. ACTH (10nM) treatment of BAC cells on day 3 of culture displayed an increase in the rate of cortisol output over the initial 6 hours, P<0.05 at each time point compared with untreated cells, followed by a marked decline in the secretion rate thereafter. The decline in the cortisol secretion rate was not due to a decrease in the number of cells as the protein content of the culture wells did not vary by ore than 10% over the 12 hour period. Nor was the decrease in cortisol secretion a result of serum-deprived culture as a comparable response was seen in serum-replete conditions. HPLC analysis of the culture medium revealed the secretion of four main steroids; cortisol, corticosterone, cortisone and 11β-hydroxyandrostenedione. Cortisol was found to account for ~ 50% of the total steroid output by BAC cells at each of the time points studied. The total steroid output increased over the initial 6 hours (6.1nmol/10⁶ cells at 6 hours) followed by a decline in levels to 4.4nmol/10⁶ cells at 12 hours. Thus, the decline in cortisol secretion was not due to the production of another steroid in preference to cortisol. Various concentrations of ACTH also invoked a similar pattern of cortisol secretion as did treatment of BAC cells with angiotensin II. Interestingly, no decline in cortisol secretion was found when BAC cells were treated with forskolin or 8Br-cAMP. Receptor desensitisation was ruled out as the cells responded to a second ACTH-treatment.

The striatum, being part of the limbic system, shows changes during depression in both structural and functional imaging studies, suggesting the involvement of dopamine. Using IBZM, a SPET ligand with high dopamine D_2/3 receptor selectivity, in-vivo striatal D_2/3 receptor availability was examined in 15 depressed in-patients and 15 matched controls. Depressed patients had increased receptor availability, implying reduced striatal dopaminergic activity in depressed patients. Increased receptor availability was related to objective measures of psychomotor retardation in the depressed group. Inter-individual IBZM binding variability may be due to a combination of state and trait effects. Two trait effects were found in controls-sexual dimorphism, independent of affective illness, with women showing higher availability, and a large effect related to temperament or personality, confirming the functional complexity of the striatum. Evidence for structural change in depression is conflicting, the strongest being for striatal and frontal lobe atrophy in unipolar depression. Patients with chronic treatment resistant unipolar depression (CTRD) were examined, as they may be expected to have the greatest likelihood of exhibiting structural change. Voxel based analysis and conventional volumetry were used to compare the high resolution MRI images of twenty patients with CTRD, with 20 matched recovered patients and 20 matched healthy controls. Only CTRD patients exhibited MRI differences. Both VBA and conventional volumetry revealed frontal lobe and right striatal atrophy. Additionally, VBA revealed reduced grey matter density in left temporal lobe and bilateral anterior hippocampi, particularly the left. Neocortical changes correlated with the cumulative illness severity. Medial frontal grey matter reductions correlated with age, psychomotor retardation as well as current and past illness severity in patients groups. Left hippocampal grey matter reductions correlated with reduced episodic verbal memory. VBA is, therefore, a sensitive, valid and complete analysis of MRIs providing results consistent with contemporary hypotheses.

