Abstract Clinical trials are at the centre of advances in our understanding of stroke and its optimal treatment. In this thesis the uses and properties of outcome assessment scales for stroke trials are described, with particular attention given to the modified Rankin Scale (mRS). Through comprehensive literature review I will show that mRS is the most frequently used functional outcome scale in clinical trials but efficacy of the scale is potentially limited by inter-observer variability. Using a “mock” clinical trial design I demonstrate that inter-observer mRS variability in contemporary practice is moderate (k=0.57). Adding these data to systematic review of published data, confirms an overall moderate inter-observer variability across ten trials (k=0.46). Differing strategies to improve mRS reliability will then be described. I will outline development of a bespoke training package, international training scores across 2942 raters again confirms suboptimal reliability (k=0.67). A pilot trial using endpoint committee review of video recorded interviews demonstrates feasibility of this approach. Attempts to improve reliability by deriving mRS from data recorded in patients’ hospital records are not successful (k=0.34). In the final chapters I present a novel methodology for describing stroke outcomes – “home-time”. This measure shows good agreement with mRS, except at extremes of disability. Finally to put mRS in a historical context, the career of John Rankin and the development of his eponymous scale is recounted.

As resistance to current therapies remains one of the major hurdles to the successful treatment of advanced colorectal cancer, we need to understand the mechanisms by which cancer cells evade therapy-induced cell death. I have investigated whether there is a link between epithelial cell adhesions, and acquired resistance to 5-fluorouracil (5-FU). I compared three pairs of human colorectal 5-FU-sensitive and -resistant cell lines, and investigated whether there was a direct role for E-cadherin and/or the Src family kinase, c- Yes (which is co-amplified with thymidylate synthase) in promoting resistance to 5-FU. I found that while knockdown of c-Yes expression had no effect, disruption of E-cadherin using a blocking antibody caused a reduction in colon cancer cell proliferation and some re-sensitisation to 5-FU. The resistant cells displayed intrinsically higher activities of putative survival pathways, namely the PI3-kinase/Akt and the MEK/MAP kinase pathways, and these were suppressed when E-cadherin function was blocked. Furthermore, the resistant cells displayed a greater dependence on signalling via the PI3- kinase/Akt pathway for their survival. Finally, preliminary experiments established a possible link between the integrity of E-cadherin-mediated cell-cell junctions, signalling through the PI3-kinase/Akt pathway and nuclear localisation of the apoptotic regulatory tumour suppressor protein p53 in modulation of 5-FU-resistance.

Proteinase-activated receptor 2 (PAR-2) belongs to a novel family of G-protein coupled receptors that are unique in their activation mechanism by which a proteolytic cleavage at N-terminus by a proteinase reveals a ‘tethered ligand’ to activate the receptor. Albeit at a low level, PAR-2 is extensively expressed in normal and pathological brains, including the hippocampus. Qualitative studies into the expression of PAR-2 in several disease conditions, including ischaemia, HIV-associated dementia, Parkinson’s disease, Alzheimer’s disease, as well as multiple sclerosis, have suggested that PAR-2 plays either degenerative or protective role depending on in which cell type an increase in PAR-2 expression is observed. However, its potential roles in modulating neuronal excitability, synaptic transmission as well as network activities remain to be determined. Utilising the whole-cell patch clamp recording technique, I demonstrate, for the first time, that the activation of PAR-2 leads to a depolarisation of cultured hippocampal neurones following application of SLIGRL (100microM), a selective PAR-2 activating peptide (5.52 ± 1.48mV, n=16, P<0.05) and paradoxically a reduction of spontaneous action potential (AP) frequency (29.63 ± 5.03% of control, n=13, P<0.05). Pharmacological manipulation reveals that the PAR-2-mediated depolarisation is most likely dependent on astrocytic glutamate release, which takes effect on ionotropic glutamate receptors. In addition, an overt depression of synaptic transmission among the cultured neurones upon PAR-2 activation is more likely to cause the reduction of spontaneous APs. In further experiments, I show, for the first time, that the activation of PAR-2 induces a long term depression (LTD) of glutamatergic synaptic transmission at the Schaffer collateral-to-CA1 synapse in acute hippocampal slices following SLIGRL (100microM) application (80.75 ± 2.54% of control at 30 minute, n=12, P<0.05). Additionally, this novel form of LTD is independent of metabotropic glutamate receptors but mediated by NR2B subunit-containing N-methyl-D-aspartic acid (NMDA) receptors. It is also suggested from these experiments that glial-neuronal signalling is contributing to this novel form of LTD. In the final set of experiments, by monitoring field potentials in the stratum pyramidale of the CA3 area in acute hippocampal slices, I demonstrate that PAR-2 activation depresses the frequency of epileptiform activities induced by the application of 4-AP/0 Mg2+, an in vitro model of epilepsy (1.53 ± 0.21Hz to 1.18 ± 0.17Hz, n=13, P<0.05, 100microM SLIGRL). In contrast, PAR-2 activation has no effect on the frequency of epileptiform activities induced by bicuculline (0.14 ± 0.03Hz to 0.13 ± 0.03Hz, n=6, P>0.05, 100microM SLIGRL). In summary, in this thesis, I demonstrate that PAR-2 modulates neuronal excitability and depresses excitatory synaptic transmission in the hippocampus. These data indicate that PAR-2 may play a regulatory role in neuronal signalling at single cell level by controlling neuronal intrinsic properties, as well as at synaptic level by tuning excitatory synaptic strength, which ultimately affects global excitability in the neural circuits as a whole. Therefore, this investigation suggests a novel physiological/pathophysiological role for PAR-2 in the brain. These data may reveal valuable clues for the development of drugs targeting a novel and potentially promising candidate.

