Around 35 million people are living with HIV-1 infection globally. Untreated infection results in progression to AIDS and mortality. Current antiretroviral treatment is not curative and does not treat the underlying inflammatory processes caused by the presence of HIV-1, a cytotoxic and tissue toxic virus. HIV-1 triggers overwhelming and dysregulated pro-inflammatory cytokine response during infection, including deranged interleukin-1 beta and interleukin-18 levels. We investigate the mechanisms underlying how HIV-1 activates the inflammasome, a multimolecular complex involved in the production of potent inflammatory markers such as interleukin-1 beta and interleukin-18. The inflammasome is often activated in a two-signal process requiring initial sensing by Toll-like receptors to generate signal 1, priming inflammasome components; and signal 2 sensed by NOD-like receptors which recruit and activate the inflammasome. Human leukocytes were stimulated with live HIV-1 or its cognate envelope proteins gp120 or gp41, or transfected with plasmids encoding HIV-1 viroporin Vpu or gp41. NLRP3 knock-down attenuated inflammasome activation and interleukin-1beta/interleukin-18 secretion following HIV-1, gp120 or gp41 stimulation. gp120 induced inflammasome activation also required the presence of TLR2 and TLR4, suggesting a degree of cooperation in effecting signal 1. Dual NLRP3/NLRC4 knockdown reduced the inflammasome response to HIV-1 gp41 stimulation which was found to be dependent on chloride anion flux across integral lipid rafts in the trans-Golgi network. The results imply a critical role for TLR2 and TLR4 in recognising HIV-1 proteins providing signal 1 for inflammasome formation. Ion fluxes in the cell due to HIV-1 infection are the catalyst that triggers inflammasome activation via NLRP3 or NLRC4, and secretion of interleukin-1 beta and interleukin-18. The findings contribute to the general understanding of HIV-1 recognition by the human innate immune system and help to further clarify precisely how HIV-1 causes striking dysregulation in cytokine profiles in people living with HIV-1 infection.

Background: There are many challenges in assessing older people in hospital and despite guidelines advocating Comprehensive Geriatric Assessment (CGA), there is a lack of clarity regarding how these patients are currently assessed in practice. Objective: To explore the assessment process on a ward for older people from the perspective of patients and health care professionals. Design: A constructivist Grounded Theory was used to understand assessment from different perspectives. Setting and subjects: Patients without cognitive impairment admitted to and professionals working on one ward for older people in an NHS University Hospital in England, UK. Methods: Data were collected between February 2015 and January 2016, including 37 interviews (15 patients, 22 professionals), a focus group (6 professionals), and 45 hours of fieldwork including observation and review of 18 sets of patient notes. Findings: The core category was ‘Navigating’, conveyed through three themes: ‘Containing complexity’, ‘Networking’, and ‘Situating the process’. Key findings were: (1) Navigating assessment is a complex, flexible, context dependent, and social process, (2) Health professionals use a combination of formal, informal, visible and invisible ways of working, (3) Registered nurses are at the centre of networking, spending most their time gathering and sharing information within the multi-disciplinary team, (4) Patients seemed to have a passive role, whilst expressing a variety of decision-making involvement preferences. Conclusions: Navigating the assessment of older people is contextually situated and involves less standardised, less visible and less formalised approaches to assessment than suggested in the guidelines. Awareness of all these aspects of navigating may result in their further utilisation and development. Nurses’ focussed on gathering and sharing information, but to move from “chasing” to “coordinating” patient care they need support from the multi-disciplinary team. Patients have limited participation within the navigating process, and their decision-making involvement preferences are not always elicited and facilitated.

