In order to investigate the CaM activation mechanism of nNOS, the effect of the conformational changes of nNOSrd and the binding of NADP(H) on the redox potential of the flavins, cofactors were assessed for the isolated FAD and FMN sub-domains by OTTLE potentiometry. The results showed that the presence of the FAD/FMN sub-domain interface does not alter the thermodynamic properties of the redox couples involved in the catalysis. This is consistent with the fact that CaM binding has a small effect on the flavins reduction potentials. Only the FMN/FMNH^· redox couple was found to be stabilised by the presence of the FAD sub-domain (increase of 80 mV). The same redox couple was also kinetically stabilised toward oxidation. The isolated FAD sub-domain was found to have similar redox potentials to the isolated nNOSrd. In the presence of NADP⁺, both the FAD sub-domain and the nNOSrd formed the charge-transfer complex with a long-wavelength absorption band centred at 780nm. Formation of this complex was found to stabilise the FADH^·/FADH^-redox couple by approx 30 mV. It is possible that in the CaM-free enzyme, the conformation of the bound NADP⁺ may control both electron transfers between the FAD and FMN and from FMN to heme by modulating the potential of the FAD hydroquinone. The accessibility of the FMN cofactor to the heme was assessed. The results showed that if the FMN, in the isolated FMN domain, is assumed to be fully accessible, then it is 100% accessible in the CaM-bound enzyme, 45% accessible in the uncompleted enzyme and only 3% accessible in the NADPH-bound nNOSrd in the absence of CaM. This suggests that the binding of CaM is responsible for a structural reorganisation of nNOS rd that “unlocks” the conformation of the enzyme and enables the FMN sub-domain motion in order to shuttle an electron from FAD cofactor to the heme. The specificity of NADP(H) in repressing the electron transfer from the reductase domain to cytochrome <i>c</i> was studied by using NADP(H) analogues. Results showed that the specificity of NADPH in inducing nNOS rd conformational change relies upon the interaction of both the tightly-bound ADP substituents and the labile nicotinamide substituents and that the tightly bound ADP substituents is essential to position the nicotinamide moieties for full electron transfer repression. It appears that the “locked” conformation of the enzyme, believed to inhibit the electron transfer to the heme, is specific for the NADP(H).

There is a requirement to demonstrate photostability of medicines early in development but a lack of clear methodologies for doing so. While the International Conference on Harmonization (ICH) has a guideline on photostability testing (ICH, 1996), it does not specify a universal method for the analysis of samples after irradiation. Moreover, the current methods used in photostability testing separate irradiation of samples from analysis, rendering sample preparation critical in the whole process. In this context, photocalorimetry (the measurement of heat changes when a sample is irradiated) offers an alternative method which addresses most of the issues in current photostability testing. Some of the advantages of this technique include the real-time collection of data, the universal character of heat as a measure of change and the “in situ” analysis of photodegradation processes. Two different photocalorimetric designs were developed in the School of Pharmacy, University of London, to study the photodegradation of pharmaceuticals in any physical form. These instruments used light-emitting diodes (LEDs), as the light source, adapted to an isothermal heat conduction microcalorimeter (TAM 227), in one case, and a Multi-Cell Differential Scanning Calorimeter (MCDSC), in the other. To test the instruments’ ability to detect photoreaction heat outputs, known photolabile pharmaceutical formulations were tested in different physical states. Solution phase studies were performed on samples of nifedipine in ethanol and the calorimetric outputs were quantitatively analysed. Solid nifedipine was also tested with the two photocalorimeters, although, data was only analysed qualitatively. Other solid drugs were tested with the photo-MCDSC; some of them are known photolabile compounds (2-nitrobenzaldehyde, benzoquinone, carbamazepine, chloramphenicol, dipyridamole and furosemide) while others are considered stable to light (paracetamol and aspirin). Parallel to the photocalorimetric studies, important progress was made regarding the analysis of calorimetric data for solid state processes and zero-order kinetics in solution.

