B cells are known to contribute to and/or influence immune response trough a variety of mechanisms. Regulatory B cells (Bregs) have recently discovered and their role in regulating autoimmune diseases has been demonstrated in different murine models. In the context of transplantation B cells are generally thought to promote graft rejection through the production of alloantibody and their capacity to efficiently present alloantigens. However, recent clinical trials with B cell depletion have suggested that B cells contribute to the regulation of immune responses to grafts. In addition the evidence that renal transplant patients that are tolerant to the graft have an expansion of B cells with a regulatory phenotype further confirm this idea. The hypothesis tested during this PhD is that murine regulatory B cells, defined as transitional-2 marginal zone precursor (T2-MZP), are contributing to transplantation tolerance. The aims of this project are: i) to investigate whether T2-MZP can transfer transplantation tolerance; ii) to analyse whether the expression of Galectin-1 (Gal-1), known to be a functional molecule for Tregs, is necessary for T2-MZP to suppress; iii) to study whether an increase in T2-MZP is observed in tolerant mice like what has been shown in tolerant transplant patients. The results obtained during this PhD have demonstrated that T2-MZP purified from naïve mice were unable to regulate the immune responses to graft antigens both in vitro and in vivo. However, their regulatory capacity was observed when T2-MZP were purified from mice kept in the conventional (CV) area of the animal house although IL-10 was not involved in their regulatory 4 capacity. A difference in the gut flora between mice kept in the Specificpathogen- Free (SPF) and CV areas was observed and maybe responsible for the differences in the T2-MZP functions. Moreover, Gal-1 was confirmed to be expressed by B cells and T2-MZP isolated from Gal-1-/- mice kept in the CV area lost the ability to suppress in vitro CD4+ T cell activation and to prolong skin graft survivals, suggesting that Gal-1 has a functional role in B cell suppressive function. One of the hypotheses was that the absence of Gal-1 influences the response of B cells to gut. Gal-1-/- B cells showed reduced IL-10 production and increased up-regulation of activation markers in response to a variety of Toll-like receptor ligands in comparison to wild-type B cells, and showed differences in the percentage of P38, ERK phosphorylation and NF-κB translocation. Finally, in trying to mimic the findings with renal transplant patients, mice were rendered tolerant to skin transplants using in vivo anti- CD40L (MR1) and donor splenocyte transfusion (DST). The number of T2-MZP increased in these mice and these cells were suppressive in vitro and, as before, their regulatory capacity did not appear to correlate with IL-10 production. Once transferred in vivo T2-MZP isolated from tolerant mice, and not animal rejecting an allograft, induced prolongation of skin transplants onto naïve recipients. These observations indicate that T2-MZP play a role in transplant tolerance, they need to be “primed” either during tolerance induction protocol or by interaction with gut flora and that Gal-1 is a functional molecule for B cells and T2-MZP B cells.

The treatment response for traumatic head injured patients can be improved by using an autonomous robotic system to perform basic, time-critical emergency neurosurgery, reducing costs and saving lives. In this thesis, a concept for a neurosurgical robotic system is proposed to perform three specific emergency neurosurgical procedures; they are the placement of an intracranial pressure monitor, external ventricular drainage, and the evacuation of chronic subdural haematoma. The control methods for this system are investigated following a curiosity led approach. Individual problems are interpreted in the widest sense and solutions posed that are general in nature. Three main contributions result from this approach: 1) a clinical evidence based review of surgical robotics and a methodology to assist in their evaluation, 2) a new controller for soft-grasping of objects, and 3) new propositions and theorems for chatter suppression sliding mode controllers. These contributions directly assist in the design of the control system of the neurosurgical robot and, more broadly, impact other areas outside the narrow con nes of the target application. A methodology for applied research in surgical robotics is proposed. The methodology sets out a hierarchy of criteria consisting of three tiers, with the most important being the bottom tier and the least being the top tier. It is argued that a robotic system must adhere to these criteria in order to achieve acceptability. Recent commercial systems are reviewed against these criteria, and are found to conform up to at least the bottom and intermediate tiers. However, the lack of conformity to the criteria in the top tier, combined with the inability to conclusively prove increased clinical benefit, particularly symptomatic benefit, is shown to be hampering the potential of surgical robotics in gaining wide establishment. A control scheme for soft-grasping objects is presented. Grasping a soft or fragile object requires the use of minimum contact force to prevent damage or deformation. Without precise knowledge of object parameters, real-time feedback control must be used to regulate the contact force and prevent slip. Moreover, the controller must be designed to have good performance characteristics to rapidly modulate the fingertip contact force in response to a slip event. A fuzzy sliding mode controller combined with a disturbance observer is proposed for contact force control and slip prevention. The robustness of the controller is evaluated through both simulation and experiment. The control scheme was found to be effective and robust to parameter uncertainty. When tested on a real system, however, chattering phenomena, well known to sliding mode research, was induced by the unmodelled suboptimal components of the system (filtering, backlash, and time delays). This reduced the controller performance. The problem of chattering and potential solutions are explored. Real systems using sliding mode controllers, such as the control scheme for soft-grasping, have a tendency to chatter at high frequencies. This is caused by the sliding mode controller interacting with un-modelled parasitic dynamics at the actuator-input and sensor-output of the plant. As a result, new chatter-suppression sliding mode controllers have been developed, which introduce new parameters into the system. However, the effect any particular choice of parameters has on system performance is unclear, and this can make tuning the parameters to meet a set of performance criteria di cult. In this thesis, common chatter-suppression sliding mode control strategies are surveyed and simple design and estimation methods are proposed. The estimation methods predict convergence, chattering amplitude, settling time, and maximum output bounds (overshoot) using harmonic linearizations and invariant ellipsoid sets.