Interpretation of laboratory tests in clinical practice is based on an understanding of the disease process within or between individuals. This is demonstrated by the variability of pathology results as compared to the previous result or against the reference range, made up from the intrinsic pathophysiological changes and also variation associated with the in vitro changes to the sample. My work is on identification and minimisation of the result variation in the pre-analytical phase, accounting for 60-70% of the errors associated with laboratory testing. The first project of my thesis is based on four studies that consider the in vitro stability of parathyroid hormone (PTH) and C-reactive protein (CRP), in which significant sample degradation is observed due to sample tube type, anticoagulant used and time to separation. The second project considers ethnic variation as a source of intra individual variation. Specifically considering intra individual ethnic variation in total cholesterol (TC) and high density lipoprotein cholesterol (HDLC), reporting significant differences were observed between Caucasian Indo-Asians in HDLC, in addition I investigated the relationship between low maternal vitamin B12 concentrations in Caucasian women and cord blood cholesterol. The third project considered the variation in laboratory results due to pre-existing conditions causing interference in common laboratory tests. I published on the effect of lipaemia on common laboratory tests, showing lipaemia does have a significant effect on laboratory tests. The following study found that the raised prolactin seen in rheumatoid arthritis is not artefactual but due to changes in cross reactivity due of prolactin subtypes. The final paper of this project shows, through a collection of case studies falsely elevated serum calcium levels in patients with paraproteinaemia.

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Introduction: paramedics conducting interfacility transfer of critically-ill patients is one of the existing models in interfacility transfer. The paramedic model is available in multiple countries, including the Kingdom of Saudi Arabia. Paramedics’ expanded scope of practice has allowed them to transport, monitor and intervene with complex patients. This PhD thesis is designed to evaluate the safety of the paramedic model in Saudi Arabia conducting interfacility transportation of critically-ill patients. Method: the PhD thesis is mixed methods. A systematic literature review was conducted to examine literature on the safety of paramedics in interfacility transfers. A retrospective chart review was conducted to examine the incidence, predictors and pattern of adverse events seen in interfacility transfers by paramedics in Saudi Arabia. Following this, a retrospective chart review of interfacility transfers by physicians to the same institution was conducted to compare in-hospital mortality and 30-days survival in both groups. Finally, an expert survey was conducted to examine the consensus of paramedics’ intervention to adverse events seen in interfacility critical care transfers. Results: the literature showed that the frequency of adverse events seen by paramedics in interfacility transfers ranges from 5.1% to 18%. The rate of adverse events in adult critical patients transferred by paramedics to a tertiary care facility in Saudi Arabia was 13.7%, in-hospital mortality was 30.4% and 30-days survival was 68.1%. There is no significant difference regarding in-hospital mortality or 30-days survival between the paramedic and physician models. The paramedics’ interventions in interfacility adult critically-ill patients were rated appropriate by the majority of the experts in 86.8% of cases; the probability of an intervention to be appropriate was 84.9%. Conclusion: paramedics with appropriate training and skill can safely transfer critical interfacility adult patients. The mortality outcomes in the paramedic model are comparable to the physician model.

Introduction : There is now compelling evidence that a significant minority of patients suffer preventable iatrogenic harm during their interactions with health care, including in UK general practice. While our understanding of the extent of the problem and the contributing factors continues to increase, it remains incomplete. Further patient safety research is therefore urgently required, particularly to develop, test and successfully implement effective improvement strategies, methods and tools. Of the main approaches currently available for improving patient safety, the general practice Trigger Review Method (TRM) is of particular interest and the main focus of this study. The TRM is, quite simply, a structured way to rapidly screen samples of random electronic patient records for undetected patient safety incidents (PSIs). It is essentially an adaptation of clinical record review, with the same underlying principles of learning from error and improving care. Development of the TRM commenced in 2007 in Scottish general practice, with subsequent testing in The Health Foundation-funded Safety and Improvement in Primary Care (SIPC) programme. In 2013, the TRM was