The change in community pharmacists’ practice from compounding and effectively unregulated medicines supply through to the highly regulated and largely automated high-volume dispensing process of today has been challenging. The economic and social standing of community pharmacy was transformed creating a need for further adaptation. This thesis explores ‘how business and professional practice models for community pharmacy in England in ten to twenty years are likely to be structured?’. It has six sections, plus an overarching discussion. A work sampling study of ten community pharmacies found that pharmacists continue to spend two-thirds of their time on dispensing related activities, compared to one tenth on counselling. The accompanying analysis links this to an increase in prescription volumes and payments that have incentivised pharmacy contractors to focus on medicines supply. A significant decrease in the average prescription duration for eight chronic disease medications over the past decade is revealed, and its desirability questioned. Using the Kingdon model of the policy process as an evaluative framework, 16 interviews with ‘policy leaders’ provided insight into how seven factors (identified from a structured thematic review of the implementation of Medicines Use Reviews) have influenced the implementation of the New Medicines Service. In addition, role theory-based thematic analysis involving 17 stakeholders in pharmacy policy highlighted the tensions between community pharmacists’ roles as shopkeepers, clinicians and businessmen, and the effects that new technologies will have on them. The analysis identifies a need for pharmacy to embrace a new strategic direction that enhances pharmacy’s contributions to health outcomes. In conclusion, community pharmacy in England should offer timelier and economically efficient ways of solving contemporary health problems. The evidence presented here suggests that without stronger internal leadership and robust external stakeholder support medicines supply will split from the provision of clinical pharmacy in the community setting, leaving community pharmacies as ‘commodity cost’, low return medicines suppliers.

Background: Inorganic nitrite, a metabolite of endogenously produced nitric oxide (NO) from NO synthases, provides the largest endocrine source of directly bioavailable NO. The conversion of nitrite to NO occurs mainly through enzymatic reduction, which is particularly favoured under hypoxia. Thus, current evidence shows that nitrite dilates small resistance arterioles where conditions of hypoxia predominate. Although organic nitrates/nitrites also mediate their principal effects via NO, they are not hypoxia dependent; hence, they selectively dilate muscular conduit arteries, lowering central blood pressures. Inorganic nitrite would be expected to lack such effects. Methods and Results: The effects of local and systemic administration of sodium nitrite on the radial artery (RA) a muscular conduit artery, forearm resistance vessels (forearm blood flow) and systemic haemodynamics in healthy male volunteers (n=43) were examined. Intra-brachial sodium nitrite (8.7 μmol/min) increased RA diameter by 28.3% (95% CI 20.3 to 36.2). Nitrite (0.087-87 μmol/min) displayed similar selectivity as glyceryl trinitrate (0.003-1 μg/min) for conduit arteries, compared to resistance arterioles. Nitrite dose-dependently increased local cGMP production from the dose of 2.6 μmol/min, by 1.1 pmol/min/100ml tissue (95% CI 0.5 to 1.8). Vasodilatation of the RA by nitrite was enhanced by administration of acetazolamide (oral or i.a.) and oral raloxifene (P=0.0248, P < 0.0001 and P=0.0006, respectively) but was inhibited under hypoxia (P < 0.0001) and hyperoxia (P=0.0006) compared to normoxia. Systemic intravenous administration of sodium nitrite (8.7 μmol/ min) dilated the RA by 10.7% (95% CI 6.8 to 14.7) and reduced central systolic BP by 11.6 mmHg (95% CI of difference 2.4 to 20.7), augmentation index and pulse wave velocity, without changing peripheral BP. Conclusions: Nitrite is a normoxia-dependent selective conduit artery dilator. The mechanism is via cGMP, and the effect is enhanced by acetazolamide and raloxifene. The selective central blood pressure-lowering effects of nitrite have therapeutic potential to reduce cardiovascular events.

Conventional wet granulation processes involve controlled coalescence of moist particles through the addition of binder liquid to dry powder particles such that the process proceeds in the direction of increasing liquid saturation. The process is terminated immediately prior to an undesirable state of uncontrolled granule growth and batch loss. The ideal conventional wet granulation process is stated to require tight control over granule nucleation conditions which are often not possible to execute commercially. Consequently, a novel reverse-phase granulation process was developed and studied involving immersion of dry powder into the binder liquid, thus eliminating the traditional granule nucleation process. The reverse-phase process proceeds in the direction of reduced liquid saturation, thus decreasing the risk of uncontrolled growth and batch loss. The effects of binder liquid quantity, binder liquid viscosity and impeller speed on the granules produced using the reverse-phase and conventional processes were compared. The conventional process exhibited induction growth behaviour and uncontrolled granule growth at elevated liquid saturation. In contrast the reverse-phase process demonstrated steady granule growth behaviour at all liquid saturations indicating greater robustness to process failure. The primary mechanism of the reverse-phase granulation process was breakage of large moist agglomerates and mechanical dispersion of the binder liquid throughout the powder formulation. The size and porosity of reverse-phase granules were controlled by the liquid saturation and impeller speed, with these physical properties being best described by the dimensionless Stokes deformation number and the growth regime map. Two potentially negative consequences associated with the reverse-phase granulation approach were evaluated. First, the compaction properties of reverse-phase granules were shown to be similar to those of conventional granules. Second, the rate and extent of hydration of the model drug anhydrous theophylline was shown to be similar for both the reverse-phase and conventional granulation processes. Based upon these findings it was concluded that the reverse-phase process may represent a feasible alternative to the conventional process, particularly should scale-up to the industrial scale prove applicable.

