Explosion is responsible for almost 80% of Coalition injuries in today’s conflicts. Haemorrhage is the leading cause of death and blast lung injury is evident in 11% of Coalition casualties surviving to reach the (UK) Field Hospital. Military prehospital evacuation times can be prolonged and the combined insults of haemorrhage and blast injury present a ‘double hit’ to oxygen delivery. Resuscitation strategies must be capable of preserving life from such trauma for several hours. Alongside fluid therapy, adjuncts to resuscitation might improve battlefield survival. This randomized controlled animal trial assessed two adjuncts: supplemental inspired oxygen and recombinant activated Factor VII (rFVIIa). Neither adjunct is currently available in the far-forward military echelon, but with modern technology, both are potentially deployable. 18 terminally anaesthetized swine were exposed to blast, controlled haemorrhage and grade IV liver laceration (uncontrolled haemorrhage). Animals were allocated randomly into three treatment groups. All animals were resuscitated with normal saline to a hypotensive systolic target (80mmHg), which continued until the 8hr end point. Thirty minutes after the onset of resuscitation each group received one of the following: single (180mcg/kg) dose of rFVIIa; supplemental oxygen (min FiO2 0.3 to maintain SaO2>95%) or the control group (breathed air throughout and received saline placebo 0.18ml/kg). 5/6 control animals died within 4 hours. Supplemental oxygen improved survival (4/6 survival to 8h endpoint, P=0.014). Single dose rFVIIa did not prolong survival compared to control (2/6 survived, p=0.65). Oxygen arrested physiological decline while control and rFVIIa animals continued to decline until death. Supplemental oxygen is a useful adjunct to fluid resuscitation in the context of haemorrhage and blast injury. Delivery of oxygen support capability to forward echelon units is recommended. By contrast, a single intravenous (pre-hospital) dose of rFVIIa was not an effective treatment for blast lung based on our model of complex battlefield injury.

People with advanced inoperable non-small cell lung cancer (NSCLC), receiving systemic therapy with a palliative intent, are at a high risk of developing distressing symptoms related to both their tumour and its treatment. Symptoms of unintentional weight loss and loss Of physical function are prognostically significant, impacting negatively upon quality, of life. This thesis outlines the potential role of palliative rehabilitation, in the form of physical activity and nutritional guidance, in the symptom management of advanced NSCLC. The MRC Guidance fot Developing and Evaluating Complex Interventions and Transtheoretical Model of Behaviour Change steered both the development and implementation of the palliative rehabilitation intervention employed within the study. Study recruitment and retention figures were gathered. In addition patient generated validated outcome measurements, functional and anthropometrical data were collected to assess acceptability and perceived or quantifiable changes in quality of life, function and symptom burden occurring during the six weeks of the intervention and six weeks post intervention. Semi structured interviews with patients and healthcare professionals explored experiences of APRIL; these were thematically analysed. Feasibility of the APRIL intervention cohort study was determined by the ability to recruit participants. Interpretation of findings regarding study feasibility and analysis of the quantitative and qualitative data were limited by low recruitment, issues with the sensitivity of outcome measures and missing data. Many people diagnosed with advanced NSCLC are both willing and interested in engaging in palliative rehabilitation research and that participation led to perceived improvements in physical and psychosocial well-being. The study supports the need for further research into early rehabilitation within this population including health care professionals' attitudes towards rehabilitation, alongside cancer treatment which is delivered with palliative intent.

Stem cells have significant potential to treat many age-related degenerative disorders that affect increasing numbers of people globally. This thesis investigated the capacity for omnicytes and human pluripotent stem cells (hPSC) to undergo directed differentiation towards neuronal cell types for the treatment of ischemic stroke and Parkinson’s disease respectively. Omnicytes express a range of markers related to pluripotency and plasticity; however they are a challenging cell source to use in the development of cell therapies. Variability in omnicyte quality was associated with patient source, disease type and cryopreservation, all of which affected the reproducibility of data. Successful generation of dopaminergic neurons was achieved using a suspension-based hPSC culture system, with modified culture medium designed to replicate endogenous signalling during development. Neurons expressing key markers of dopaminergic neurons were generated and were capable of producing dopamine in response to KCl challenge. The work also showed that transfection of saRNA could enhance the expression of key genes i.e. foxa2, lmx1a and TH, relative to mock transfected cultures, although not significantly. Results also showed that the specific hESC line used (Shef6) had a greater propensity for differentiation toward dopaminergic neurons than MSUH001 hiPSC. This work successfully used saRNA to enhance gene expression, but shows that transfection efficiency is a limiting factor to its use. However, if transfection efficiency can be addressed, saRNA will become a powerful tool in the generation of cell therapies, particularly if it can be applied to suspension cell cultures.

