Background: Liver ischaemia reperfusion injury (IRI) occurs after prolonged ischaemia followed by reperfusion in the clinical setting, such as liver resection surgery and liver transplantation and is associated with increased morbidity and mortality. Ischaemic preconditioning (IPC) is a therapeutic strategy to lessen liver IR injury. Liver IPC is a mechanical technique whereby a short period of occlusion of the blood supply to the liver confers protection against IR injury. Understanding the molecular mechanisms of IPC offers the possibility of developing techniques and pharmacological agents that will reduce IRI and improve clinical outcomes. Hypothesis, Aims and Objectives: The molecular mechanisms of liver IPC are not clearly established. Nitric oxide is an important mediator, but the role of the activation and expression of nitric oxide synthase (NOS) and its isoforms endothelial NOS (eNOS) and inducible NOS (iNOS) is unclear. Haem oxygenase-1 (HO-1) protects, but it is unclear if this depends on NOS. There appear to be two phasese of liver IRI and IPC: an early and late phase. There is evidence that NOS has roles in both phases, but that HO-1 has a role only in the late phase. The principal aim was to develop a transgenic eNOS knockout (eNOS-/-) model of liver IRI and IPC to specifically probe the in vivo physiological roles of eNOS and its interactions with HO-1 these processes. Methods: An in vivo mouse model of partial (70%) warm hepatic ischaemia reperfusion (IR) was used. Normal and transgenic double knockout for eNOS (eNOS-/-) mice were used. A partial warm liver IR model was used where ischaemia was applied to cephalic lobes only followed by reperfusion. Ischemic preconditioning (IPC) consisted of ischaemia applied directly to the cephalic lobes followed by reperfusion then IR to the cephalic lobes. Final reperfusion was either for 2 hours (normal and eNOS-/- mice) representing early phase IRI or for 24 hours (normal mice) representing late phase IRI after which the animals were terminated for tissue and blood samples. The endpoints measured were surface laser Doppler flow to assess liver 14 microcirculation during the experiment and blood and liver tissue samples at the end of the experiment for serum ALT, liver histological injury scores, Western blotting for eNOS, iNOS, phosphorylated eNOS (p-eNOS), HO-1 protein and RT-PCR for HO-1 mRNA. Results: 1. In this model of early phase partial hepatic IR consisting of 45 minutes ischaemia to the cephalic lobes and 2 hours reperfusion (index IR), there was IRI in normal mice across the three endpoints for IRI of serum ALT, histological injury and microcirculatory dysfunction. IPC consisting of 5 minutes ischaemia and 10 minutes reperfusion (IPC 5/10) preceding index IR reduced IRI across the three endpoints in normal mice. In eNOS-/- knockout mice, there was also IRI across the three endpoints, with greater hepatocellular and histological injury than normal mice, but no difference in the microcirculatory dysfunction compared to normal mice. In eNOS-/- knockouts IPC 5/10 did not reduce IRI across the three endpoints. This indicates that eNOS is a mediator of the protective effects of IPC in early phase liver IRI and baseline eNOS reduces the severity of IRI even without IPC. Based on the differences on the effects on the endpoints between all the experimental groups, it appears that IPC protection is mediated by eNOS in hepatocytes and sinusoidal endothelial cells (SECs) and baseline eNOS protection in liver IR without IPC is mediated by hepatocyte eNOS only. 2. It was demonstrated that in the early phase partial hepatic IR model that both IR and IPC increased eNOS protein expression and eNOS activation by phosphorylation with no additional effect of IPC. This indicates that the protective effect of eNOS with IR alone may at least be partly mediated by increased eNOS protein expression and activation by phosphorylation, but the benefits of IPC in reducing early phase IR injury are mediated by eNOS activation by other mechanisms. Expression of iNOS protein does not play a role in IR injury in our model. Haem oxygenase-1 (HO-1) protein was not expressed in our model, but HO-1 mRNA was expressed in both normal and eNOS-/- animals following liver IR and IPC, indicating that HO-1 expression is not dependent on eNOS. HO-1 may therefore still have a protective effect in the late phase of IR injury acting in a parallel pathway to eNOS. 15 3. In the model of late phase partial (70%) hepatic IR consisting of 45 minutes ischaemia, recovery from anaesthetic and 24 hours reperfusion (index IR) developed using normal mice only, there was liver IRI across two endpoints of serum ALT and histological injury (microcirculatory dysfunction was not studied in the late phase) with progression of histological injury from mainly sinusoidal congestion to hepatocyte necrosis. IPC consisting of 5 minutes ischaemia and 10 minutes reperfusion (IPC 5/10) preceding index IR reduced IRI across the endpoints. HO-1 protein was detected with late phase IR and IPC, indicating timecourse of early phase HO-1 mRNA expression followed by late phase HO-1 protein expression. This is consistent with the possibility of HO-1 potentially having a protective role in late phase liver IR and IPC. Conclusions We have developed a model of early and late phase liver IRI and IPC, which has demonstrated that eNOS is a protective mediator in IRI and IPC in the early phase, HO-1 is activated independently of eNOS and may have a role in late phase IRI and IPC. This model should prove useful in investigating the roles of eNOS and HO-1 in liver IPC and IR injury. This would ultimately be used to identify pathways for development of pharmacological agents that would effectively reduce liver IR injury in the clinical setting and improve patient outcomes related to this.