No description available.

Much of the variation seen in surgical outcomes can be explained by differences in the quality of management of post-operative complications and ward-based care. The surgical ward round (WR) is critical to determining post-operative care and serves as the primary point of interaction between clinician and patient. Despite this, it is an area not subject to training or assessment at present. This thesis demonstrates the high degree of variability which exists in the conduct of WRs. It establishes the link between suboptimal patient assessment and increased risk of preventable post-operative complications. These place patients not only at risk of short-term deterioration, but result in reduced long-term survival as well. In order to quantify WR quality, a novel assessment tool has been developed and validated within a simulated environment. Ward simulation is a nascent branch of simulation which has been only preliminarily explored to date. A simulation environment was developed to take advantage of the known benefits of simulation such as controllability, reproducibility, and recordability, whilst maintaining a high level of fidelity and realism. An evidence-based curriculum for surgical WR training was designed and implemented in a simulation-based course. By focusing on structured generic processes of patient assessment and management, this resulted in significant improvement of trainee performance in routine WRs. To ensure standardised and optimum management of specific conditions, checklists have proven themselves to be of great value in a number of surgical and medical disciplines. Surgical complications are common, yet their management often suboptimal. As part of this thesis, evidence-based protocols for the management of the six most common complications were designed and validated. The implementation of these in a simulation-based randomised, controlled trial has resulted in greatly increased adherence to evidence-based standards of care, as well as improved communication and clinician performance. This thesis explores the variance currently present in surgical ward rounds, and the potentially grave consequences of this for patient outcomes. To date, WRs have been one of the last areas of surgical care still dependent on the Halstedian principle of experiential learning alone. The tools have now been developed with which to assess, improve, and standardise critical structures and care processes in the assessment and management of the post-operative surgical patient. Future implementation of these and integration into surgical curricula will benefit clinician training, patient care, and surgical outcomes alike.

Previous studies aimed at identifying biomarkers of tolerance in kidney transplant patients have revealed an expansion of peripheral blood B cells and over- expression of B cell-related genes. In humans, Memory, Naïve and Transitional B cells are the main B cell subsets found in circulation, and it is has been shown that the Transitional subset, defined by its regulatory properties, is expanded in tolerant recipients compared to non-tolerant kidney transplant patients. However, the role this population plays in kidney transplantation tolerance remains unclear. Here, we report three different mechanisms to explain the contribution of B cells in transplantation tolerance. Kidney transplant patients were divided into five groups; tolerant, stable, monotherapy, patients who lost tolerance and chronic rejector. In addition, this study also included a group of age/gender-matched healthy volunteers. B cells from each group of patients were tested for antigen presentation, antibody production, cytokine production, and co-stimulatory function. B cells from tolerant patients produced higher levels of IL-10 and lower levels of TNF-α than B cells from chronic rejector after CD40 and CpG activation. Moreover, B lymphocytes from tolerant patients also exhibited a failure in the BCR signalling pathway, suggesting a certain degree of anergy or responsiveness by these cells. Donor- specific assays revealed that B cells from tolerant patients were inefficient to recognise donor-antigens, compared to B cells from chronic rejector. This impairment prevented the triggering of the Th1 response by recipient CD4+ T cells and donor-specific antibody production by Plasma cells. Finally, Transitional B cells were the lowest CD4+ T cell- activating cells, compared to Naïve and Memory B cells. This reduced CD4+ T cell activation was due to low cell viability, reduced CD86 expression and high IL-10 production. In conclusion, these data suggest that B cells from kidney tolerant recipients contributed to maintaining organ acceptance and graft survival by donor-specific and non-specific regulatory properties exhibited by all their subsets, especially Transitional B cells.

