Regeneration of human central nervous system (CNS) neurons is limited due to the inhibitory environment that forms post injury known as the glial scar. Reactive astrocytes within the glial scar produce both inhibitory and permissive extracellular matrix molecules (ECM) into the local environment. Chondroitin sulfate proteoglycans (CSPGs) are a component of the ECM which have been shown in vitro and in vivo to inhibit neurite regeneration. Physiologically relevant in vitro models of the glial scar are essential in developing new therapeutics and understanding the cellular processes that underpin neural regeneration. In this study human pluripotent stem cells were differentiated using the highly potent and stable synthetic retinoid EC23. A concentration dependent profile of the action of EC23 on stem cell differentiation was determined, furthermore, the mechanisms for the enhanced biological activity of EC23 were investigated. This study used the well described small molecule EC23 to form aggregates of neural progenitors which were characterised and used in a two dimension (2D) and three dimension (3D) model of neurite outgrowth. Next, the neurite outgrowth substrate was manipulated to represent the inhibitory ECM of the glial scar using the CSPG Aggrecan. The presence of Aggrecan inhibited neurite development and was used to assess small molecules that could enhance outgrowth in 2D and 3D. Small molecule modulators of; rho-associated protein kinase (ROCK); retinoic acid receptor β2; glycogen synthase kinase 3β and protein tyrosine phosphatase σ were shown to at least partially enhance neurite outgrowth on Aggrecan in 2D and 3D. Furthermore, the bacterial enzyme Chondroitinase ABC was used to cleave chondroitin sulphate glycosaminoglycan side chains (GAG) from Aggrecan to further aid neurite outgrowth in this model. In addition a 2D and 3D co-culture system was developed using the human astroglioma cell line U118MG and human stem cell-derived neural progenitors described previously. This model demonstrated inhibition of neurite outgrowth by U118MG which could be overcome by ROCK inhibition. This thesis describes the development of a novel model of neurite outgrowth using human stem cell-derived neurons. The model described was used to investigate Aggrecan induced neurite inhibition and to investigate pathways involved in neural regeneration.

Simulation or scenario learning is an integral part of student paramedic development and, despite the increasing amount of paramedic research, very little is known about how students and tutors experience it. Current literature regards simulation as invaluable without exploring why this may be the case and this study aims to address this. This is a compressed time mode ethnographic approach study that incorporates data from student paramedics during and immediately after simulation learning events and tutor views of facilitating the simulation experience. This, along with a comprehensive literature review, provides an overview of simulation in the student paramedic development pathway. This thesis exposes how student paramedics find the simulation process anxiety provoking and explores the many reasons for this. The performance aspect of scenarios is echoed in the dramaturgical language used when talking about simulation learning events and the similarities between simulation learning events and simulation assessment events merely adds to this stress. Using the lens of critical pedagogy, issues of power (control and hierarchy) within the educational and organisational structures are examined and offered as another possible explanation for the high levels of anxiety in simulation learning. The thesis ends with the question of whether simulation learning can be changed for the better and if so, how.

Graft-versus-host disease (GvHD) remains the main complication associated with haematopoietic stem cell transplantation (HSCT). GvHD is caused by allo-reactive donor T cells mounting an attack against specific target tissues. CD4+CD25HiFoxp3+ regulatory T cells have been shown to modulate GvHD in vitro and also in vivo animal models. More recently early stage clinical trials have described the successful use of Treg to reduce the incidence of GvHD following HSCT. The aim of this study was to investigate further the suppressive mechanisms by which Treg are able to modulate GvHD and assess the influence of Treg on the beneficial graft-versus-leukaemia (GvL) effect therefore providing further insight into the use of Treg in the therapeutic management of GVHD. Data presented in this thesis demonstrates the successful isolation and expansion of a highly pure Treg population which maintained suppressive capacity throughout culture. We also confirmed that Treg retain suppressive capacity following cryopreservation resulting in reduced workload and increased consistency when used for in vitro functional studies. We also provide the first human in vitro evidence that Treg are able to prevent cutaneous GvH reaction by blocking the migration of effector T cells into the target tissues. The presence of Treg during allo-stimulation caused reduced effector cell activation, proliferation, IFNγ secretion and decreased skin homing receptor expression. Further investigation into the Treg modulation of dendritic cells demonstrated, for the first time in experimental in vitro human GvHD, that this was due to ineffective effector T cell priming in the presence of Treg caused by impairment of dendritic cell functions. Comprehensive phenotypic and functional analysis of Treg treated moDC showed their decreased antigen processing ability and allostimulatory capacity, resulting in a less severe GvH reaction in the skin explant model. Furthermore, this work has revealed that despite Treg impairing in vitro GvL mechanisms at a cellular level there was no association observed between increased Treg levels and the incidence of relapse in a small clinical cohort of HSCT patients. In conclusion this study has provided further insight into the mechanisms by which Treg are able to modulate GvHD. This would inform future clinical trials using Treg as a therapeutic alternative to current GvHD treatment and prophylaxis.