Bio-scientific research has relied heavily on models of cell monolayers cultured on plastic. For most cell types, this does not represent their in vivo tissue organisation well. As a result they behave differently in vitro from in vivo, leading to poorly predictive data. Plastic compression (PC) of collagen is used to engineer constructs with more tissue-like conditions. The aim of this study was to test the feasibility of using these constructs as a three-dimensional tissue model for assessing the fate of hyaluronan nanoparticles (HA-NP). Collagen hydrogels were seeded with cells and HA-NP and subjected to PC. Due to their small size, HA-NP retention following PC was investigated. HA-NP uptake by cells was then compared to conventional monolayer cell cultures. 19.1±1.2% of the initial HA-NP load was retained following PC, which could be increased to 31.1±3.1% by multi-layering. This entrapment was found to be largely physical as HA-NPs were released from the construct following cellular remodeling, but not without it. Cells in monolayer reached their maximum HA-NP uptake in 3 days whilst cells in collagen peaked at 7 days. This maximum uptake was 60.1 a.u., twice as large as that of 3D-cultured cells (32.8 a.u). A novel method was developed to analyse local collagen densities which revealed particular collagen distributions in micro-patterned constructs depending on the shape of template used; round grooves had a 21.4±4% increase in collagen density at their bases, whilst rectangular grooves displayed two peaks corresponding to their internal corners, which were 15.2±4% and 16.9±3% denser than the unpatterned regions. This work has enabled greater understanding of the PC and micro-moudling which will aid in creating more complex tissue constructs in a predictable and controlled way. The importance of 3D tissue organisation in in vitro models, particularly for nanoparticle testing, has also been demonstrated in this work.

Abstract Background - High levels of anxiety and pain during conscious surgery are associated with poorer post-surgical outcomes. This thesis aimed to compare the effectiveness of intraoperative distraction interventions for anxiety and pain management during minimally invasive surgery under local anaesthetic, the impact on 3 month outcomes and patient's experiences of conscious surgery. Methods - Literature and systematic reviews explored the current literature surrounding conscious surgery. Two randomised controlled trials investigated the impact of intra-operative distraction interventions on pain and anxiety during conscious surgery. Analyses of postoperative outcomes including multiple regression and analyses of covariance explored the relationships between intra-operative and post-operative outcomes at 3 months post-surgery. A qualitative analysis explored patient experience of minimally invasive surgery under local anaesthetic. Results - Intra-operative distractions including touch, audiovisual stimuli and interaction with nurses resulted in significantly lower reports of intra-operative anxiety and pain than treatment as usual. Participants who interacted with nurses during surgery reported significantly greater improvements in quality of life at 3 months post-surgery than those who received treatment as usual. Intra-operative distractions had no impact on the course of post-operative pain, return to work or satisfaction with treatment. The symptoms of varicose veins negatively impacted upon quality of life and participants felt unprepared for the experience of surgery. Relationships improved patient experience during surgery and improvements in physical and psychological wellbeing were reported at 3 months following surgery. Conclusions - The use of simple distraction techniques, particularly interacting with nurses, intra-operative touch or audiovisual stimuli used during surgery significantly improved patient experience. Participant satisfaction with surgery was high but there is scope to improve the provision of pre-operative information patients receive to increase their understanding of surgery. Relationships with others influenced all stages of the operative process. (c) Briony Hudson 2014

A milk preparation has been shown by several previous workers to have potential as a coagulable analogue fluid for the study of flow related thrombosis <I>in vitro</I>. Christy used bodies of revolution in pulsed and steady milk mixture flows, in an attempt to identify the hydrodynamic causes of blood clotting in determinate and simple flow situations. The varying extent of deposition on the downstream-side of test-bodies of different shapes indicated stasis and some aspect of agitation in the vicinity of a surface as the concomitant conditions necessary for clot to occur on that surface. The principal aim of the research reported here was to develop Christy's milk coagulation experiments further, by obtaining more information about deposit growth hence to shed light, by inference, on mechanisms of flow-related thrombus formation. To this end, milk experiment reproducibility was improved, ways to eliminate bubble and wall clot formation were attempted, and the feasibility of ultrasonic imaging as a means of continuously measuring the thickness of deposited clot was assessed and subsequently employed. The use of real-time ultrasonic imaging enabled wall clot deposition, and its influence on milk experiments, to be continuously monitored macroscopically. It may also provide a way of assessing various methods aimed at preventing wall clot formation. The prospective advantages of ultrasonic imaging were not fully realized by the arrangement adopted in these preliminary experiments, but it now appears that ultrasonic imaging could be of use for studying curd deposition on test-objects, with certain recommended modifications to the current arrangement.

