Shape-based approaches have many potential areas for development in the future for application to in silico pharmacology. Further exploration of the role of molecular shape may lead to better understanding of the substrate specificity of enzymes and the possibility to reduce toxic effects that may be caused by ligands binding to undesired target proteins. Methods exploiting molecular shape for activity and toxicity prediction might have a great influence on the drug discovery process. There are different approaches that might be used for this purpose, e.g. shape fingerprints and shape multipoles. Both methods describe the shape of molecules, discarding any chemical information, using numerical values. Focusing only on shape can lead to identifying novel core structures of molecules, with improved properties. Molecular fingerprints are binary bit strings that encode the structure or shape of compounds; shape is measured indirectly by alignment to a database of standard molecular shapes – the reference shapes. The Shape Database should represent a wide range of possible molecular shapes to produce accurate results. Therefore, this was the main focus of the investigation. The shape multipoles method is a fast computational method to describe the shape of molecules by using only numbers and therefore it requires low storage needs and comparison is performed by simple mathematical operations. To describe the shape, it uses only 13 values (3 quadrupole components and 10 octupole components). The performances of both methods in grouping compounds based on shared biological activity were evaluated using several test sets with slightly better results in case of shape fingerprints. However, the shape multipole approach showed potential in finding differences in shape between enantiomers. Among the possible applications of the shape fingerprints method are solubility prediction (on comparable level as well-established methods) and virtual screening.

In HER-2 positive metastatic breast cancer, the proto-oncogene Bcl-3 was found to be overexpressed. Bcl-3 acts as a transcriptional co-activator: it forms a ternary complex with DNA and homodimer (p50)2 and stimulates the transcription of a different panel of genes in the metastatic progression of the breast cancer. Its exact role in endogenous tumours is still unknown. In vivo knockdown studies shown, that Bcl-3 deficiency did not affect the primary tumour, but it reduced the occurrence of metastases by 80% without any effects on the normal mammary gland function. Patients with this type of tumour have a poor prognosis, do not respond to the typical treatment or show resistance and side effects to the usual therapeutic options. Hence, there is an urgent need to find new candidate drugs. A virtual screening targeted against a newly identified Bcl-3 binding pocket allowed the identification of 10 molecules, tested in vitro cell based assays. Four molecules were active. In this thesis, different analogues of these four hit compounds were synthesised. Based on docking studies, another two scaffolds were designed. The non-cytotoxic profile of the new analogues was assessed using the cell titer blue assay. The activity of the compounds was established using the colony forming assay (CFA). To prove the Bcl-3/p50 interaction, immunoprecipitation and co-immunoprecipitation were performed. None of the compounds was cytotoxic. Some of them exhibited a promising activity in the CFA. One molecule exhibited an interesting activity profile both in vitro and in vivo and in pharmacokinetic studies and it has been forwarded into full pre-clinical development.

Hydrogen sulfide (H2S) is the simplest endogenously produced thiol and has an indispensable role in cardiovascular homeostasis. It has been shown that exogenous H2S supplementation protected the heart against myocardial ischaemia/reperfusion injury through a mechanism which is yet to be defined. In this thesis, it was hypothesised that controlled application of thiol/H2S donors at reperfusion would mitigate acute myocardial infarction. We sought to characterise the cardioprotection and molecular targets of three H2S donors (mesna, GYY4137 (a slow-releasing, non-mitochondrial targeted H2S donor) and AP39 (a mitochondria-targeting H2S donor)). This characterisation was conducted using a broad range of experimental models and techniques including anaesthetised rat model of ischaemia/reperfusion injury, Western blotting and mitochondrial studies using isolated cardiomyocyte mitochondria, namely subsarcolemmal and interfibrillar mitochondria. Mesna did not limit infarct size when it was given pre-ischaemia or at reperfusion. GYY4137 and AP39 significantly limited infarct size when given specifically at the time of reperfusion through different mechanisms. Cardioprotection established by GYY4137 was mediated mainly by triggering of PI3K/Akt/GSK-3β signalling at reperfusion with partial dependency on eNOS activity. Selective mitochondrial delivery of H2S at reperfusion using AP39 had no effect on Akt, eNOS, GSK-3β and ERK1/2. In isolated mitochondria, AP39 inhibited Ca2+-sensitive opening of PTP in subsarcolemmal and interfibrillar mitochondria through attenuation of mitochondrial reactive oxygen species generation. The studies presented in this thesis provided novel mechanistic insights into cardioprotection by H2S. These studies suggest that targeted delivery of H2S represents a novel and effective adjunctive therapy to ameliorate the injurious effects of reperfusion which contribute to acute myocardial infarction.