There is a growing concern that adverse events occur frequently in operating theatres. Adverse events such as wrong site surgery and surgical site infections have a severe detrimental impact on not only the patient but also the healthcare staff and the services. Institute of Medicine's report, 'To err is human', highlighted that teamwork failures are a leading cause of death and suffering. Yet, in surgery, measuring teamwork and designing interventions to improve teamwork and patient safety in operating theatres remains an area of research that is largely unexplored. This thesis aims to measure and improve teamwork in operating theatres to ensure safer surgery. In this project, the WHO surgical safety checklist was evaluated for its impact on patient safety. The WHO checklist improved patient safety processes in operating theatres but its impact on teamwork, intra-operative problems and theatre efficiency was not clearly understood. Therefore, a framework was developed to measure teamwork failures, equipment problems and technical failures as surrogate markers of teamwork, patient safety and efficiency in operating theatres. Equipment failures emerged as a sensitive measure of teamwork in operating theatres. Teamwork failures were also associated with technical failures, delay in case progress and patient harm. It emerged that the WHO checklist can improve teamwork and theatre efficiency and reduce equipment problems in operating theatres when it is used in its true spirit rather than a tick-box exercise.

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No description available.

This thesis focusses on the dominant theme at my research activity, the triboiogy of bearings for use in Total Hip Replacement. This theme covers the majority of my research projects and hence publications to date. Restricting the research that is covered has the benefit of focusing the summary into a narrative which formed part of the worldwide research effort in this area. It does however mean that some of the more esoteric projects, such as the work on baby's dummies, were not included. . For those interested in this field or merely curious, the reference (6) has been included in the list of citations attached to this document.