Many studies have shown that mesenchymal stem cells (MSCs) can be used as immunosuppressants to treat graft-versus-host disease (GVHO) and autoimmune diseases. The injection of MSCs prolonged allogeneic solid organ transplantation in animal models. The current project aimed to investigate whether tissue-engineered cartilage derived from MSCs could be transplanted allogeneically. We mainly looked at murine MSCs (mMSCs) in this project. mMSCs are more difficult to isolate than human MSCs. We tried several methods to isolate mMSCs and found that a FACS isolated subpopulation of mMSCs, i.e. PaS cells (POGFRa+, Sca-1 +), had clear chondrogenic differentiation potential. The mechanisms involved in the immunosuppressive effect of MSCs are still under debate. Our in vitro data showed that the immunosuppressive effect of MSCs is through a negativefeedback loop. Pro-inflammatory cytokines from proliferating Iymphocytes activate MSCs to produce immunosuppressive molecules that suppress the proliferation of Iymphocytes. This immunosuppressive effect is shared with other stromal cells. A species difference exists in terms of the immunosuppressive effect of MSCs.

This thesis explores the practice of sedation in hospice palliative care. Internationally this has been a controversial subject for over 20 years, with the use of sedation considered to be on a spectrum between euthanasia and symptom control at the end of life. This is a complex area of study, incorporating not only technical details regarding drugs and doses, but also relating to underlying values regarding end of life care. In the UK end of life care has developed from the ‘hospice movement’ of the 1960s, into the broad and far reaching approach of palliative care. Alongside this development, palliative care has espoused its own ‘ethos’ and values, evident in much of the literature in this area. This thesis presents the data from an ethnographic study in a UK hospice. The aim of the study was to develop a normative understanding of the use of sedation in hospice palliative care. The ethnography allowed an in depth understanding of this practice through prolonged immersion in the field of study. This enabled the practice of sedation to be understood as a process, or series of decisions, based upon a tacit understanding of a patient’s proximity to death. This was driven by the desire of hospice staff to bring about a comfortable and peaceful death, which was in turn motivated by the underpinning values, of the individual, the organisation, and of the approach of palliative care. This thesis has important implications for the future: for the specific use of sedative drugs in hospice palliative care, as well as for the broader issues in palliative care concerning decision-making at the end of life. A new definition for sedation at the end of life is constructed, relating particularly to, as it is derived from, the practice and underpinning values of hospice palliative care in the UK. Furthermore, as the evolving and changing nature of UK palliative care is considered, the capacity for hospice palliative care to enable the expression of different values, which manifest as a result of the changes in palliative care, represents a challenge to one of the core principles of the approach; patient centred care. This thesis introduces and considers values based practice as an approach which may facilitate the identification of values in decision-making, and reorientate care towards a more ‘patient-values-centred’ approach.

Refinements were devised for husbandry systems and routine experimental techniques with each tested against current best practice. Hand picking of male mice for experimental group formation was found not to be random. A random number generator provided a more consistent dispersion of dominant males throughout groups. Environmental enrichment reduced aggressive interactions between male mice, suggesting that these animals can be group housed with a reduced risk of injury. The addition of oxygen to euthanasia mixtures of carbon dioxide was found to cause lung haemorrhage in mice, suggesting this refinement is not appropriate when killing mice. The adverse effects of sodium pentobarbitone killing of mice and rats were found to be reversible when lignocaine hydrochloride was added to the euthanasia mixture. Administering acetyl-promazine on completion of surgery eliminated post-operative disturbance following ketamine/medetomidine anaesthesia of rats. Rats showed a clear preference for a darkened environment during recovery from anaesthesia but only preferred 'warmed environments if they had become hypothermic while anaesthetised. Transponder chips were shown to otfer no welfare benefits over rectal probes for determining rat body-temperature. Warming or chilling the injectate reduced injection pain. A novel temperature datalogger was tested and found have potential for use in laboratory animal welfare science.