included as one of the three core components of the Scottish Government’s Patient Safety Programme for Primary Care (SPSP-PC). Scottish general practices were also financially incentivised through the Quality and Outcomes Framework (QOF) to routinely apply the TRM and report their findings. However, despite the increasing and national interest in the TRM, many unanswered questions remained: what is its potential value, how acceptable and feasible is it and to what extent (if any) will, or should, it become part of routine general practice? The aims of this study were therefore to: (i) describe the patient safety perceptions of general practice clinicians and staff; (ii) determine the usefulness of the TRM; (iii) explain how the TRM worked; and (iv) identify the main factors that facilitated or hindered its implementation. Methods: This study has a mixed-methods design. It was undertaken in the West of Scotland region in two NHS Health Boards: Greater Glasgow and Clyde (GGC) and Ayrshire and Arran (A&A). Convenience samples of 12 general practice teams and 25 GP Specialty Trainees (GPST) were recruited. Data were collected through: semi-structured interviews (n=62) with a range of general practice clinicians and staff; and cross-sectional trigger reviews of selected electronic patient records. Normalisation Process Theory (NPT) underpinned all stages of the research. NPT is a socio-technical, middle-range theory about the ‘work’ people do collectively and as individuals to implement and sustain complex health care interventions such as the TRM. The majority of the qualitative data were analyzed thematically and a NPT framework was applied to the remaining data. Quantitative data were analysed using recognised statistical tests. Results: A total of 47 primary care clinicians reviewed 1659 electronic patient records and detected 216 PSIs. A substantial minority of these were considered to have led to moderate or more substantial harm (29.2%), while the majority (54.8%) were rated as being preventable or potentially preventable. The most common type of PSI related to ‘medication’ (40.7%) and the most commonly implicated drug was Warfarin. The participants reported considering or undertaking specific improvement actions during and after approximately two thirds of trigger reviews. The most common action was ‘feedback to colleagues’. More specific actions included: undertaking significant event analyses (SEAs) and clinical audits, designing or redesigning practice protocols and including their findings in their appraisal documentation. The vast majority of participants identified four main factors as being particularly important for the successful implementation of the TRM, and by extension its potential normalisation. The first and most important factor was provision of adequate resources and protected time to conduct trigger reviews. The second factor was whether senior leaders in the practice teams, the government and professional bodies practically demonstrated their support for the TRM through, for example, contextually integrating it into existing general practice processes. The third and fourth factors related to the characteristics of participants. Successful implementation required knowledgeable clinicians to remain engaged with the TRM, and to perceive it as useful, acceptable and feasible – which the vast majority of participants were, and did. Discussion: This study is the first known attempt to investigate how the TRM is implemented and perceived from the perspective of general practice clinicians and staff. The main findings are that most participants experienced the method as acceptable, feasible and useful. It is clear that the TRM is uncovering important patient safety concerns and also driving improvements in related care systems and processes at the individual practice level. The implication is that this is making significant and demonstrable differences to patient care, while impacting positively on local safety culture. On the evidence presented, normalisation of the TRM in general practice can therefore be recommended. However, while the usefulness of an intervention is an important factor in determining whether it is normalised or not, the study findings also clearly indicate – consistent with the international literature – that there are other factors that are at least equally important for normalisation. At the time of writing, there are no formal mandates or financial incentives for general practice clinicians or teams to perform regular trigger reviews. It therefore seems likely that normalisation of the TRM in Scottish general practice will be gradual and piecemeal, if it happens at all. Nevertheless, the lessons learnt from this study can be incorporated in the ongoing efforts to further improve the safety of care in general medical practice. In particular, researchers and policy makers should pro-actively identify and address the main factors that are known to facilitate or hinder the implementation of improvement initiatives; the existing knowledge and ‘engagement’ of clinicians should be recognised and harnessed; and the lessons learnt from PSIs should be more widely disseminated.