The aims of this thesis were: 1) to establish the role of nitric oxide (NO) and cyclic GMP in neutrophil chemotaxis and superoxide anion generation (SAG), 2) to investigate the effects of a novel NO donor GEA 3162, the A_2A receptor agonist 2-HE-NECA, and the PGI₂ analogue cicaprost on neutrophil accumulation and myocardial injury <i>in vivo</i>, in a rat model of MI-R, and 3) to identify the role of neutrophil activation in the formation of stable platelet aggregates and whether heparin, which is used systematically to anticoagulate for CPB, contributed to platelet dysfunction during CPB by interfering with neutrophil-platelet interactions. Effects of heparin <i>in vitro</i>, on neutrophil SAG and myeloperoxidase release were also determined. The mechanisms responsible for chemotaxis and neutrophil activation are not fully understood. Selective inhibitors of the NO and cyclic GMP pathways have been used to elucidate their roles in the activation and inhibition of human neutrophils. In addition, the ability of NO donors to inhibit neutrophil chemotaxis was compared with their ability to increase neutrophil nitrate/nitrate and cyclic GMP levels. The results confirm that neutrophil activation results from the stimulation of several signal transduction systems. It appears that chemotaxis can occur via a NO-dependent as well as NO-independent pathway. Similar pathways appear to operate in SAG. The results also suggested that the small concentrations of NO and cyclic GMP induced by fMLP activated neutrophils while large concentrations of NO and cyclic GMP are inhibitory. The effects of GEA 3162, 2-HE-NECA, and cicaprost on neutrophil accumulation and myocardial injury in a rat model of MI-R were investigated. Myocardial ischaemia was induced by occlusion of the left main coronary artery (45 min) and then reperfused (120 min). Drugs or saline vehicle were infused intravenously for 130 min beginning 10 min before reperfusion. Neutrophil accumulation in the area at risk and normal area was assessed by myeloperoxidase assay.

The hypothesis that 5αR1 plays a role in the regulation of aromatase activity by limiting substrate availability to the latter in the human prostate was tested in a co-transfection model and in primary cultures of prostatic cells. In COS-1 cells transiently co-transfected with plasmids encoding 5αR1 and aromatase, inhibition of 5αR1 led to an increase in aromatase activity but not vice versa. This pattern of interaction between the two enzymes was also shown to occur with endogenous enzymes expressed in primary cultures of prostatic fibroblasts. No aromatase activity was detectable in epithelial cells. By regulating aromatase activity, 5αR1 may therefore contribute to the local control of oestrogen concentration within the prostate gland. In this study, prostatic cells were revealed to be oestrogen target cells, not only expressing mRNAs of both subtypes of oestrogen receptor (ERα and ERβ) but also responding to oestradiol with increased proliferation rtes. This oestrogen stimulation of proliferation was antagonised by the antioestrogen ICI 182,780. Moreover, prostatic fibroblasts aromatised androgen substrates to oestrogens (oestrogens formation). Both fibroblast and epithelial cell cultures also converted oestradiol to oestrone (oestrogen catabolism) but not vice versa. Primary cultures of prostatic cells therefore responded to oestrogen stimulation and expressed major components for oestrogen action including oestrogen receptors and oestrogen-metabolising enzymes; they would serve as a useful model to further study oestrogen action, regulation and signalling pathways in the prostate. In addition to the above enzyme inhibitors, the effects of Permixon® (a phytotherapeutic agent for the management of prostatic hyperplasia) were investigated. The mechanism of action of Permixon® is not entirely clear; however, in previous studies in inhibited both 5α-reductase isozymes in prostatic cells. Permixon® was herein shown to have oestrogenic properties. It not only indirectly increased aromatase activity in fibroblasts by inhibiting 5αR1, but also inhibited the conversion of oestradiol to the weaker oestrogen oestrone. It is therefore conceivable that Permixon® exerts its therapeutic effects by affecting the metabolism of both androgens and oestrogens within the human prostate.