The receptor for advanced glycation endproducts (RAGE) is a 35-kDa polypeptide of the immunogloblin superfamily that has been implicated as a mediator of both acute and chronic vascular inflammation. RAGE has also recently been implicated in the pathology of pulmonary hypertension (PH): a rare, progressive disease of the small pulmonary arteries characterised by pulmonary vascular remodelling, thrombosis, vasoconstriction and increased pulmonary vascular resistance. A ligand for RAGE, the calcium binding protein MTS1/S100A4, is expressed in occlusive vascular lesions of patients with advanced PH. MTS1/S100A4 is upregulated and secreted by pulmonary arterial smooth muscle cells (PASMCs) in vitro on activation of the 5HT1b receptor and 5HT transporter (5HTT). Additionally, the proliferative effect of 5HT on these cells, which is mediated by 5HT1b and 5HTT, may be inhibited by antagonism of RAGE or reduced bioavailability of MTS1/S100A4. These data suggest that MTS1/S100A4, through its action at RAGE, is a key mediator of 5HT-induced hPASMC proliferation. Transgenic mice overexpressing MTS1/S100A4 are observed to develop obliterative pulmonary vascular disease and possess increased right ventricular pressure at baseline and after hypoxia when compared to wildtype mice (WT). These increases occur in the absence of an increase in pulmonary vascular remodelling suggesting that MTS1/S100A4 overexpression is associated with some other structural or functional change in the pulmonary circulation. We sought to further our understanding of the role of RAGE in pulmonary hypertension through treatment with a small molecule inhibitor of monocyte chemoattractant protein 1(MCP-1), a marker of downstream of RAGE rage activation; through further characterisation of the MTS1/S100A4 mouse in a chronic hypoxic model of PAH; and through treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability in vivo. In each case systolic right ventricular pressure (sRVP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling were measured in normoxic conditions or after a two week chronic hypoxia challenge to induce PH. These in vivo experiments were supplemented with functional studies in isolated intrapulmonary arteries to assess vascular reactivity and vascular elastance as well as studies of pulmonary fibroblast proliferation in vitro. Treatment with the MCP-1 synthesis inhibitor Bindarit produced no detectable effects upon the pulmonary response of mice to chronic hypoxia, though this study may have been hampered by difficulties with the methylcellulose vehicle. MCP-1 produced no degree of proliferation in pulmonary fibroblasts and neither augmented nor inhibited proliferation induced by 5HT. We found little evidence for the exacerbation of PH in MTS1/S100A4 mice in normoxia, hypoxia or after 4 weeks of normoxic recovery. Mean RVP was elevated above that in WT mice exposed to hypoxia. However, MTS1/S100A4 mice appeared protected against hypoxia-induced vascular remodelling and decreases in vascular elastance. No other significant differences in sRVP, RVH or remodelling were observed between strains. Vessels isolated from MTS1/S100A4 mice tended towards an enhanced contractile response to 5HT in normoxia compared with vessels in WT mice but were also more sensitive to the nitric oxide donor SNP. These differences in vasoreactivity were largely abolished by exposure to hypoxia. Treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability produced a significant reduction in sRVP after hypoxia in comparison to vehicle-dosed mice -possibly associated with the prevention of a hypoxia-induced decrease in proximal vascular elastance. However, no benefit upon the development of remodelling or the extent of RVH was observed. Vessels isolated from mice treated with sRAGE and challenged with hypoxia showed a marked increase in contractility. Further work demonstrated that sRAGE produces a small, slowly developing contraction in isolated vessels and that the maximal force of contraction to 5HT was markedly augmented in the presence of sRAGE. Finally, treatment with sRAGE did not inhibit fibroblast proliferation in vitro as induced by 5HT but was observed to cause a small degree of proliferation alone and to augment hypoxia-induced proliferation. In summary, we have reported a number of seemingly contradictory findings associated with RAGE in pulmonary hypertension. Treatment with sRAGE produced a beneficial reduction in hypoxia-induced PH associated with protection against decreased proximal vascular elastance but produced no change in hypoxia-induced RVH or remodelling as well as greatly increasing vascular contractility. MTS1/S100A4 mice show some evidence of deleterious changes to the pulmonary circulation, but these may be offset by beneficial compensatory mechanisms such as increased sensitivity to nitric oxide and protection against vascular remodelling. MTS1/S100A4 stimulates smooth muscle cell proliferation suggesting that it may involved pulmonary vascular remodelling. However, inhibition of RAGE was observed to enhance fibroblast proliferation in response to hypoxia here. Fibroblasts are important regulators of SMC proliferation in vivo. These findings therefore suggest a more complicated relationship between RAGE, its ligands and the remodelling process. Since both MTS1/S100A4 overexpression and sRAGE treatment in vivo produced findings which are difficult to reconcile using the currently employed techniques, it is clear that furthering our understanding of RAGE will require study with greater focus upon the interaction of different cell types in the pulmonary vasculature and the manner in which the disturbance of this may lead to alterations in the physical and physiological properties of the pulmonary circulation.

