Multiple receptor tyrosine kinases (RTK) have been shown to be over-expressed in the malignant Hodgkin Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). However, the activation status of many of these RTKs has not been studied. Furthermore, the contribution of aberrant RTK activation to the pathogenesis of HL is currently unknown. In chapter three, I have shown using a human phospho-receptor tyrosine kinase array that HL cells are characterised by the activation of multiple RTK. I have confirmed the over-expression and activation in HRS cells of two of these RTK, MET and RON and provided preliminary evidence that MET is negatively regulated by LRIG1 in these cells. In chapter four, I have shown for the first time that DDR1 is over-expressed in primary HRS cells. Furthermore, I have shown that in many cases, DDR1-expressing HRS cells are intimately associated with collagen, the ligand for DDR1. However, knockdown of DDR1 in a HL cell line in which DDR1 appeared to be constitutively phosphorylated revealed no detectable change in phenotype and few transcriptional changes. While exploring possible reasons for this, I identified that HL cells express multiple DDR1 isoforms including several novel transcripts. Finally, in chapter five, I have shown that HL cells are sensitive to the RTK inhibitor, dasatinib. Furthermore, consistent with the aberrant activation of multiple RTKs in HL cells, I observed that these cells were also sensitive to lestaurtinib and dovitinib, two next generation multiple-target RTK inhibitors.

This thesis examines the history of murder by poison in Scotland during the nineteenth and early twentieth centuries, in the context of the development of the law in relation to the sale and regulation of poisons, and the growth of medical jurisprudence and chemical testing for poisons. The enquiry focuses on six commonly used poisons. Each chapter is followed by a table of cases and appendices on the relative scientific tests and post-mortem appearances. The various difficulties in testing for these poisons in murder and attempted murder during the period are discussed and the verdicts reached by juries in poisoning trials considered. It is argued that murder by poison during the nineteenth and early twentietrh centuries raised particular legal and medical problems, as not only were symptoms often not recognised by doctors, but chemical testing was inadequate, and juries as arbiters of fact often did not understand the evidence that was presented to them in court during trials for poisoning. Further, the ease with which these poisons could be purchased for very small sums of money, the rise of the insurance industry, and the prominence of burial clubs all contributed to providing opportunity and motive for murder. Since poisons were easy to obtain and difficult to detect, it seems probable that poisoning was much more common than is usually accepted.

Depression is one of the most common causes of periods of disability. There is evidence suggesting that the serotonin system is involved in the pathophysiology of depression. It has been suggested that synaptic serotonin levels are reduced in depressed patients, and that pharmacological blockade with antidepressants of the serotonin transporter (SERT) would result in alleviated symptoms of depression by enhancing serotonin neurotransmission. Since depression can be treated with antidepressants that target SERT, and a recently discovered 5-HTT gene-linked polymorphic region (5-HTTLPR) of the SERT gene has been shown to predispose to depression, the SERT assumes a key role in depression. Traditionally, depression severity was assessed using psychological testing of patients. However in the last 20 years, neuroimaging techniques using magnetic resonance imaging (MRI) of brain structures and molecular single photon emission computed tomography (SPECT) evolved which appear promising to better understand the pathophysiology at the tissue level. However, preclinical data on abnormalities that involve the serotonin system are limited. The studies presented in this thesis attempt to shed more light on the feasibility of using the novel MRI technique diffusion tensor imaging (DTI), and SPECT to detect disturbances of the serotonin system. Firstly, in order to elucidate the capabilities of DTI as a research tool in the detection of conceivably mild changes in white matter involving the serotonin system, a mouse model of life-long SERT deficiency was studied. Secondly, in order to validate DTI image processing methodology, a mouse model with reportedly profound myelin dysfunction was examined. Histology techniques were applied to the same mouse brains in order to explore the tissue correlate of the DTI signal changes. Thirdly, as myelin was hypothesised to interact with the serotonin system, in vitro autoradiography of SERT in mice with widespread hypomyelination was conducted in order to test this hypothesis. Lastly, in a rat model of SERT depletion, the relative abilities of a well established SPECT radioligand, [125I]βCIT (2β-carbomethoxy-3β-(4-iodophenyl)tropane), and a relatively novel SERT tracer, [123I]ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine) were examined using micro-SPECT. The data demonstrate that DTI did not detect any changes in white matter organisation in SERT-deficient mice. Surprisingly, subtle changes in white matter microstructure were detected in mice that were haploinsufficient for SERT, i.e. heterozygous null mice, displaying a 50 % SERT reduction compared to WT as detected using DTI. On the other hand, profound hypomyelination was detected using DTI in another mouse model with white matter pathology, and correlations between DTI and histopathological markers were present, indicating that this technology provides good indications of severe pathology, while small changes, if present, may be missed. In addition, the SERT availability appeared not to be affected in mice with widespread hypomyelination. While post mortem autoradiography of SERT-depleted rats showed widespread reductions in SERT binding using dedicated specific SERT ligands, micro-SPECT using [125I]βCIT and [123I]ADAM did not show any differences. [125I]βCIT delivered good quality brain SPECT images, however analysis of [123I]ADAM scans was hampered by the poor definition of structures. Thus this thesis provides important information on the feasibility, and sensitivity of current neuroimaging modalities. In addition, methodological flaws and uncertainties in the current literature were identified, which underpins the need for improving and standardising methodological approaches, particularly in SPECT imaging.