A STUDY OF THE ROLE OF MICRORNAS IN INFLAMMATORY BOWEL DISEASE: THE EFFECT OF MIR-31 DYSREGULATION IN THE EXPRESSION OF TSLP IN ULCERATIVE COLITIS By Andrew Cia ridge Our current understanding suggests that Inflammatory Bowel Disease (lBD) is an immunological mediated disease triggered by unknown environmental factors in those who are genetically susceptible. MicroRNAs (miRNAs) are a class of small non coding RNAs that play a critical role in many immunological pathways by regulating gene expression. To date over 70 miRNAs have been identified as having a differential expression in IBD although their roles remain unlmown. Using microarray and RT-qPCR we have identified the differential expression of 7 miRNAs; miR-31, -146b, -194, -200b, -223, -375 and -422a, in the sigmoid mucosa of treatment na'ive Ulcerative Colitis. Of these, the expressions of miR-31 and -223 are also increased in mucosal Iymphocytes and the CD4+CD25in •• rm.dia •• subfamily. Using in silica analysis, thymic stromal Iymphopoietin (TSLP) is identified as a target for 4 of the 7 miRNAs; miR-31, -223, -194 and -422a. TSLP has been shown to be increased in TH2 mediated disorders of the lung and skin and implicated in epithelial barrier homeostasis and Iymphocyte function in response to luminal flora. Here we show for the first time the expression of TSLP by healthy Iymphocytes and a decreased expression in mucosal biopsies and mucosal Iymphocytes in active uc. In addition, using a human Iymphocyte cell model we demonstrate an increased expression of miR-31 in stimulated Iymphocytes which is inversely related to a decreased expression of TSLP mRNA and an even larger Abstract effect on the expression of TSLP protein in the same cells. Selective inhibition of miR-31 prevents the decrease in TSLP in stimulated Iymphocytes. The increased expression of miR-31 in UC actively decreases the expression of TSLP, a factor associated ' with epithelial homeostasis and less destructive inflammation. Manipulation of the miR-31 / TSLP pathway offers a potential therapeutic strategy for uc.

The aim of this research was to account for the meaning practitioners and clients gave to Guided Human Equine Interactions (GHEI). GHEI is a term that covers a range of interactions between human and horse for the purpose of facilitating emotional and social development in the human participants. There is paucity of research to evidence assumptions inherent in practice and little is known about the meaning participants give to GHEI. Despite this, practice is rapidly expanding in Britain following similar growth in America where GHEI originated. An underlying premise in this thesis is that the discourses employed to account for equine involvement in therapy and learning will construct practice. Therefore, language samples were obtained from interviews with ten GHEI practitioners, two GHEI websites, and from the personal experience of one client through their diary entries. The data were interrogated, analysed and interpreted according to a discursive strategy based on work by Ballinger and Cheek (2006). Five dominant discourses (metaphysical, sensory, social, mental distress and symbolic) were surfaced across three studies. These discourses served differing functions and woven together, formed a discursive framework that accounted for the meaning practitioners and clients gave to GHEI. This incorporated not only the cognitive, affective and behavioural elements seen in mental health interventions such as Cognitive Behavioural Therapy (CBT), but also included the added dimensions of spiritual, sensory and symbolic meaning. These added factors provided for a more holistic experience for the client than office based therapy alone. The insights gained suggest the horse has an impact, not only on the spatial distancing between the client and practitioner, but also on their relational distancing. This distancing enables the client to be positioned as the expert at the centre of the intervention. However, expert client positioning was only apparent as long as the practitioner did not draw on a dominant mental distress discourse, or tried to market GHEI. There was a lack of risk discourse across all studies, which was considered an important added factor in positioning the client as expert. Spatial and relational distancing may assist GHEI practitioners in being alongside individuals who decline to engage in more formal interventions It was noted that adaptation had taken place in the discursive construction of GHEI from practice developed in America. This had led to culturally relevant practice being accounted for in Britain. It was concluded that the inclusion of a horse in therapeutic and learning practice is at an intersection between past discourses where the metaphysical, such as spirit and healing, were privileged and contemporary discourses where social care and treatment of mental illness are authoritative. There is a symbiotic relationship between the function of the horse in accounting for the meaning given to GHEI and the function of dominant discourses. This results in a centaur-like enmeshment of human and horse.