A milk analogue for clotting blood was developed by Lewis. This was then used by Christy, and subsequently Marosek, to investigate the clot deposited in the region of a series of bluff bodies and heart valves in both steady and pulsatile flows. Bodies of revolution, such as a teardrop, were used to simplify the flow structures present in the flow, making the relation of flow features to the deposition of clot more amenable to analysis. Christy concluded that stasis was a necessary but not sufficient condition for thrombus deposition. A quantitative flow visualisation technique known as particle image velocimetry (PIV) has been developed and applied to the flows investigated by Christy but with water as the test fluid. Steady flow PIV investigations around a Björk-Shiley valve showed significant three dimensional structure which varied markedly over time. In pulsatile flows, both at and below physiologically relevant flowrates, significant variation was observed between recordings made at the same point in successive pulse cycles. This may indicate that the assumptions underlying the sampling methods used in LDA investigations to estimate the Reynolds stresses downstream of heart valve prostheses in pulsatile flows are flawed. In pulsatile flows of a period of 1 Hz and a mean volumetric flowrate of 6.7x10<SUP>-5</SUP> m<SUP>3</SUP>/s, the maximum bulk viscous shear stresses determined using PIV were of the order of 0.05 N/m<SUP>2</SUP> with maximum values of 0.5 N/m<SUP>2</SUP> near the wall, well below the value expected to cause lysis of red blood cells even for extended exposure. In the same physiologically relevant flow conditions, the maximum viscous shear stress at the trailing tip of the valve designed by Dr Norman Macleod was again of the order of 0.5 N/m<SUP>2</SUP>. This level of wall shear is at the lower end of the range over which behavioural and shape changes are induced in endothelial cells and the adhesion of platelets to endothelial cells may be promoted. Particle image velocimetry is a powerful augmentation to the range of techniques available for assessing the performance of heart valve prostheses <I>in vitro. </I>

The human nose is a complex 3-dimensional organ that in the event of damage or disease can dramatically alter an individual’s profile. In recent decades, rhinoplasties have become one of the most sought after cosmetic procedures, evidencing a public tendency to view the nose as being crucial to the perception of facial attractiveness. For a subset of patients who suffer extensive damage to the nose, the lasting physical effect has devastating consequences. Severe disfigurement of the nose can cause patients to suffer from psychosocial issues, ranging from poor body image and low self-esteem, to depression and suicidal thoughts. For centuries dating back to 600BC, reconstructive surgeons have developed methods for transplanting tissues from different parts of the body to reconstruct this important organ. The overall aim has been to produce a nose as close to normal anatomy and function as possible, using a series of staged operations, together termed a Total Nasal Reconstruction. In recent years however, the field of tissue engineering has opened up a wealth of opportunities to solve the dilemmas of the reconstructive surgeon. Major advancements in cell biology and scaffold technology have meant that the application of tissue engineering methodology to clinical problems is nigh. The ability to engineer bespoke tissues for patients, would in theory minimize the need for donor tissue transplantation, simplify the surgery, and therefore improve clinical outcome. For this reason, this study aimed to apply the paradigm of tissue engineering to nasal reconstruction, developing a cartilaginous construct that could be used to advance this surgical procedure.

The prostate has a zonal anatomy, with differing susceptibilities to disease (benign prostatic hyperplasia originates from the transition zone, prostate cancer largely arises in the peripheral zone). The molecular reasons for this are not understood. Previous prostate cancer microarray studies have used whole benign, diseased or tissue adjacent to the carcinoma as normal controls, for what is an epithelial disease. This study provides a gene expression profile of normal, non-diseased prostate, or a ‘reference prostate gene expression profile’. This has been compared to prostate cancer to identify novel biomarkers of disease. This study also investigates zonal differences in gene expression between different anatomical zones of the prostate. I used normal, human donor prostate tissue, laser capture microdissection (LCM), and Affymetrix gene expression arrays to achieve these aims. Eight LCM prostate epithelial samples from 3 donor prostates were used. The gene expression data was validated by low density real-time PCR and immunohistochemistry on a prostate tissue microarray. Major differences in gene expression were discovered between whole tissue and LCM epithelium only prostate using homology tables. Novel prostate adenocarcinoma genes were identified using a publicly available LCM prostate cancer gene expression array dataset. 9318 genes showed significant differential expression in normal vs. cancer datasets. Three targets, MCM2, NR1D1 and ABCA1 were validated at the protein level. Expression of NR1D1 and ABCA1 were increased in cancer, suggesting they are novel epithelial biomarkers of prostate cancer. An analysis of zonal differences in gene expression found significant differences between zones. Zonal specific markers included TGM4 (central zone), LPL (peripheral zone), and COL9A1 (transition zone). This study provides: (i) a gene expression profile of the normal prostate epithelium (ii) novel, prostate adenocarcinoma specific gene and protein markers and (iii) the first gene expression profile of normal epithelium on the basis of zonal anatomy of the prostate.