Circadian rhythms optimise health by ensuring that the internal rhythms of metabolism and cardiovascular physiology are synchronised to daily variations in the light/dark cycle and other recurrent environmental challenges. Disruption in in the light/dark cycle (photoperiod disruption; PD) which occurs during shift work has been associated with changes in metabolism or physiology, (e.g. hypertension, hyperglycaemia) that may influence outcome after stroke. Evidence from pre-clinical studies indicates that hyperglycaemia and hypertension are associated with increased lesion growth and final infarct after focal cerebral ischaemia. Therefore, it was hypothesised that PD would impact on physiological parameters and increase sensitivity to focal cerebral ischaemia. Investigating the effect of chronic photoperiod disruption on outcome following permanent focal cerebral ischaemia For this purpose, a PD protocol was employed to simulate shift work patterns of light/dark (LD) exposure. Male Wistar rats were exposed to either a 12:12 LD cycle or a 6-hour phase advance protocol for 9 weeks. This was done by switching the lights on 6 hours earlier than the previous photoperiod every 3 days. T2-weighted MRI was performed at 48 hours after permanent middle cerebral artery occlusion. Chronic PD for 9 weeks did not significantly affect food intake, body weight and key physiological parameters such as blood glucose and blood pressure and did not increase sensitivity to ischaemic damage in young, normotensive rats. This suggests that light alone is not a single factor to induce circadian disruption. The potentially adverse impact of shift work on stroke outcome may require additional factors such as high fat/high sugar diet or pre-existing co-morbidities. Characterisation and optimisation of animal model for transient focal cerebral ischaemia Results from the previous study of permanent ischaemia raised the question as to on how PD impacts on stroke outcome in the presence of reperfusion. In addition, since the majority of stroke patients present with co-morbid factors, subsequent studies aimed to determine the impact of PD in the presence of pre-existing hypertension following transient focal ischaemia. Prior to this a pilot study was conducted to optimise the time of middle cerebral artery (MCA) occlusion and to characterise the associated neurological and functional outcomes. The following information was used to inform the subsequent studies; 1) the optimal MCA occlusion time in spontaneously hypertensive rats (SHR) was 30 min and 2) transient MCA occlusion resulted in reproducible functional impairments in both neurological score and the adhesive label test assessed at 7-days. Impact of photoperiod disruption on sensitivity to focal cerebral ischaemia and microglia activation in spontaneously hypertensive rats Adult male SHR underwent 30 min transient MCAO following the 9-week PD protocol. Reperfusion resulted in significant tissue salvage. However, PD in the presence of pre-existing hypertension did not exacerbate ischaemic lesion evolution assessed by diffusion-weighted MRI, and by measuring the final infarct volume. Furthermore, PD alone did not induce significant changes in microglia activation in the brains of SHR that were not subjected to ischaemia. Poor collaterals and pre-existing hypertension in SHR may explained to lesser effect of PD on lesion evolution, as there may have been less penumbral tissue available for PD to exert its detrimental effect. Conclusion The studies presented in this thesis demonstrated that PD did not increase sensitivity to focal cerebral ischaemia in normotensive rats. Similarly, PD in the context of major stroke co-morbidity/risk factor did not exacerbate ischaemic damage. This suggest that the primary mechanism of cardiometabolic disturbances among shift workers may not primarily mediated by shifting in the light/dark cycle.

The incidence of end-stage renal disease is increasing in Western Society. Renal transplantation is known to be the optimal treatment for ESRD, being associated with significant reduction in morbidity, mortality for patients and cost for wider society when compared to remaining on dialysis. Unfortunately, the growing number of patients listed for renal transplantation has occurred without a matched supply in the number of suitable organs. This has led to longer average waiting times for increased numbers of patients, who consequently suffer adverse outcomes at considerable cost to the National Health Service as a result of organ shortage. One strategy employed by clinicians to meet demand for organs has been to transplant ‘suboptimal’ kidneys’ historically rejected as unsuitable for transplantation, which are usually retrieved from older and less fit donors. Sometimes referred to as ‘extended criteria’ or ‘marginal kidneys’, such allografts are more prone to damage in the peri-transplantation period, with the major pathological process recognised to be ischemia-reperfusion injury (IRI). Although functioning ‘marginal’ allografts have been shown to confer benefit to recipients, early transplant failure is associated with negative outcomes. Consequently, there is a real need to develop treatments to mitigate renal IRI, especially since the use of ‘marginal’ kidneys is likely to increase. Stem cell therapy has been shown to protect solid organs from IRI in a number of different animal models. Consequently, there