Exogenous surfactant therapy has dramatically improved survival in extremely preterm infants, however the turnover of exogenous and synthesis of endogenous surfactant components are still poorly understood in this group. Additionally there is evidence for this patient group that improving nutrition improves long-term outcomes in respiratory function, growth and neurodevelopment. Phosphatidylcholine (PC) is the dominant phospholipid in both surfactant and in plasma and can be synthesised from choline by one of two pathways: the CDPcholine pathway, which is present in all nucleated cells, or by three sequential methylations of phosphatidylethanolamine in the PEMT pathway, which is localised to hepatocytes and is the primary source of polyunsaturated PC species and de novo synthesis of choline. This study quantified choline phospholipid metabolism and pulmonary surfactant kinetics in preterm infants in vivo. Children aged between 23 and 28 weeks gestation and in receipt of exogenous surfactant were intravenously infused with [methyl-D9]choline chloride within 48 hours of birth. Lipid extracts from sequential plasma and endotracheal aspirate samples were then analysed by electrospray ionisation tandem mass spectrometry (ESIMS/ MS). Fractional incorporation into newly synthesised PC species is demonstrated rapidly in plasma samples at a higher rate than previously reported in adults, indicating a high level of hepatic activity for CDP-choline. Analysis of the PC species derived from the PEMT pathway shows significantly lower flux in this pathway than reported in adults. Finally incorporation into surfactant PC species is very low initially before rising slowly over several days and with the rapid changes in other acidic phospholipids suggests a rapid recycling of components of the exogenous surfactant not equilibrating with the CDP-choline pathway thereby providing evidence for the first time of differing rates of exogenous surfactant recycling versus de novo synthesis in the human preterm infant. This study proves that the technique works in the clinical environment, is sensitive and rapid enough to provide data in a clinically relevant timeframe, opening the possibility for translational use to identify biomarkers for disease progression.

Targeted and controlled delivery of therapeutic agents on demand is pivotal in realising the efficacy of many pharmaceuticals. The design and fabrication of a novel, electrically-addressable, porous structure-based drug delivery device for the controlled release of therapeutic proteins and peptides, are described in this thesis. The initial prototype microdevice design incorporates a porous polysilicon (PPSi) structure as a drug reservoir. Two alternative methods were investigated to fabricate the PPSi structure: i) the chemical stain etching method; ii) a reactive ion etching (RIE) method through a masking template. Random pores, with irregular pore shape and size in the micro- to mesoporous regime (< 50 nm), were obtained using the stain etching method but this method suffered from poor reproducibility and non-uniformity. Two novel RIE approaches were investigated to fabricate ordered PPSi structures; two different masking templates were investigated – a porous anodic alumina (PAA) and a metal mask with hexagonally arranged holes produced by a novel nanosphere lithography (NSL) technique. A quasi-ordered PAA template with pore diameters in the region of 50 nm was fabricated but was not suitable for the subsequent proposed RIE process. By using the NSL technique, quasi-ordered PPSi structures with tapered pore profiles, were obtained. This is the first demonstration of the fabrication of PPSi with ordered pores of sizes in the macropore range of ~ 370 nm. A revised silicon-based prototype microdevice was designed and fabricated. The microdevice incorporates a nanostructured, quasi-ordered porous silicon (PSi) as a drug reservoir and an integrated heater and temperature sensor as an active control mechanism. The PSi structure was fabricated using a modified NSL technique and a Bosch-based RIE process. Hexagonally arranged cylindrical pores with diameters between ~75 nm and ~120 nm, and depths in the range of ~330 nm and 500 nm, were obtained. The novel fabrication techniques investigated here are simple and versatile; both p-type and n-type PSi structures have been successfully fabricated. Proof-of-concept studies, using the revised prototype drug delivery microdevices, suggested that the nanostructured PSi would be suitable for the passive release of an intermediate-sized (~23 000 Dalton) model protein. It is envisaged that the microdevice has the potential to deliver osteoinductive growthfactors, on demand, to the site of fracture, in a controlled and sustainable manner, as a first step to an intelligent therapeutic system for skeletal regeneration.