This developmental research study concerned how trainers, drawn mainly from the commercial (pharmaceutical) sector of the field of clinical research, shared understandings of practice in a professionally localised community, as part of their continuing professional development. Trainers in this community had a heterogeneous range of identities including full-time and part-time trainers: clinical research trainers, training managers; clinical research managers, clinical research associates, compliance managers, auditors and others. The main aim was to explain conditions shaping this community and its concept of practice. The study involved observing practice from an interlocutory position, using Cultural- Historical Activity Theory (CHAT), to reveal the cultural complexity of the concept of practice within this community. Two competing rationalities, expressed within contrasting pedagogies with associated cultural standards of compliance or conscience, were established for training:- • as a restricted technical function focussed on the transmissive delivery of content, or • as an expansive approach to organisational learning focussed on deliberative enquiry. These competing rationalities reflected the struggle of an emergent profession to establish autonomy of standards, with implications for the field of practice and wider society: establishing the moral order through a culture of conscience, based on standards of excellence or because a system of regulatory governance dominates the drive to uphold standards through a culture of compliance. A conceptual-analytical framework, substantiated by empirical evidence, was proposed to describe and analyse the concept of practice embodied in the community’s object of activity. Through demonstrating CHAT at the level of declarative conceptions, procedural models, and social discourses/interactions, a link was established between the dominant concept of practice (expressed within a transmissive pedagogy) in the community and the larger sociocultural context (compliance culture rooted in the system of regulatory governance). The contribution of this study is to show how CHAT can be applied with theoretically formulated and empirically tested evaluative tools, to reveal the richness of human experience and the complexity of human activity in terms of its cognitive and cooperative social elements, identified as objective regularities unique to the activity system under investigation.