Every year there is 2% increase in the reported cases of glioma. It is important to treat glioma from materials that are readily available in nature. One of the interesting properties of liposomes is their ability to target tumours and facilitate the cellular uptake of therapeutic agents compared to the agent alone. In this project, liposomes were prepared from SPC (Soy phosphatidylcholine) and HSPC (Hydrogenated soy phosphatidylcholine) phospholipids. Drug-free liposomes prepared by proliposomes were compared with the conventional method of producing liposomes. Conventional liposomes are biocompatible and biodegradable, however, they are physically and chemically unstable. The instability problems were avoided by formulating liposomes using the ethanol-based proliposome technology. In ethanol-based proliposome method, aqueous phase (e.g. water) was added to an ethanolic solution of phospholipid to generate liposomes. In this work, natural anticancer materials such as Paclitaxel (PTX) and Momordica charantia extracts (Whole fruit, Fruit alone and Seed alone) from Africa, China and India were incorporated in liposome formulations. These liposomes were analysed by investigating, the resultant size, size distribution and zeta potential of the vesicles. Either, the anticancer drug or extract of Momordica charantia was mixed with liposomes and checked for the efficacy of the anticancer-liposome formulations on the viability of glioma cell lines and the molecular mechanism of the cell death were also investigated. The results show that liposomes prepared by the conventional thin- film method were comparatively large in size as compared to liposomes generated from proliposomes. Liposomes generated from proliposomes (made from SPC or HSPC), when generated by hydration with Momordica charantia extracts (WF – Whole fruit, FA – Fruit alone or SA – Seed alone) from Africa, China or India respectively, or when prepared using PTX in the lipid phase had significantly larger size and wider size distribution as compared to drug free liposomes. Liposomes generated from proliposomes were neutral or negatively charged and were a mixture of oligolamellar and multilamellar vesicles regardless of formulation. Particle size and size distribution of HSPC liposomes were larger than SPC liposomes on inclusion of either Paclitaxel or Momordica charantia extracts. Liposome generated by proliposome method using the Momordica charantia extracts (FA, SA and WF) exerted cytotoxic effects against glioma cells 1321N1, Gos-3 and U87-MG at the higher concentrations with or without liposomes. Momordica charantia extracts showed either slight or no significant effect on the normal glial cells. The liposome formulations were more effective against glioma cells as compared to drug-free liposomes. FA extract of Momordica charantia was very effective with and without liposomes but less than PTX liposomes. The activities of caspase 3/7, caspase 9 and cytochrome c release were elevated in cancerous glial cell line indicating apoptosis via mitochondrial cell death or intrinsic pathway. In conclusion, the study showed that liposomes generated from proliposomes were appropriate to target cancerous glial cells by binding plant extracts Momordica charantia and PTX to SPC and HSPC phospholipids. The cell death was induced by apoptosis via mitochondrial cell death.

International initiatives, such as the Nagoya Protocol to the United Nations Convention on Biological Diversity (the Protocol), have created (or are creating) “access and benefit sharing” rights which seek to ensure that genetic resources and traditional knowledge associated with such genetic resources (“TKAGR”) cannot be used without the consent of rights holders. These initiatives (including the Protocol) are unclear on how far non-consensual “use” extends to man-made downstream derivatives of the products of genetic expression. It also gives no guidance as to the degree to which control over TKAGR should extend throughout the drug discovery process. This work demonstrates how such TKAGR entering into a drug discovery process will be diluted with other information, used as an inspiration for further research, or for the development of research tools which may, in turn, lead to further discoveries and highlights how useful drugs may be very distal from the original inspiration provided by the TKAGR. This work also examines the causal link between an original piece of TKAGR and remote “downstream” uses of that information within drug discovery. It identifies “serendipitous” discoveries of unexpected second uses as a potential point at which the causation in law link to distal use may potentially be broken. This thesis examines the high level normative justifications for these rights, and in particular uses consequentialist/utilitarian, contribution/desert claims and distributive justice (Rawlsian maximin) claims to test their justifiable scope.