is great interest in researching the ability of stem cell-based therapies to ameliorate solid organ damage and perhaps to encourage organ regeneration. However, debate exists regarding the exact mechanism by which stem cells produce their effects. Some researchers suggest that stem cells directly differentiate to replace specialised cell types in damage organs. Other investigators conclude that stem cells produce their effects in a paracrine fashion via the release of extracellular vesicles with the horizontal transfer of genetic material between cells. Unfortunately, no therapies are currently in widespread use to reduce damage to allografts in the peri-transplant period. In part, this reflects the lack of robust small animal models for screening potential renal IRI therapies before testing in large animal models. Furthermore, clinical application has been limited by safety concerns, and particularly by the risk of stem cells undergoing malignant transformation and subsequent tumour formation in recipients. However, investigators hypothesise that the use of stem cell-derived, extracellular vesicles may confer similar beneficial therapeutic efficacy, but lack many of the side effects associated with stem cells themselves. This thesis describes experiments in which stem cell-based therapies are tested in conventional and novel animal models of renal IRI and renal transplantation. In Chapter 3, initial experiments unexpectedly demonstrated the potential of ex vivo expanded stem cells to undergo malignant change and induce tumour formation in recipient animals. Therefore, the subsequent research investigated the effects of freshly isolated stem cells or those of novel extracellular vesicle preparations. In Chapter 4, experiments unexpectedly demonstrated the shortcomings of a conventional rat model of renal IRI. Therefore, Chapter 5 describes the development of a novel rat of model of renal IRI, in which stem cell-based therapies may be tested. Using this animal model, Chapters 6 and Chapter 7 describe the investigation of novel stem cell-based therapies and their effects on renal IRI. Some of these treatments were found to protect kidneys from IRI damage with preservation of renal function and structure in the medium to long-term. Chapter 8 describes a rat model of renal transplantation, in which therapies were investigated after being screened for efficacy in the novel rat IRI model. Although no functional difference was demonstrated, renal histology was preserved by treatment, although the mechanisms by which this effect occurred remain unclear. These findings suggest that stem cells and their extracellular vesicles have the potential to reduce peri-transplantation renal IRI and hence improve long-term outcomes of ‘marginal’ allografts. However, clinical translation requires the long-term efficacy and safety of these novel therapies to be investigated in large animal models of renal transplantation, before further testing in pilot studies.

The risk of hip fracture in people with type one diabetes mellitus (T1DM) is reported to be 7 to 12 times greater than in those without T1DM, and this increased risk is evident in both children and young adults. This fracture risk is higher than expected bone mineral density (BMD) measurements, which indicates the likelihood that other skeletal factors, not captured by DXA, may contribute toward increased fracture risk. There is increasing evidence that alteration in trabecular bone microarchitecture and increased bone marrow adiposity (BMA) are causes for excess skeletal fragility, yet these data are lacking in people with T1DM. Recent technological advances in magnetic resonance imaging (MRI) have allowed the quantification of trabecular bone architecture. In addition, MRI can quantify the amount of intra-abdominal fat, and magnetic resonance spectroscopy (MRS) can also be used to assess BMA. These advances may enhance our understanding of the underlying causes of diabetic osteopathy which may lead to improved fracture risk predictors and preventive measures in patients with T1DM beyond that provided by dual energy x-ray absorptiometry (DXA). The overall objective of this thesis was to improve the understanding of the bone pathology of young adult women with childhood-onset T1DM by using high resolution MRI. A cross-sectional study was first carried out to assess trabecular bone microarchitecture of the tibia, vertebral BMA and abdominal adiposity in patients with childhood onset T1DM (n=30) compared with healthy controls (n=28). Additionally, the biochemical markers of bone turnover, adiposity and GH/IGF-1 axis (IGF-1, IGFBP3, and ALS) were examined to evaluate the underlying mechanism that might result in bone deficit in this group of people. We found that young women with childhood onset T1DM had reduced apparent trabecular bone volume (appBV/TV) and apparent trabecular number (appTbN) and greater apparent trabecular separation (appTbSp) than women without T1DM. Interestingly, these differences remained significant after adjustment for multiple confounders. Furthermore, these abnormalities were markedly obvious in those with microvascular complication compared with those without microvascular complication. Although women with T1DM had greater abdominal adiposity compared with healthy controls, there was no significant difference in BMA between the groups. However, BMA showed positive significant association