The aim of the research was to investigate the concepts of, and attitudes towards, people with physical disabilities held by occupational therapy (OT) students, so that a theory of professional attitudes and professional action could be developed. The research was building on previous research by the author, which found that OT students tended to have a maternalistic and nurturing view of disabled people, and also as a response to issues raised by the social model of disability which questioned whether OT was an oppressive or empowering profession. Utilising an integrated methodology, the research sought to address the following research questions: what, amongst OT students, is a 'professional' attitude towards disabled people? are the attitudes of OT students towards disabled people any different from those of other students? do these attitudes change over time? are there any differences in the 'personal' and 'professional' attitudes of OT students towards disabled people? how accepting of disabled people are OT students, would they be willing to work with disabled people as colleagues? is there an hierarchy of relationships for people with different impairments? what does the 'professional' attitude mean in practice? how does this 'professional' attitude develop? what factors influence its development? does contact with disabled people have any effect on attitudes? do OT students express attitudes and values which oppress or empower their disabled clients? A case study approach was used with a variety of data collection methods. The main focus of the study was the collection of data, using a questionnaire and a series of interviews, from a cohort of OT students throughout the 3 years of their OT degree. The questionnaire included the Attitudes Towards Disabled People Scale, a suitability for OT training scale, and a semantic differential exploring stereotypes of disabled people. Data were also collected from other groups of OT students comparing personal and professional attitudes and attitudes in terms of social distance, using the Disability Social Distance Scale. Comparative data was collected from non-OT students. In order to explore attitudes in greater depth a small group of students was selected from the main OT cohort and interviewed about their attitudes and approaches to disabled people at 3 points during their studies. Analysis of the data revealed that the OT students held highly positive personal and professional attitudes towards disabled people. These attitudes were also demonstrated by the use of an empowering, client-centred approach to OT interventions. However, the OT students had a tendency to focus on an individualistic and personal tragedy approach to disability. This individualistic approach might result in oppressive practice. The findings were used to develop a conceptual framework for OT interventions with disabled people which should allow therapists to articulate and develop their practice within an empowering framework.

Based on the study of 34 healthy and 19 osteoarthritic knees in three different age groups (early, middle and late adulthood), this thesis reports the discovery of the potential of knee acoustic emission (AE) as a biomarker for quantitative assessment of joint ageing and degeneration. Signal processing and statistical analysis were conducted on the joint angle signals acquired using electronic goniometers attached to the lateral side of the legs during repeated sit- stand-sit movements. A four-phase movement model derived from joint angle measurement is proposed for statistical analysis, and it consists of the ascending- acceleration and ascending-deceleration phases in the sit-to- stand movement, followed by the descending-acceleration and descending-deceleration phases in the stand-to-sit movement. Through the quantitative assessment of joint angle signals based on the four-phase model established, statistical differences of different knee conditions related to age and degeneration were discovered based on cycle-by- cycle variations and movement symmetry. For AE burst signals acquired from piezo-electric sensors attached to the knee joints during repeated sit-stand-sit movements, the statistical analysis started from the quantity of AE events in the proposed four movement phases and extended to waveform features extracted from AE signals. While the quantity of AE events was found to follow certain statistical trends related to age and degeneration in each movement phase, detail statistical analysis of AE waveform features yielded the peak amplitude value and average signal level of each AE burst as two most significant features. An image based knee AE feature profile is presented based on 2D colour histograms formed by the peak amplitude value and average signal level in four movement phases. It provides not only a visual trend related to knee age and degeneration, but also enables visual assessment of the

Mucosal delivery of biotherapeutics as a non-invasive means of delivery could potentially be enhanced using nanoscale therapeutic carriers. However, nanoparticles do not readily cross the mucosal barriers, with the epithelium severely restricting their translocation into the systemic circulation. Translocation of nanocarriers across the mucosae may be improved by employing ligands capable of exploiting receptor-mediated cell uptake processes. This work explores the potential of vitamin B12 transport pathway for mucosal delivery of B12-decorated model nanoparticles and investigates the cell trafficking pathways involved in these processes. Cyanocobalamin (vitamin B12) was chemically modified to produce the α-ω-aminohexylcarbamate B12 derivative – as a suitable bioconjugate – which was then conjugated to fluorescent, carboxy-functional nanoparticles (<200 nm). These systems were applied to intestinal Caco-2 monolayers, expressing the relevant proteins involved in B12 trafficking and endocytic processes. Vitamin B12-conjugated nanoparticles demonstrated notably increased cell uptake and transport capacities in Caco-2 monolayers, compared to their unconjugated counterparts. Importantly, the cell uptake of B12-conjugated nanoparticles occurred via a pathway that was different to that used by both soluble B12 and unmodified nanoparticles. B12-conjugated nanoparticles circumnavigated the lysosomal compartment and were transported by a route perturbed by caveolae-specific inhibitors, unlike the clathrin-mediated trafficking of soluble vitamin B12. These previously unreported observations are important and have potential implications in the field of bioconjugate and nanocarrier-mediated drug delivery. Epithelial cell uptake and transport of B12-conjugated nanoparticles was also investigated in airway-derived Calu-3 cells, shown to express the B12-intrinsic factor receptor, cubilin. B12-nanoparticles showed markedly larger cell uptake and transport capacities in Calu-3 layers, with B12-conjugation dramatically influencing the intracellular trafficking of the particles in a similar way to Caco-2 cells. The B12 endocytotic machinery therefore shows potential for delivery of nanocarrier-associated therapeutics across the airways. Present work also aimed to establish methods for the production of stable nano-sized protein crystals displaying a slow drug release profile, based on evidence that protein therapeutics which are formulated in this manner, offer beneficial drug-delivery properties and can be targeted using biological ligands. Nano- and micron-sized insulin crystals were prepared by an adaptation of the batch crystallisation approach. The crystals were stabilised using a chemical crosslinker, namely β-[Tris(hydroxymethyl) phosphino] propionic acid (THPP). The resulting insulin crystals were generally stable in the absence of crystalisation buffer, displayed a slow-release profile, with the released insulin retaining its biological activity. This study therefore shows that formulating protein bioactives in this form is possible and may provide a promising strategy to develop biotherapeutics with improved drug delivery properties.