Organophosphate compounds have been widely developed as pesticides (e.g. paraoxon) and also as chemical warfare agents (nerve agents, e.g. sarin). These compounds rapidly inhibit the enzyme acetylcholinesterase (AChE), causing overstimulation within the cholinergic nervous system. If left untreated, this can be fatal. Current medical countermeasures to nerve agent poisoning consist of pretreatment with pyridostigmine and an emergency therapy comprising atropine, diazepam and pralidoxime. As well as a replacement pretreatment for pyridostigmine, physostigmine has been proposed as a component of a next generation of therapy to nerve agent poisoning, along with scopolamine and HI6. In animal studies, this therapy has been shown to lessen the level of incapacitation and increase survivability post poisoning with nerve agent. The exact mechanism of action of physostigmine in this combination is as yet unclear. The primary aim of this study was to test the hypothesis that the beneficial effect of physostigmine in the proposed therapy is due to reversible inhibition of AChE, thereby protecting it from irreversible inhibition by nerve agent. To test this, extracellular field potentials were recorded from the molecular layer of the dentate gyrus in an in vitro slice model developed from the guinea pig. This response was shown to be modulated by the application of physostigmine and the nerve agent sarin and interactions between the two inhibitors were characterised. The results provided evidence for protection of ChE by physostigmine. The functional response (field potential) was related to cholinesterase activity measured in slices exposed to sarin. This is the first evidence of the mechanism of action of physostigmine protection against nerve agent in the CNS. Not only will these results support the use of physostigmine as a pre-treatment, it also supports its use as a possible immediate therapy.

Occupational therapy (OT) pre-registration education in the United Kingdom (UK) stands at the intersection of the fields of higher education (HE) and professional practice. It is subject to various government agendas including an ongoing commitment to widening participation in HE and to diversifying the health and social care workforce to reflect modern cultural diversity. Both have contributed to a changing profile in the OT student population and in 2005, 67 percent of the intake was mature (College of Occupational Therapists, 2007b), and increasing numbers are entering with ‘non-traditional’ academic backgrounds, an umbrella term which subsumes a variety of entry qualifications. The early weeks of study in HE can prove challenging to students as they settle into the new learning environment and begin to comprehend the expectations held of them (Yorke, 2005). It has been suggested that those from non-traditional academic backgrounds may find this transition, particularly the need to take a high level of responsibility for their own learning, difficult as a result of the skills, experiences and expectations accumulated throughout their pre-entry education (Sambell and Hubbard, 2004). While small-scale studies suggest that OT students from such backgrounds are as academically successful as traditional school-leavers at graduation (Howard and Jerosch-Herold, 2000), there is little evidence offering insight into how they actually experience and negotiate the demands of their programme. Recognising that learning and teaching are embedded within the milieu in which they occur, this longitudinal research adopted a case study methodology to capture complexity and understand the issue within its natural context (Yin, 2003). In an instrumental single-case design (Stake, 1995), a neither unique nor extreme undergraduate OT programme became a vehicle for exploring the educational experiences of students with non-traditional academic backgrounds. Thirteen volunteer participants were drawn from a single cohort in one of the UK’s research intensive universities. Data were collected via initial focus groups exploring pre-entry educational experiences and expectations of studying in HE, reflective diaries recording educational experiences that participants considered significant or meaningful, and one-to-one semi-structured interviews conducted towards the end of participants’ first and third years of study which focused on exploring their learning experiences. Supplementary and contextual data were provided by analysis of institutional, school and departmental documents to provide insight into the culture and practices of the learning context and a progression routes study which considered the entry qualifications, progression and exit awards of four cohorts of OT students from a range of educational backgrounds. The nature of students’ entry qualifications or academic background were found to have no statistically significant impact on whether they passed at Level 4, 5 or 6, or achieved a ‘good’ (upper second or first class) honours degree, although male students and those from amongst the lower socio-economic groups had significantly poorer academic outcomes at all levels of analysis. Theoretical thematic analysis (Braun and Clarke, 2006) of qualitative data underpinned by Bourdieu’s (1990b) theory of practice highlighted that students’ educational experiences were much less influenced by the nature of their academic backgrounds than by the congruence of individual dispositions or habitus, born out of social provenance, with the dominant culture of the particular field of HE they had entered. Emerging codes converged to represent themes suggesting clusters of shared experience amongst some participants, while examination of each individual dataset revealed varying positional tendencies and trajectories within the field. This research highlights the important roles played by academic, linguistic, social and practice-oriented capital in the way that students developed a feel for and learned to play ‘the game’ and present knowledge and understanding in the form ‘legitimated’ by the field. Juxtaposing the nature and expectations of the new field in relation to those previously occupied by individual participants and the established habitus each brought with them helped to illuminate the situation and adds a new dimension to understanding individual experiences of learning in HE.