Total knee arthroplasty is a common elective orthopaedic procedure. The surgery itself causes soft tissue and bony trauma leading to a systemic response which includes endocrinological, immunological and haematological events. This thesis aims to investigate the potential association between total knee arthroplasty and such markers of inflammation, endothelium and coagulation. The study consisted of 4 groups; group 1 underwent an uncemented total knee arthroplasty; group 2 underwent a cemented total knee arthroplasty; group 3 underwent an uncemented total knee arthroplasty but received an intra-operative infiltration of local anaesthetic; group 4 underwent an uncemented total knee arthroplasty but had a post-operative drain for 24hours. Blood sampling was undertaken pre-operatively and at day 1 and day 7 post-operatively for the white cell count, platelets, neutrophils, C-reactive protein, interleukin 6, e-selectin, soluble CD40L, tissue plasminogen activator, von Willebrand factor, CD40 and CD1442a. Statistical analysis was undertaken in the form of pair sampled t-tests between group 1 and each of the other three groups. Although there some significant changes in one or two of the variables between the groups the only variable which demonstrated a significant difference in all comparisons was the CD1442a count. The exact role of CD1442a is unclear but there evidence to suggest that it may reflect the inflammatory and thrombotic process or contribute directly to the ongoing atherothrombogenesis. During the statistical analysis it was noted that the majority of the variables showed no clear statistical difference between the groups. In chapter 7 an ANOVA / Freidman analysis demonstrated that all but one of the variables, the CD1442a count, showed no statistical difference between all four groups. This allowed all the variables to be collated and presented as the single largest cohort study to date demonstrating the effects of total knee arthroplasty on the markers of inflammation, endothelium and coagulation. All the variables assessed showed a statistically significant change from pre-operative levels to day 7 post operation. 3 In summary our studies demonstrate that total knee arthroplasty results in activation of common markers of inflammation, endothelium and coagulation. These changes may explain the increased incidence of venous thrombosis and thrombo-embolism post-operatively as well as a potential risk of venous thrombo-embolism.

This current PhD Thesis in Neuropathology focuses on the development and assessment of in vitro simulation approaches to intracerebral haemorrhage. The PhD Thesis provides a clinical and experimental neuropathological overview of intracerebral haemorrhage as well as an account of the in vitro simulation approaches to the disease, before proceeding to the presentation of the experimental work designed and performed by the author. The development of the herein presented in vitro simulation approaches to intracerebral haemorrhage was based on the use of an immortalized embryonic murine hippocampal cell-line (mHippoE-14) and its response to oligomycin-A and ferrum or haemin under appropriately selected conditions (aiming to simulate the natural history of the disease in a more reliable manner). The PhD Thesis provides a characterization of the mHippoE-14 cell-line (through a real-time cellular response analysis and a cytomorphological characterization), before proceeding to the actual experimental justification of the conditions chosen for the development of the herein presented in vitro simulation approaches to intracerebral haemorrhage, and their assessment. The latter was performed through the undertaking of: (a) real-time cellular response analysis, (b) cytomorphological assessment, (c) profiling of neuronal markers’ expression, (d) neurochemical assessment, and (e) proteomic profiling. All experiments were performed at the University of Glasgow. The current PhD Thesis also provides a critical appraisal of: (a) the utility, novelty and limitations of the developed in vitro simulation approaches, and (b) the positioning of the developed in vitro simulation approaches within the neuropathopoietic context.