Over the past fifteen years there has been increasing interest in the environmental occurrence, fate and effects of substances used as pharmaceuticals or personal care products. While the understanding of the environmental fate and ecotoxicity of pharmaceuticals is now well developed, less information is available on the uptake of pharmaceuticals and personal care products into aquatic organisms and, in particular, into sediment-dwelling organisms. This study was therefore performed to develop an understanding of the factors and processes affecting the uptake of pharmaceuticals and personal care products into the sediment dwelling oligochaete worm, Lumbriculus variegatus. The study combined experimental studies into the distribution of a range of pharmaceuticals and personal care products in sediment-water systems and studies into the uptake of the study compounds under a range of conditions. The results were used to parameterize and evaluate a model for estimating uptake of pharmaceuticals and personal care products into benthic organisms. Adsorption of the study compounds from water to sediment solids increased in the order diclofenac < chloramphenicol < salicylic acid < naproxen < caffeine < sulfamethazine < triclosan < fluoxetine. Comparison of the sorption results with estimations from available models for predicting sorption from chemical properties indicated that relationships developed for neutral organic chemicals were not appropriate for use on ionisable pharmaceuticals and personal care products. While predictive models, developed specifically for ionisable chemicals, produced improved predictions of sorption, even these predictions were not perfect. Bioconcentration factors for the study compounds from water into L. variegatus were found to increase in the order chloramphenicol < diclofenac < salicylic acid < fluoxetine < naproxen < triclosan. The differences in bioconcentration factors could not be explained by differences in log Kow and log Dow which are descriptors that have previously been used to predict the uptake of neutral organic substances and ionisable substances in other species of invertebrates. There was also disagreement between the uptake measurements and predictions obtained from models developed for estimating the uptake of ionisable chemicals into aquatic organisms. The uptake of four of the study compounds (caffeine, diclofenac, fluoxetine and triclosan) was further evaluated at different water pH values. For three of these compounds (diclofenac, fluoxetine and triclosan), the potential for metabolism by L. variegatus was also assessed as was the uptake and route of uptake from whole sediments. Uptake of diclofenac and fluoxetine was found to be highly sensitive to changes in pH with bioconcentration factors varying by over two orders of magnitude (diclofenac) and four orders of magnitude (fluoxetine) across three pH units. Tissue analysis indicated that while diclofenac is not metabolized by the worms, fluoxetine and triclosan are heavily metabolized. The whole sediment studies demonstrated that uptake of diclofenac and fluoxetine occurs primarily from the sediment pore-water whereas for triclosan, sediment ingestion provides a small contribution to the uptake. Results from the different components of the study were used to parameterize and evaluate a model for estimating uptake of pharmaceuticals and personal care products from sediments into benthic organisms. Comparison of predictions from this model for diclofenac, fluoxetine and triclosan were compared to measurements from whole sediment studies. While the model was found to under-predict the uptake of triclosan, good predictions were obtained for diclofenac and fluoxetine. With further development and evaluation, the uptake modeling approach could provide a valuable tool for use in the risk assessment of ionisable compounds such as many pharmaceuticals and personal care products.

This project developed, assessed and improved an existing virtual screening technology; the Investigational Novel Drug Discovery by Example (INDDEx) package; and uses the technology to discover new drug hits and leads. INDDEx performs ligand-based virtual screening: learning from molecules with known activity and fragmenting them into substructural elements. A model is built based on logical rules defining required distances between elements of substructure. In the optimisation phase of this project, the program's speed was increased, a support vector machine method was implemented and a graphical output for the generated rules was added. In the investigation and screening phase, INDDEx screened the ZINC database to find new inhibitors for SIRT2, a poorly investigated target with few known inhibitors. The top ranked molecules were docked with GOLD to produce a consensus score. A new molecule was found with 50% inhibitory concentration of 0.67μm against SIRT2. In the assessment phase, the performance of INDDEx as a virtual screening tool was assessed by benchmarking it on the DUD database and comparing it with the performances of eHiTS LASSO, PharmaGist and DOCK. INDDEx gave 1% Enrichment Factors of 69.2, 82.7 and 90.4, and 0.1% Enrichment Factors of 492, 631 and 707, both when learning from 2, 4 and 8 active ligands. A strength of INDDEx is its scaffold-hopping ability. Scaffold-hopping is important for developing new drug leads, and is regarded as a challenge. Considering only ligands structurally dissimilar to ones in the benchmarking learning data, INDDEx gave 1% Enrichment Factors of 52.3, 63.6 and 66.9 when learning from 2, 4 and 8 active ligands. In the improvement phase, an algorithm was implemented to take hits found by INDDEx and explore the synthetic space resulting from synthetic modification of those hits. This was realised using a library of virtual reactions and a method of predicting which molecules have the most potential for modification. Two assessments quantified the additional molecular space made available to search and qualified the new method as a significant improvement on a naïve approach.