Xenon is a general anaesthetic gas with neuroprotective properties. Inhibition of the N-methyl-D-aspartate (NMDA) receptor glycine co-agonist site has been shown to mediate xenon neuroprotection against ischemic injury in vitro. Site-directed mutagenesis was used to produce point mutations in the GluN1 subunit of rat NMDA receptors. These were then expressed in HEK293 cells and the responses of mutant GluN1/GluN2A receptors to glycine and anaesthetics assessed using patch-clamp electrophysiology. Two mutations of the phenylalanine 758 site were found to eliminate xenon binding to the receptor without altering glycine affinity or the binding of sevoflurane and isoflurane. These selective mutations will allow for knock-in animals to be used to dissect the mechanism(s) underlying xenon neuroprotection and anaesthesia in vivo. The ability of the other noble gases (helium, neon, argon, and krypton) to influence two known molecular targets of xenon - the NMDA receptor and the TREK-1 channel - was also assessed using patch-clamp electrophysiology. These were found to have no influence on either NMDA receptors or TREK-1 channels. Finally, xenon's neuroprotective efficacy against traumatic brain injury (TBI) in vivo was assessed using the rodent controlled cortical impact model of TBI. Focal contusion injury was administered to male C57 mice and animals administered either 75% xenon or control treatment for three hours. Xenon-treated animals performed better in functional tests and displayed favourable outcomes in brain histology, as compared to control animals. This data provides the first evidence of a neuroprotective effect for xenon against TBI in vivo.

Background: The noble gases xenon and argon are neuroprotective in models of brain injury. This thesis investigated the neuroprotective mechanisms of the inert gases xenon, argon, krypton, neon and helium in in vitro models of traumatic brain injury and ischemia. Methods: This study used an in vitro model of focal mechanical trauma and oxygen and glucose deprivation using mouse organotypic hippocampal brain slices. Injury was quantified by propidium iodide fluorescence. Results: Xenon (50.6 kPa) and, to a lesser extent, argon (50.6 kPa) are neuroprotective against traumatic injury when applied after injury (xenon 0.43 ± 0.03 protection at 72 h after injury [N = 104]; argon 0.30 ± 0.0% protection [N = 44] vs control injury 1.0 ± 0.05 [N=144]; mean ± SEM). Helium, neon, and krypton are devoid of neuroprotective effect. Xenon (50.6 kPa) prevents development of secondary injury after trauma. Argon (50.6 kPa) attenuates secondary injury, but is less effective than xenon (xenon 0.50 ± 0.05 reduction in secondary injury at 72 h after injury [N = 104]; argon 0.34 ± 0.08 reduction [N = 44] vs control 0.86 ± 0.05 [N = 144]; mean ± SEM). Glycine reverses the neuroprotective effect of xenon, but not argon in both models of TBI and OGD, consistent with competitive inhibition at the N-methyl-d-aspartate receptor glycine site mediating xenon neuroprotection against traumatic brain injury. Conclusions: Xenon neuroprotection against traumatic and ischemic brain injury can be reversed by elevated concentrations of glycine, indicating a key role of inhibition of the NMDA receptor glycine co-agonist site in mediating neuroprotection against these injuries. Argon does not appear to have any effects on NMDA receptors and is neuroprotective via a mechanism distinct to that of xenon. Krypton and neon are devoid of neuroprotective effects in either injury model.

Alzheimer’s Disease (AD) is the most common form of dementia currently affecting more than 35 million people worldwide. Hypometabolism is a major feature of AD and appears decades before cognitive decline and pathological lesions. This has a detrimental impact on the brain which has a high energy demand. Current models of AD fail to mimic all the features of the disease, which has an impact on the development of new therapies. Human stem cell derived models of the brain have attracted a lot of attention in recent years as a tool to study neurodegenerative diseases. In this thesis, neurons and astrocytes derived from the human embryonal carcinoma cell line (NT2/D1) were utilised to determine the metabolic coupling between neurons and astrocytes with regards to responses to hypoglycaemia, neuromodulators and increase in neuronal activity. This model was then used to investigate the effects of Aß(1-42) on the metabolism of these NT2-derived co-cultures as well as pure astrocytes. Additionally primary cortical mixed neuronal and glial cultures were utilised to compare this model to a widely accepted in vitro model used in Alzheimer’s disease research. Co-cultures were found to respond to Aß(1-42) in similar way to human and in vivo models. Hypometabolism was characterised by changes in glucose metabolism, as well as lactate, pyruvate and glycogen. This led to a significant decrease in ATP and the ratio of NAD+/NADH. These results together with an increase in calcium oscillations and a decrease in GSH/GSSG ratio, suggests Aß-induces metabolic and oxidative stress. This situation could have detrimental effects in the brain which has a high energy demand, especially in terms of memory formation and antioxidant capacity.