Background Sepsis is a condition characterised by a massive acute inflammatory response and insulin resistance. Several inflammatory mediators involved in the immune response during sepsis have been identified. Recently it has become clear that adipose tissue contributes to the production of pro- and anti-inflammatory mediators, which have been termed adipokines. Adiponectin is an adipokine that has anti-diabetic, anti-atherogenic and anti-inflammatory properties. Its role in chronic inflammatory diseases, such as type II diabetes mellitus (DM) and obesity has been extensively studied. Generally, adiponectin is down-regulated in these conditions which are characterised by insulin resistance. Adiponectin, previously thought to be exclusively expressed in and secreted from adipocytes, has now been shown to be released from other tissues such as skeletal muscle, cardiac muscle and bone. Adiponectin from adipose tissue is down-regulated in mouse models of sepsis, however, no information is available about the role of adiponectin receptors. In chronic insulin resistance, adiponectin receptor gene expression is decreased, suggesting a down-regulation of the ‘adiponectin system’. Adiponectin gene expression appears to be partially regulated by NFκB, a transcription factor co-ordinating the release of inflammatory mediators in response to an appropriate stimulus, such as lipopolysaccharide. Other signalling mechanisms may also be involved, in particular the HIF-1α pathway. HIF-1α is another transcription factor with a large number of target genes, many of which are involved in the inflammatory process. Although HIF-1α was initially discovered as a cellular regulator of hypoxia, the pathway has now been shown to be activated by other non-hypoxic mechanisms of up-regulation, including bacterial infection. HIF-1α is expressed in immune cells, however, its role in adipose tissue during sepsis remains unclear. Methods Three different lines of experiments used in this thesis. The animal model used high dose LPS injected intra-peritoneally (under general anaesthesia) into 8-10 week old male C57BL/6J mice. Mice were killed at 4 or 24 hours after injection and tissues (Peri-renal, subcutaneous and epididymal fat, liver, muscle, small bowel and spleen) were removed for analysis. Adiponectin and adiponectin receptor gene expression was determined by quantitative real-time PCR (qPCR). The cell culture model used cell lines, 3T3-L1 adipocytes and C2C12 myocytes, grown in culture and then treated with varying concentrations of LPS. Cells were harvested at 4 and 24 hours and qPCR was performed to ascertain adiponectin and adiponectin receptor gene expression. The same animal and cellular models were utilised for the HIF-1α investigations with protein determination carried out using ELISA. Finally, twenty-one septic patients were recruited from the Intensive care unit at the Royal Liverpool University Hospital, following ethical approval and written consent. Blood samples were taken on days 1 and 2 and day of discharge and serum levels of total and HMW adiponectin were determined by ELISA. Results Alterations in adiponectin and its receptors expression in murine endotoxaemia Adiponectin receptors were down-regulated following LPS injection. The greatest changes acutely were in muscle, liver and peri-renal fat (adipoR1) and liver, muscle, peri-renal and sub-cutaneous fat (adipoR2). There were no significant changes in the other tissue depots. After 24 hours, there were fewer changes in gene expression with adipoR1 being down-regulated in liver and skeletal muscle and AdipoR2 in skeletal muscle only. Down-regulation of adiponectin gene expression following LPS was confirmed in the adipose tissue depots. We demonstrated that the adiponectin gene was expressed in skeletal muscle and sequencing of the PCR product confirmed a 100% match for adiponectin mRNA. C2C12 myocytes were then used to verify the presence of adiponectin mRNA in skeletal muscle cells. In tissue depots, adiponectin gene expression was significantly reduced in skeletal muscle in both the 4 and 24 hour cohorts respectively. Alterations in adiponectin and its receptors expression in cell lines In the cell lines, the inflammatory response to LPS was confirmed using IL-6 as a reference gene. This also confirmed methodological success. Adiponectin gene expression from 3T3-L1 adipocytes was acutely reduced following treatment with high dose LPS but there were no changes in expression in cells treated with lower concentrations of LPS. There were no changes at 24 hours. Adiponectin receptors were down-regulated but not consistently with dose and these changes were only observed in the cells harvested after 4 hours. In C2C12 myocytes, there was a significant reduction in adiponectin gene expression following high doses of LPS but there were minimal changes in adiponectin receptor expression in the C2C12 myocytes. Human Study There was a significant increase in both total and HMW adiponectin between day 1 and day of discharge and the ratio of HMW adiponectin to total adiponectin also increased between admission and discharge. There were no changes in total or HMW adiponectin or their ratio between day 1 and day 2 of admission. HIF-1α HIF-1α gene expression was up-regulated in liver and spleen 4 hours post LPS injection. The changes persisted 24 hours after LPS treatment with increased expression in liver, small bowel and spleen. Protein levels were elevated in skeletal muscle after 4 hours and liver after 24 hours and spleen. Discussion These results increase the evidence that adipose tissue is not an inert storage medium for fatty acids but a sophisticated endocrine organ. The ‘adiponectin system’, including adiponectin and its two receptors, is down-regulated in-vivo and in-vitro models of sepsis. This may play a role in the metabolic derangements such as hyperglycaemia and insulin resistance. In addition, hypoadiponectinaemia may have a significant role in the disordered inflammatory process known to occur in sepsis, possibly impacting on mortality as shown in some animal studies. Adiponectin is not exclusively adipose tissue derived and interestingly we have demonstrated the presence of adiponectin mRNA in other tissue such as skeletal muscle. The effect of reduced gene expression from extra-adipose tissue depots is yet to be established but may have a paracrine or autocrine effect rather than an endocrine role. Low total and HMW adiponectin levels during human sepsis have also been identified. Whilst hypoadiponectinaemia in sepsis has been observed in previous studies, increases in HMW adiponectin associated with clinical improvement have not been previously demonstrated. A further signalling pathway investigated in these models was HIF-1α. These results demonstrate a global up-regulation of HIF-1α gene expression across tissue depots and cellular models. This may reflect tissue hypoxia but also may reflect non-hypoxic up-regulation by LPS and inflammatory mediators. HIF-1α is known to play a part in the inflammatory process and, like adiponectin, has links to the NFκB signalling pathways.