Central pulse pressure (cPP), a product of ventricular-arterial interaction, is an important determinant of cardiovascular outcomes in hypertension. The aim of this thesis is to advance the understanding of pulsatile haemodynamics and to explore mechanisms that may selectively reduce cPP. The conventional view is that cPP comprises a component determined by the direct interaction of myocardial contraction with the impedance of the proximal arterial tree (closely related to pulse wave velocity, PWV) and a component ‘augmentation pressure’ generated by pressure wave reflections from muscular conduit arteries. Surprisingly little is known regarding regulation of conduit artery tone despite its potential influence on cPP. In the first part of this thesis, muscular large arterial tone was examined using a human forearm blood flow model. Vasoactive substances were infused locally into the brachial artery and vasodilator responses of the radial artery, as a muscular conduit artery, and forearm resistance microvasculature were examined. Nitric oxide donors, in particular, glyceryl trinitrate (GTN) were found to have the most selective action on conduit arteries compared to other vasodilators. In the second part of the thesis, I examined whether the action of GTN to reduce augmentation pressure could be accounted for by this selective dilation of muscular arteries. GTN was given systemically and by intra-coronary infusion in patients undergoing cardiac catheterisation. Invasive aortic blood pressure and flow velocity were analysed in the time domain by wave intensity analysis. This allows separation of pressure into a forward component generated by myocardial contraction and a backward component generated by ‘reflection’ from the peripheral arterial tree. A surprising finding was that changes induced by GTN were mainly attributable to a reduction in forward rather than backward pressure waves. That this resulted from a change in myocardial contractility was confirmed by local intracoronary injection of GTN. The final part of the thesis examines the relative contribution of forward and backward pressure waves in hypertension. An elevated cPP in hypertensive compared to normotensive subjects was accounted for primarily by an increased forward pressure wave. That this was due to increased myocardial contractility was confirmed by examining whether the pattern of wave intensity seen in hypertension could be reproduced, in normotensive subjects, by the inotrope dobutamine (when compared to the vasoconstrictor norepinephrine used as a control). This thesis thus provides novel insight into a) regulation of conduit artery tone, and b) pulsatile haemodynamics, highlighting the contribution of left ventricular ejection characteristics in determining pressure augmentation and cPP.

Three longitudinal case studies were conducted to investigate developmental language impairment (1)1.1) in Egyptian Arabic (EA). While there have been descriptions of adult acquired aphasic deficits in Arabic, this study details the linguistic characteristics of children with impairments specific to language. To select the subjects, an exclusionary checklist was used based on the criteria used for specific language impairment (SLI, Ixronard, 1998). The subjects consisted of two males and one female, first seen at less than 5 (X) years and recorded longitudinally (21 -36 months). Data from 12 normally developing children, aged between 1 00 4,04 years, was also collected for comparative purposes and to detail normal developmental errors in EA. Patterns of language impairment and development were investigated using spontaneous language measures and specific structured tasks. The language samples were phonetically transcribed from video tapes during non-directive therapy and parent child play sessions. The spontaneous language measures included, Mean Morphemes per Unit (MPU), percent structural errors, functional analysis of utterances and an error analysis of specific grammatical morphemes. 'Ihe three EA-DLI children shared similar patterns of errors although cognitively they had different strengths. 'Their patterns of impairment reflected primarily morpho-syntactic difficulties. Many of the linguistic characteristics observed in the EA-DLI children's language were also produced by the controls, but less frequendy. The EA-DLI children's MPUs were found to be restricted with higher percentages of morphological errors than the language matched controls. An unmarked default verb form resembling the Imperfective-stem was a frequent substitution error. The functional analysis revealed that the EA-DLI children were similar to the controls in their use of requests and labels, however they produced more Learnt Repetitive phrases and disordered sentences and fewer Intravcrbals due to their difficulties with abstract verbal reasoning. 'ihe difficulties described in this study compnse of some linguistic features specific to EA and other features that have been reported in cross-linguistic studies of SLI. The shared features included difficulty with grammatical morphology, lack of master)' at expected developmental stages and limited use of inflectional morphology leading to agreement errors. Verbs were difficult, percentages of errors were high and fewer verbs were produced than nouns. In contrast to the findings of SLI in other languages Tense and Aspectual marking was not problematic, but difficulty was with subject verb agreement for gender, number and person. Prepositions, pronouns, plurals and negative particles were either omitted or substituted resulting in error patterns. The grammatical theories developed to account for SLI reported in English, German and Swedish (Hakansson et al., 2003 Clahsen and Hansen, 1997 van der Lely, 2002) were judged against the evidence acquired in this study on the three EA-DLI children. The limitations of these theories are discussed and alternative interpretations are provided.