Guanylyl cyclase - cyclic guanosine monophosphate (cGMP) signalling has been demonstrated to play an important role in the endogenous cardioprotective signalling of the myocardium during early reperfusion. It is proposed that infarct limitation is afforded by elevating cGMP and activating protein kinase G and its distal targets. It was hypothesised that increasing the activity of soluble guanylyl cyclase (sGC) would limit myocardial ischaemia-reperfusion injury. Primarily using the rat isolated perfused heart method, the experiments reported in this thesis investigate the role of exogenous targeting of sGC during early reperfusion, specifically exploring targeting different redox states of the enzyme and their effects on myocardial infarct size. The novel sGC stimulator BAY 41-2272 and activator BAY 60-2770 were selected to investigate this hypothesis. Both administration of BAY 41-2272 and BAY 60-2770 during early reperfusion significantly limited infarct size compared to controls. This was associated with elevated total tissue cGMP levels. Inhibition of nitric oxide could not completely abrogate this protection, but exogenous perfusion of nitric oxide along with BAY 41-2272 showed synergistic action. Oxidation of the prosthetic haem group by ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidised forms of sGC together did not afford additive protection, in fact it reduced the protection afforded compared to the individual treatments. Preliminary data also suggest that targeting the particulate form of guanylyl cyclase increases activity of Akt signalling during early reperfusion suggesting common signalling between soluble and particulate guanylyl cyclase. These data suggest that targeting sGC during early reperfusion can afford cardioprotection by limiting infarct size. The relationship between cGMP elevation and infarct size needs to be investigated further. Nevertheless, these studies suggest that sGC may be a tractable target for the therapeutic management of acute myocardial infarction.

Tamoxifen has made a significant contribution in decreasing breast cancer related deaths for over 30 years and until recently was the gold standard for treatment of ER positive breast cancer (Fisher et al, 1998). Resistance to tamoxifen is however a considerable issue with cells utilising a number of molecular mechanisms to bypass the growth inhibition caused by blocking ER activity. This move towards an anti-hormone resistant state from an antihormone responsive state is associated with the transition to a much more aggressive phenotype including increased proliferation and also invasiveness. Thus unfortunately, acquisition of tamoxifen resistance is not only associated with a recurrence of breast cancer, but this cancer is also much more aggressive in nature with fewer treatment options available than the initial cancer. This study has identified Lyn kinase as increased in tamoxifen resistant breast cancer cells compared to oestrogen-responsive breast cancer cells. Subsequent removal of Lyn kinase from tamoxifen resistant breast cancer cell lines using RNAi technology led to a significant decrease in cell proliferation, increased apoptosis and also a decrease in migration and invasion. A mechanism has been suggested whereby Lyn kinase is involved in a calcium dependent zinc wave which ultimately leads to the activation of tyrosine kinases. Metastasis to other sites in the body is ultimately responsible for fatalities due to breast cancer and so being able to block its action is key to treating breast cancer in the clinic. Therefore identifying Lyn kinase as a gene target that leads to the advancement of breast cancer to a more aggressive state provides a powerful tool for treating breast cancer in the clinic.