A series of life and death decisions, in a high-tech hospital unit, made by a multidisciplinary team of specialists and the patient's family - intensive care is the epitome of modern medicine. What better encapsulates the ambivalence of medicine in the twentieth century? From the beginnings of intensive care units in the 1950s to the institution of specialist societies, journals and training by the mid-1980s, so many of the tensions of modern medicine have been articulated in this story. The development of British intensive care has involved collaboration and conflict - the benefits of multidisciplinary knowledge and experience, with the competition for status and 'ownership' of patients. It has utilised the high-tech - striving to advance therapeutic capabilities balanced against the risks of iatrogenesis and loss of patient identity and autonomy, as well as the low-tech - the importance of clocks and windows with a view of the outside world for temporal orientation and sensory stimulation, for example. At times, the 'scientific' pathophysiology of critical illness has been pitched against 'intuitive' nursing care. At other times, the nurse has been acknowledged as the primary therapist. Critically ill patients have been the subject of a hospital specialty based on generalism rather than a single organ system or age group. Expanding and contracting notions of reversibility and salvageability have informed decision-making in a resource-intensive field. Using archives, journals, newspapers, oral history interviews, films and museum objects, this thesis challenges the view that intensive care originated from the 1952 Copenhagen poliomyelitis epidemic or the postoperative recovery room. Instead, it argues that intensive care was not just a response to illness, but that it grew alongside a new category of illness created by practitioners and policymakers - critical illness. Changing notions of critical illness in turn shaped the practice of intensive care. Until the mid-1960s, critical illness was seen as phase of illness, with the highest degree of nursing dependency. As therapy developed, it began to be measured as the highest degree of medical and technological dependency. With research and symposia on pathophysiology, critical illness came to be regarded as a physiological state, imbalanced and in need of correction. Such notions informed questions of who should care for the critically ill, where, and how.

The relationship between spatiotemporal patterns of spontaneous activity and functional specialisation in developing neuronal networks is complex and its study is crucial to our understanding of how network communication is initiated. This project quantifies transitions between structural and functional states in embryonic stem cell cultures during differentiation. The work also focussed on the role of γ-aminobutyric acid (GABA), known to be vital for neuronal network development. The work used many techniques, including carbon nanotube (CNT) -patterned substrates to manipulate network architecture, multi-electrode arrays (MEAs) and calcium imaging to quantify function. An embryonic stem cell line (CC9) was used to generate ‘de novo’ neuronal networks and these were monitored over 13 – 22 days in vitro (DIV), while network structure forms and stabilizes. On CNT-patterned arrays, differentiating CC9s migrated and sub-clustered on CNT islands showing that network structure could be manipulated. No spontaneous electrophysiological (unit) activity was found in these cultures. However, intracellular calcium responses were readily induced and seen spontaneously at 13-20 DIV. Activity rate, kinetics and number of active cells increased between 16-18 DIV, correlating with changes in network clustering. Post 17 DIV, activity transformed from near-random to periodic and synchronous. Many events were initiated by ‘hubs’ and degrees of critical behaviour were observed, moving towards more efficient information processing states with development. Blockade of GABAA receptors lead to elevated spontaneous activity and supercritical behaviour, depending on developmental stage. Application of exogenous GABA induced large, slow calcium transients in a developmental stage-dependent manner, suggestive of a mixed excitatory/inhibitory role. These findings begin to show how activity develops as stem cells differentiate to form neuronal networks. GABA’s role in controlling patterns of activity was more complex that previously reported for neuronal networks in situ, but GABA clearly played a vital role in shaping population behaviour to optimise information processing properties in early, developing networks.