with current glycaemic control (r= 0.45, p=0.02). Women with T1DM had lower bone turnover and decreased GH/IGF axis compared with healthy controls. Osteocalcin and ALS were negatively correlated with trabecular separation in women with T1DM. III Next, a one-year prospective study was conducted in a subset (n=28) of the participants involved in the cross-sectional study. The aim of this study was to compare one year changes in trabecular bone microarchitecture and BMA in women with and without T1DM. Additionally, the study aimed to evaluate the effect of glycaemic control on these changes over this period. After adjustment for relevant confounders, the cases (n=17) had a lower median appTbN and a higher median appTbSp at baseline and 12 months compared with healthy controls (n=11). Although the sample size was small at follow-up, the trabecular bone deficits were clearly noticeable in those with retinopathy compared with those without retinopathy. Similarly, there was no difference in median BMA which was 26.2% (12.1, 62.1) and 22.4% (9.6, 41.9) in cases and controls, respectively (p=0.57). Additionally, over the 12 month period, there was no significant change in MRI-measured parameters in cases or in controls, and no differences in the change of these variables between the two groups. Mixed model effect analysis showed that age was a negative predictor of percent changes of appBV/TV, appTbN and appTbSp in both cases and controls (p=0.02, p=0.02, p=0.002, respectively). Interestingly, there was a strong correlation between change in HbA1c and change in BMA (r=0.8; p=0.002). In the third study, we aimed to assess adiposity-based determinants of bone mineral density and bone microarchitecture in healthy young women and women with T1DM. Additionally, we aimed to compare the feasibility of using DXA and MRI-measured bone parameters to differentiate women with and without T1DM. In addition to high resolution MRI we used DXA scans to measure BMD and body composition from the same participants (n=26) involved in the longitudinal study. Vertebral BMA was positively correlated with VAT. Additionally, we demonstrated evidence of an inverse association of vertebral BMA and DXA-measured bone parameters of femoral neck, lumbar spine and total body independent of demographics and body composition in healthy young women and women with T1DM. These finding support the hypothesis that BMA is linked with low bone density, and may contribute to excess bone fragility. Moreover, this study suggested that MRI-measured trabecular bone measurements were able to differentiate between T1DM with and without microvascular complication compared with DXA-measured BMD. In summary, differences in MRI-measured trabecular microarchitecture parameters identified in this body of work provide preliminary explanations for elevated fracture risk in young women with childhood onset T1DM. Additionally, these findings provide potential insight into a number of possible underlying mechanisms of diabetic osteopathy.

Background As one of the most common types of shoulder pain, subacromial impingement/rotator cuff tendinopathy (SIS/RCTendinopathy) is frequently treated with physiotherapy. However there is uncertainty as to which patients do and do not improve with this approach. Aim To identify baseline factors that predict outcome in SIS/RCTendinopathy patients following treatment with physiotherapy. Method Patients with a clinical diagnosis of SIS/RCTendinopathy referred for physiotherapy were recruited. Baseline potential prognostic factors (demographics, clinical history, patient reported measures, clinical measures and structural pathology) were recorded immediately prior to the first physiotherapy appointment. Treatment was pragmatic as determined by the treating clinician. Outcome was determined by the change in a patient reported outcome measure (Oxford Shoulder Score, OSS) from baseline. Time points for analysis were at discharge and three months post-discharge. Findings Seventy-six patients fulfilling the inclusion and exclusion criteria were recruited and outcome data were available from 73 subjects at discharge and 62 subjects at three months post discharge. The number of candidate variables were trimmed using a sequential decision process comprising methodological, conceptual and statistical methods. Multivariate regression analysis comprising forward selection and backward elimination was applied. Baseline function (measured by the total SPADI; higher SPADI = greater pain and disability) and (younger) age predicted 15.7% of the outcome (greater improvement in function) from baseline to discharge. Total SPADI (higher SPADI = greater pain and disability) predicted 9.6% of the outcome (greater improvement in function) from baseline to three months post-discharge. Conclusion The study findings provide evidence of a limited ability to predict outcome. Potential reasons for this include the multifactorial nature of the condition and the high degree of heterogeneity in the treatment of the cohort. The limited sample size and lack of consideration of variable types such as patient or clinician expectation are also noteworthy limitations of the study. Nonetheless, the regression findings indicate the value of considering baseline function and age for informing the likely change in patient reported pain and disability across the period of treatment and up to three months post discharge.