The flow of people between urban Britain and rural Bangladesh is longstanding and continuous. In addition to people; food, plants and seeds are transported between the countries. The exchanges are both practical and highly symbolic processes and while transnational in nature have a significant impact at a local level in both places. The PhD thesis explores the nature of food and plant exchange between Sylhet (Northeastern Bangladesh) and the UK among women at a household level, how these transnational exchanges impact on the food-scape and medicinal plant knowledge in each place and how differences are played out across generations. The PhD adopted a mixed methods approach (participant observation, interviews, focus groups and questionnaires) with research conducted in Cardiff, London and Sylhet among two generations of Bengali women. Research into ethnobotanical practices of migrant communities in industrialised countries has found a rich and distinct body of knowledge. However, among the substantial and well-established UK Bengali population there is a lack of research on ethnobotanical medicinal knowledge. Furthermore how they are influenced by on-going links with Bangladesh is unknown. The therapeutic use of plants among Bengalis exposes a significant overlap between food and medicine, this is an area largely ignored in public health, medical and food studies of Bengalis in the UK. This thesis builds on previous research and begins to address some of the gaps in literature. The research it presents indicates that the Bengali community in the UK remains connected to their place of origin (Sylhet), not least through the exchange of food and plants, however what these food and plants mean vary according to place and generation. If academics and professionals are to understand therapeutic food-plant use among diaspora it is essential to look at the existing links with 'home' countries and changes in knowledge and practice across generations and ages. The implications of this research are important for ethnobotany, migrant and international health studies, public health and food studies.

This thesis addresses the dynamics between drug, solvent and membranes and the challenges in overcoming the stratum corneum barrier using different chemical penetration enhancers. Although the mechanisms of skin penetration have been subject of research for many decades, there are a lot of questions yet to be answered. The purpose of this investigation was to give insight about the mechanisms of partition and diffusion of a drug into the membrane. Synthetic membranes are a useful and cheap approach to understand those mechanisms owing to their low complexity compared to human tissue. Solubility, solvent uptake and evaporation behaviour of the solvents were performed to study interactions between solvents, actives and membranes. These interactions will dictate the permeation and delivery of actives. Furthermore, spectroscopic techniques such as ATR-FTIR were of extreme importance for the further investigation of solvent-membrane interactions and also to study the diffusion of different species (solvents and actives) across silicone membranes. Assessment of permeation using more complex membranes such as porcine ear skin, which have been shown to be good models to assess drug permeation, includes in the experiment, the complexity inherent to biological tissue. Understanding the permeation of actives and solvents in vitro is the first step to understand drug delivery in vivo. A recent spectroscopic technique allows the non-invasive investigation of real time drug delivery in vivo. Confocal Raman spectroscopy allows the profiling of actives inside the skin and is able to differentiate between formulations therefore expanding our understanding of the mechanisms involved in drug absorption after its application to the SC. The findings indicate the formulation components play a crucial role in the delivery of actives in vitro and in vivo and an informed selection of solvents is one of the strategies to design optimal topical formulations.

Glioblastoma is the most common and most biologically aggressive primary brain tumour in adults. In spite of tremendous investment into research which has led to the development and application of novel diagnostic and therapeutic measures in the management of glioblastoma, the prognosis is still dismal with median survival time of about 12 – 15 months. Also, the success of most cytotoxic drugs clinically employed in the treatment of glioblastoma is greatly limited by their dose-limiting toxicity which typically manifests as clinically significant reduction in blood cell counts. The aim of this study is to demonstrate that a high dose nanoparticle formulation of the cytotoxic drug lomustine using a self-assembling chitosan amphiphile, quaternary ammonium palmitoyl glycol chitosan would lead to improved survival outcomes without a commensurate increase in toxic effects. The novel nanoparticle based lomustine formulation employed in this study enabled the administration of a lomustine dose (13 mg kg-1) 10 times higher than the dose (1.2 mg kg-1) achievable with an ethanolic formulation of lomustine. Human glioblastoma tumour bearing mice treated with the high dose formulation had a mean survival time of 33.1 days while the mice treated with the low dose formulation had a mean survival time of 22.5 days after intravenous administration of the drug once daily for 10 consecutive days. The increased (1.5 times longer) survival time resulting from treatment with the nanoparticle based high dose formulation was not accompanied by an increase in gross toxic effects. Thus, the nanoparticle based formulation afforded the administration of lomustine in a continuous high dose schedule which led to beneficial therapeutic outcomes. In addition, three self-assembling peptide amphiphiles were synthesised and characterised for potential application in the transport and delivery of therapeutic molecules to the brain for the treatment of intracranial tumours.