It is well-established that in vitro differentiation of Embryonic Stem Cells (ESCs) can recapitulate embryonic development. In this project a novel, step-wise, serum-free differentiation system was developed using specific recombinant growth factors for investigating the differentiation of mESCs, through formation of a primitive streak-like population expressing Brachyury and specification of a subpopulation of mesodermal progenitors expressing both paraxial and lateral plate mesoderm markers. These cells subsequently differentiated efficiently in monolayer cultures to chondrocyte and osteoblast lineages marked by cell morphology, Alcian blue/ALP staining as well as by chondrocyte and osteoblastspecific gene expression. The role of the Rho kinase (ROCK) pathway in cartilage and bone cell differentiation is controversial. Exposure of ESC cultures to the ROCK inhibitor, Y-27632, at mesoderm enrichment and/or monolayer differentiation phases revealed that continuous exposure to Y27632 modulated differentiation in a developmental phase-dependent manner, with up to a 7-fold and 2-fold increase in chondrogenesis and osteogenesis, respectively. In contrast, temporal exposure of Y27632 favoured chondrogenic over osteogenic differentiation. This was confirmed by qPCR analysis of chondrocyte (Sox9, Sox5, Acan, Coll II, Col X) and osteoblast (Runx2, Osx, ALP, BSP, OC)-specific gene expression. Furthermore, temporal exposure to FGF2 and BMP4 together with phase-specific addition of Y-27632 enhanced differentiation/expansion of hypertrophic chondrocytes and mineralising osteoblasts. Finally, renal capsule grafting studies showed that the mesoderm-derived ESCs mimicked endochondral ossification, which was enhanced by Y27632 treatment. This study established a novel ESC model system, which generated defined, manipulatable and expandable chondro-osteoprogenitor populations that will provide insights into the molecular basis of bone/cartilage development. Moreover, a phase-dependent inhibition of ROCK signalling modulated early chondro-osteoprogenitor lineage commitment and enhanced cartilage and bone formation. These studies provide a novel targetable pathway for generating specific populations for potential bone and cartilage tissue repair and replacement.

Ventilator-associated pneumonia (VAP) is the commonest nosocomial infection in Intensive Care and is associated with significant morbidity and mortality. Biomarkers offer the potential to speed up diagnosis and differentiate pulmonary from nonpulmonary infection. We postulated that measurement of cell surface receptors in addition to soluble proteins, in dual sites (blood and BAL) and calculation of an index ratio of BAL / blood would increase the discriminative utility and differentiate pulmonary from non-pulmonary infection. Our body of work included paired blood and BALF obtained from 91 patients in a pilot study: 27 with VAP, 15 ventilated patients with non-pulmonary sepsis, 18 ventilated patients with no evidence of infection and 31 non-ventilated non-infected patients. In each sample, the monocytic and neutrophilic surface proteins TREM-1, CD11b and CD62L were assessed using flow cytometry. Soluble proteins (IL-1β, IL- 6, IL-8) were assayed using ELISA in addition to Procalcitonin, CRP and white cell count. The levels of soluble TREM-1, IL-1β and IL-8 were significantly raised in the BAL of patients with VAP. BAL monocytic surface TREM-1 was also significantly higher in VAP. The BAL/blood ratio increased the discrimination of patients with VAP from non-VAP. Furthermore, the BAL/blood ratio of patients differentiated VAP from non-pulmonary infection. Monocytic and neutrophilic TREM-1 were assessed during the development and resolution phases of VAP. Monocytic surface TREM-1 and its BAL / blood ratio accurately mirrored the changes with infection, indicating them to be putative biomarkers of infection. Finally, we constructed and validated a biomarker panel to discriminate patients with VAP from non-VAP. The panel comprised the BAL/blood ratios of monocytic TREM-1 and CD11b, the BAL levels of soluble TREM-1, IL-8 and IL-1β together with the blood levels of IL-6 and CRP. It had high utility in identifying patients with VAP.

Somatic stem cell populations display a remarkable capacity to self-renew and generate specialised cell types throughout the life of the organism. In my thesis I examined extrinsic and intrinsic factors that regulate stem cell quiescence, a reversible state of growth arrest crucial to the preservation of somatic stem cell number and function in many systems. Skeletal muscle-specific stem cells, known as satellite cells (SCs) are responsible for skeletal muscle regeneration. The ability of skeletal muscle to regenerate declines with age. I identify fibroblast growth factor 2 (FGF2) as a potent mitogenic factor that is up-regulated in the aged muscle fibre and causes a loss of SC quiescence and depletion of the stem cell pool. Deletion of a negative regulator of FGF signalling, Sprouty1 (Spry1), in SCs increases stem cell loss, whereas over-expression of Spry1 partly prevents depletion. These experiments show that an age-associated change in the SC niche is partly responsible for stem cell depletion during ageing. In the adult forebrain, new neurons produced from neural stem cells (NSCs) in the hippocampus play an important role in learning and memory formation. I show that deletion of the chromatin remodelling enzyme chromodomain helicase DNA-binding protein 7 (CHD7) in NSCs results in a severe reduction in neurogenesis. I identify CHD7 as an essential regulator of NSC quiescence and self-renewal. Collectively, my results suggest that the regulation of the intrinsic chromatin landscape and the extrinsic niche environment are essential for somatic stem cell function, and may contribute to ageing when disrupted.

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