Introduction: CardioPulmonary Resuscitation (C.P.R.) is a critical clinical intervention widely recognised (Laws, 2001) to evoke stress in attending clinicians. Little is known about how junior clinicians (doctors) understand their early experiences in performing C.P.R., or whether their preparation could be improved. Problem: Undergraduate medical students have traditionally reported anxiety (Duns et al., 2008) at participating in CardioPulmonary Resuscitation. A recent systematic review of best practice in C.P.R. education focused on clinical knowledge and skills, but not emotional preparation (Mosley et al, 2012). No study has critiqued whether doctors’ pre-qualification anxieties align with their clinical reality. Less is known about the extent of their post hoc support needs. Methodology: Previous studies of doctors’ experience (Morgan and Westmoreland, 2002) have used exclusively quantitative data collection. Early qualitative data on young nurses’ experience of C.P.R (Ranse and Arbon, 2008), which used a focus group method, has identified: the experience of a chaotic environment; inadequate post-C.P.R. debrief; and unrealistic rehearsal in training. This qualitative study has used 1:1 interviewing and a Philosophical Hermeneutic (Gadamer 1975) lens to explicate how young doctors experience (and make sense of) their early attempts at C.P.R. The sociological framework of Symbolic Interactionism (Blumer, 1969, Charon, 2010, Mead, 1934) was deployed to offer a human interaction based interpretation of participants’ accounts. An experiential learning theory (Jarvis et al., 2003) offered further insights into the dimension of experiential learning. 3 Results: Eighteen participants were interviewed over 18 months. Using NVIVO 9 software, a thematic analysis technique, and a hierarchical analysis ladder (Spencer et al., 2003), four major themes were identified: 1. Current C.P.R. education is, at a skills and knowledge level, comprehensive and adequate. 2. Simulation rehearsal practises higher responsibilities than those clinically experienced, and usually fails to accommodate the “ambient” conditions of the real event. 3. C.P.R. offers novice clinicians a variety of experiential learning opportunities about leadership and about professional expectations of personal resilience (stoicism) as a doctor. 4. Participant support needs are usually unique, contextually generated, and largely unrecognised. Almost invariably unidentified, these needs reflect a variety of emotional states experienced during C.P.R.: surreality; exhilaration; satisfaction; or distress. Implications: This study has demonstrated the feasibility of 1:1 interviewing to generate deep, rich and granular accounts. Analysis through the lenses of Philosophical Hermeneutics, the sociological framework Symbolic Interactionism and the revised experiential learning theories of Jarvis offered unique perspectives and understandings of these experiences. The influence of “ambient” contextual conditions during C.P.R. has been partially, though not exhaustively, explicated. Whilst educational rehearsals should attempt simulation of reality, not all realities can be simulated. Post hoc support needs are unrecognised and educational responses unquantified. A modern duty of care to staff should require high quality interventions in three areas: pre hoc preparation; intra hoc conduct; and post hoc support.

Seizure onset prediction in epilepsy is a challenge which is under investigation using many and varied signal processing techniques, across the world. This research thesis contributes to the advancement of digital signal analysis of neurophysiological signals of epileptic patients. It has been studied especially in the context of epileptic seizure onset prediction, with a motivation to help epileptic patients by advancing the knowledge on the possibilities of seizure prediction and inching towards a clinically viable seizure predictor. In this work, a synchrony based multi-stage system is analyzed that brings to bear the advantages of many techniques in each substage. The 1st stage of the system unmixes and de-noises continuous long-term (2-4 days) multichannel scalp Electroencephalograms using spatially constrained Independent Component Analysis. The 2d stage estimates the long term significant phase synchrony dynamics of narrowband (2-8 Hz and 8-14 Hz) seizure components. The synchrony dynamics are assessed with a novel statistic, the PLV-d, analyzing the joint synchrony in two frequency bands of interest. The 3rd stage creates multidimensional features of these synchrony dynamics for two classes (‘seizure free’ and ‘seizure predictive’) which are then projected onto a 2-dimensional map using a supervised Neuroscale, a topographic projection scheme based on a Radial Basis Neural Network. The 4th stage evaluates the probability of occurrence of predictive events using Gaussian Mixture Models used in supervised and semi-supervised forms. Preliminary analysis is performed on shorter data segments and the final system is based on nine patient’s long term (2-4 days each) continuous data. The training and testing for feature extraction analysis is performed on five patient datasets. The features extracted and the parameters ascertained with this analysis are then applied on the remaining four long-term datasets as a test of performance. The analysis is tested against random predictors as well. We show the possibility of seizure onset prediction (performing better than a random predictor) within a prediction window of 35-65 minutes with a sensitivity of 65-100% and specificity of 60-100% across the epileptic patients.