Ageing is associated with the functional decline of both haematopoietic stem and endothelial progenitor cells (HSPCs and EPCs), leading to an imbalance between cellular damage and repair. Telomeres are the end caps of chromosomes that maintain chromosomal integrity and shorten with age. Telomerase is the enzyme responsible for telomere replication. The absence of telomerase leads to premature ageing. Oxidative stress as well as metabolic stress and short telomeres are key contributors to the manifestation of different age-related diseases. However, the exact effect of these factors on HSPCs and EPCs is not clear. The effects of metabolic stress were studied by the addition of different glucose concentrations to low passage (early) cells in culture. Metabolic stress impaired the growth of EPCs and CD34+ HSPCs. There was no change in telomerase enzyme activity under metabolic stress in CD34+ HSPCs. However, metabolic stress upregulated the metabolic co-activator PGC-1α in EPCs but not in CD34+ HSPCs. The effects of oxidative stress were investigated by incubating peripheral blood EPCs, and cord blood CD34+ expanded HSPCs under 40% O2 in culture. While early EPCs show resistance to oxidative stress, CD34+ HSPCs showed impaired growth and differentiation potential. This impairment was associated with increased telomerase activity, no changes to TERT or TERC expression, and maintenance of telomere length. Oxidative stress limited CD34+ HSPC myeloid differentiation. In particular, CD15+ granulocytes were more sensitive to oxidative stress than CD14+ monocytes. Furthermore, CD15+ granulocytes reduced the expression of TERC during myeloid differentiation. In contrast to CD34+ HSPCs under growth conditions, there was no increase in telomerase activity during myeloid differentiation under oxidative stress. To investigate the effects of ageing in vivo with telomerase dysfunction, HSPCs from bone marrow of aged telomerase deficient TERT-/- and TERC-/- first generation mice were studied. Ageing resulted in the accumulation of Lineage-Sca-1+CKit+ stem cells and CFU-GM colonies in wild type mice. TERT-/- mice without telomere shortening showed a normal phenotype at young age (1.5-7.5 months) and augmented ageing of bone marrow with increased age (22 months). On the other hand, TERC-/- mice with short telomeres led to a premature ageing bone marrow phenotype, even at young ages (8-12 months). Interestingly, both TERT-/- and TERC-/- showed more erythroid progenitor colonies. Furthermore, short- (7 months) and long-term (16 months) dietary restrictions ameliorated the ageing bone marrow phenotype. Together, the data presented demonstrates the damaging effect of oxidative and metabolic stress on humans in early EPCs and CD34+ HSPCs. In a mouse model, normal ageing disrupted HSPCs. Telomerase deficiency augmented normal ageing, whilst short telomeres appear to be a major determinant of ageing. These ageing phenotypes in mice can be ameliorated by dietary restriction.

Background: The management of depression in palliative care is impeded by inadequate understanding of the nature of depression in this context. Estimates of the prevalence of depression in palliative care vary widely and very few studies have assessed remission. Further, there is concern that somatic symptoms common in advanced disease may confound diagnosis. Whilst antidepressants are the mainstay of treatment in the general population, their efficacy and acceptability in palliative care are unclear. Aim: To provide new knowledge on the clinical epidemiology and pharmacological treatment of depression in palliative care, and consolidate this evidence in a clinical guideline to inform and improve future practice. Methods: A cross-sectional survey with 4-week follow-up was conducted to determine the prevalence and remission of depression in patients newly referred to specialist palliative care. Presence of depression was assessed using the PRIME-MD PHQ-9. The predictive value of somatic symptoms was assessed by calculating the PPV and NPV of EORTC-QLQ-30 items on sleep disturbance, poor appetite and fatigue. A systematic review and meta-analysis was undertaken to determine whether antidepressants are appropriate for palliative patients. Due to the dearth of data in advanced disease, the efficacy and acceptability of antidepressants and placebo were compared in RCTs of patients with any physical illness. In addition a subgroup analysis of patients with life-threatening illness was performed. Outcomes were assessed at 4 time-points (4-5, 6-8, 9-18 and & 18 weeks). Data from the survey and meta-analysis informed the development of a clinical guideline on the management of depression in palliative care, and a Delphi study was conducted to elicit expert opinion where evidence was absent. The GRADE system was used to indicate the quality of evidence and strength of recommendations.