Natural products have been the source of numerous leads for several drugs. As these natural products are often isolated in small quantities, it is necessary to produce them synthetically to allow testing for biological activity. Furthermore, synthesis allows the preparation of unnatural analogues for SAR studies. Cyclic peptides represent an important family of biologically active natural products. The hepta- and octacyclopeptides sanguinamide A and sanguinamide B were recently isolated in submicromolar amounts by the Molinski group. The lack of material prevented biological evaluation of the natural products. For this reason and to confirm the structural elucidation we have targeted the total synthesis of sanguinamides. In addition to two proline residues, sanguinamides A and B include heterocycles and natural L-amino acid residues. We have completed the total syntheses of sanguinamides A and B; however the synthetic rotamers differed in both cases from the natural rotamers. We have investigated the influence of macrocyclisation on cis/trans conformational preference of the proline residues for the synthesis of sanguinamide A. We attempted several isomerisations and calculated the relative energies of the different sanguinamide conformers. [D-Ile]-Sanguinamide A, Cys(tBu) analogue of sanguinamide A and the synthetic sanguinamide B displayed antibacterial activity while the synthetic trans, trans-sanguinamide A displayed mild tyrosine kinase inhibitory activity. While extracted stylissamide A showed inhibition of translation during the elongation step, even though being structurally identical to the natural product, the synthetic compound prepared by macrocyclisation from a linear precursor was found to be totally inactive. Histones undergo different types of covalent modifications on the N-terminal tails such as acetylation, phosphorylation and methylation. Histone modification is a major mechanism of regulation in gene expression, replication and repair. Deregulation of histone modifications leads to cancer progression and therefore, inhibitors of enzymes which are able to catalyse the addition and removal of these epigenetic marks have therapeutic potential for treating cancer. An enzyme of particular interest is the family of zinc-dependent histone deacetylases (HDACs) that remove acetyl groups from acetylated lysine residues. Depsipeptides were prepared as HDAC inhibitors. We will ii present our total synthesis of largazole along with a range of analogues and discuss the SAR obtained from HDAC and cell proliferation assays. We elucidated the stereochemistry of burkholdac B by total synthesis of three diastereomers. The diastereomers made along with the natural product were tested as HDAC inhibitors. We are interested in inhibitors of lysine-specific demethylase 1 (LSD1) which is a different kind of epigenetic enzyme involved in demethylation of histone proteins in chromatin. Tranylcypromine is known to be an LSD1 inhibitor. Analogues of PCPA have been synthesised in order to explore the structure-activity relationships of this inhibitor. Analogues were also prepared and tested as LSD1 inhibitors.