Background: Rheumatoid arthritis (RA) is a symmetric polyarthritis arising from autoimmune dysregulation leading to severe disability and increased risk of co- morbidities and death. A chronic disproportionate inflammatory process lies at the heart of disease pathogenesis. Breach of self-tolerance, subsequent immune effector cell activation in the context of abundant expression of effector cytokines all contribute to uncontrolled inflammation. Molecular safeguards that normally operate to promote immune regulation appear defective in RA. Intensive basic and translational research over the last 30 years have contributed the emergence of an array of new therapeutics for the treatment of RA, which has transformed patient outcomes. The identity of the cytokine targeting treatments that have been most successful elucidates a functional hierarchy that implicates elements of both innate and adaptive immunity. In particular, dysregulation of TNFα and IL-6 biology are at the core of effector pathways and as such unravelling their detailed regulation is of critical importance. Moreover their primary synthesis places myeloid cells, and, in particular, blood-derived monocytes at the heart of pathogenic circuitry. Best current clinical practice is to treat early disease and deploy aggressive treatments directed towards restoration of immune balance in virtually all patients. However only a proportion of such patients will actually have poor prognosis disease and in reality merit such aggressive interventions - the identification of such clinical endotypes is a major challenge for the next decade. The field of epigenetics and consequent regulatory control of inflammatory cells offers rich potential in this regard. Examples of such regulatory elements are small RNA species – microRNAs (miRs), which serve as negative regulators of cellular transcription and thereby repress protein translation. Importantly they do so across functionally integrated pathways, operating beyond individual moieties. A growing body of evidence implicates a significant role of miRs in the regulation of inflammatory processes in the context of RA. Objectives: To identify miR species that are differentially regulated in patients with poor response to therapeutic intervention, compared to patients with well- controlled disease and healthy controls. Thereafter, to characterise candidate 2 miRs arising from these investigations to thereby determine their functional significance. Together these studies will shed light on a substantially ignored area of RA biology, namely the underlying mechanisms that subserve drug resistance in RA. Key Results: Microarray profiling of CD14+ monocytes derived from patients with drug resistance upon receipt of DMARDs or biologic treatments, compared with good responders or matched healthy controls identified the miR-23a~24-2~27a cluster to be significantly repressed in monocytes from resistant RA. Further analysis identified that two members of the cluster, miR-23a and miR-27a are implicated in a feedback loop regulating the IL-6 pathway. Thus IL-6 stimulation of primary monocytes suppresses the expression of this miR cluster, permitting expression of their direct molecular target, namely IL-6R, thus sensitising cells to further IL-6 signalling. I also observed that cells lacking miR-23a and miR-27a express higher levels of the pro-inflammatory cytokines TNFα and IL-6 when stimulated with LPS, further confirming that lack of these miRs has direct implications for chronic inflammatory processes. The remaining member of this miR cluster, miR-24, was shown to directly target methylene tetrahydrofolate reductase but not dihydrofolate reductase enzymes, implicating it in the target pathway of methotrexate (MTX), the most commonly used anchor DMARD. Although this is unlikely to confer disease resistance, this interaction suggests that miR-24 levels could be predictive of tolerability of methotrexate use. The potential biomarker capabilities of miR-24 in relation to MTX use, or miR-23a and miR-27 with regards to responsiveness to anti-IL-6 or JAK signalling inhibition therapeutics will be evaluated in my future work. Conclusion: This series of studies has elucidated highly novel pathways that mediate amplification of inflammatory responses in blood-derived monocytes through feedback pathways operating via regulatory miRs. Furthermore, analysis of a distinct cohort of RA patients allowed identification of miR species that have the potential to be utilized as clinical biomarkers for treatment efficacy or tolerability evaluation. Although a separate validation study is required, the detailed investigation of the role of these miRs performed here provides a clear mechanistic insight into their function and will certainly support future discovery.

O presente trabalho tem como objetivo conhecer o comportamento do aço inoxidável austenítico AISI 304L para o processo de Estampagem Incremental de Chapas (ISF - Incremental Sheet Forming), baseado nos parâmetros: raio da ferramenta RT, ângulo de parede a e passo vertical dz. Os experimentos baseiam-se na variante da Estampagem Incremental denominada Estampagem Incremental com Ponto Simples (SPIF - Single Point Incremental Forming). Foram realizados 29 ensaios, em três ferramentas com raios de 5, 8 e 10 mm. Basicamente, a estrutura empregada foi um dispositivo para fixação da chapa, um centro de usinagem vertical e um software de CAD/CAM. O melhor acabamento da superfície conformada medido através da média da rugosidade Rz foi alcançado com ferramentas de raio de 10 mm. Após a conformação, os corpos de prova apresentaram um significativo encruamento de seus grãos, o que conduziu parte da microestrutura originalmente austenítica a uma transformação martensítica induzida por deformação (efeito TRIP). Pode-se constatar também, que existe uma tendência do aumento do ângulo de parede aumentar a microdureza da região encruada. Os ensaios de Estampagem Incremental nesta pesquisa mostram que os gráficos das deformações exibem a tendência de que o modo como ocorrem as deformações, aproximam-se muito da deformação plana (Q2 0). / The objective of this work is to study the behavior of austenitic stainless steel AISI 304L during the ISF process (Incremental Sheet Forming). The study was based on the following parameters: tool radius RT, wall angle and vertical depth dz. The tests were based on a variation of the ISF process, called SPIF (Single Point Incremental Forming). A total of 29 tests were performed with 5, 8 and 10 mm of tool radius. Basically, the structure used was a rig for attachment of the sheet, a Vertical Machining Center and a CAD/CAM software. The best surface finish formed, measured by parameter RZ, was obtained with 10 mm of tool radius. After the forming, the specimens presented a significant mechanical hardening of the grains, which induced part of the microstructure originally austenitic to a martensitic transformation induced by deformation (TRIP effect). It can also be verified that there is a tendency that the increase of the wall angle increases the micro hardness of the mechanical hardened area. The graphs of strains show a trend: the deformation mode is very close to plane strain conditions (Q2 0).

O presente trabalho tem como objetivo conhecer o comportamento do aço inoxidável austenítico AISI 304L para o processo de Estampagem Incremental de Chapas (ISF - Incremental Sheet Forming), baseado nos parâmetros: raio da ferramenta RT, ângulo de parede a e passo vertical dz. Os experimentos baseiam-se na variante da Estampagem Incremental denominada Estampagem Incremental com Ponto Simples (SPIF - Single Point Incremental Forming). Foram realizados 29 ensaios, em três ferramentas com raios de 5, 8 e 10 mm. Basicamente, a estrutura empregada foi um dispositivo para fixação da chapa, um centro de usinagem vertical e um software de CAD/CAM. O melhor acabamento da superfície conformada medido através da média da rugosidade Rz foi alcançado com ferramentas de raio de 10 mm. Após a conformação, os corpos de prova apresentaram um significativo encruamento de seus grãos, o que conduziu parte da microestrutura originalmente austenítica a uma transformação martensítica induzida por deformação (efeito TRIP). Pode-se constatar também, que existe uma tendência do aumento do ângulo de parede aumentar a microdureza da região encruada. Os ensaios de Estampagem Incremental nesta pesquisa mostram que os gráficos das deformações exibem a tendência de que o modo como ocorrem as deformações, aproximam-se muito da deformação plana (Q2 0). / The objective of this work is to study the behavior of austenitic stainless steel AISI 304L during the ISF process (Incremental Sheet Forming). The study was based on the following parameters: tool radius RT, wall angle and vertical depth dz. The tests were based on a variation of the ISF process, called SPIF (Single Point Incremental Forming). A total of 29 tests were performed with 5, 8 and 10 mm of tool radius. Basically, the structure used was a rig for attachment of the sheet, a Vertical Machining Center and a CAD/CAM software. The best surface finish formed, measured by parameter RZ, was obtained with 10 mm of tool radius. After the forming, the specimens presented a significant mechanical hardening of the grains, which induced part of the microstructure originally austenitic to a martensitic transformation induced by deformation (TRIP effect). It can also be verified that there is a tendency that the increase of the wall angle increases the micro hardness of the mechanical hardened area. The graphs of strains show a trend: the deformation mode is very close to plane strain conditions (Q2 0).