Heart valve replacements are among the most widely used cardiovascular devices and are in rising demand. Currently, clinically available devices are restricted to the mechanical and bioprosthetic valves which are associated with significant complications and drawbacks. Synthetic leaflet heart valves based on newly emerged superior materials represent an attractive alternative to the existing prostheses, merging superior durability of mechanical valves and enhanced hemodynamic function of bioprosthetic valves. A new nanocomposite polymer based on polyhedral oligomeric silsesquioxane (POSS) and poly(carbonate-urea)urethane (POSS-PCU) has been developed for biomedical application. In this thesis, application of POSSPCU for development of a novel synthetic leaflet heart valve has been fully investigated. Mechanical and surface properties of this material in both room and body temperature were investigated as well as durability, fatigue properties, and anticalcification potential under accelerated physiological condition. Endothelialisation potential of the polymer was also elicited by isolation, characterisation and culture of endothelial progenitor stem cells extracted from human peripheral and umbilical cord blood. Haemocompatibility of the material was evaluated in terms of thrombogenenicity, palatelet activation and induced inflammatory response. Based on the favourable outcomes of these experiments, a novel trileaflet valve prosthesis was developed using advanced design and manufacturing strategies. The valve prototypes were then assessed in terms of hydrodynamic performance using a pulse duplicator system. The results indicate that POSS-PCU nanocomposite is an optimal material to be used in the fabrication of new generation synthetic leaflet heart valves with enhanced durability and superior performance.

Background Patients with diabetes have a 10% life-time risk of developing diabetic foot ulceration (DFU). DFU is the commonest cause of severe limb ischaemia in the western world. In diabetes mellitus, anaemia is frequently unrecognized. Recently, cell-to-cell interactions, via gap-junctional communication have been shown to play a key role in the molecular biology of wound-healing. Gap-junction channels are assembled of transmembrane proteins called connexins (Cx). Over-expression of Cx43 in wounded skin is associated with both a direct negative effect on keratinocyte and fibroblast migration and delayed wound-healing. Aims The aim of this thesis was to examine the problem of anaemia in patients with poorly-healing DFU. Further, to investigate the prevalence and type of anaemia seen in the high-risk severe DFU patient group, and to determine the current service provision required. Finally, to gain further scientific understanding of the wound-healing process and roles of anaemia and Cx43. Findings In severe DFU patients, >75% have iron deficiency anaemia, although a significant spread of indices was observed. Functional Iron Deficiency (FID) was seen in 25.5%. With current clinical markers, it is incredibly difficult to determine causal relationships. An in-vivo full-thickness excisional wounding study demonstrated that Hb decline correlated with slower re-epithelialisation (p=0.0060). Full-thickness skin biopsies taken from severe limb ischaemia patients demonstrated abnormal histological features in association with increased epidermal Cx43 expression at the wound-edge. 80% of unwounded surgically-viable skin showed persistence of these abnormal features not previously described. Conclusion In severe DFU patients, a high incidence of anaemia and FID was observed Identification of anaemia, AID and FID in this patient group is necessary to assess the role of therapeutic strategies. This is the first detailed in-vivo histological study to demonstrate that iron-deficiency anaemia may play a role in delayed wound-healing. Abnormal Cx43 dynamics in severe limb ischaemia may also represent a novel future therapeutic target.

Tissue engineering of small intestine aims to provide a cure to patients suffering from short bowel syndrome by increasing the absorptive surface through neo-intestinal mucosal tissue. So far, preliminary in vivo attempts by a research team in the USA have shown regeneration of neo-intestinal mucosa in rat models with some success; however experiments in this complex field of tissue engineering still remain in infancy and far from clinical use. A fresh perspective is required to further investigate all the three aspects of tissue engineering, namely, the polymer scaffold, the cell supply, and the biomolecules. The concept of nanocomposite polymer is rapidly emerging and has generated a lot of enthusiasm in tissue engineering due to their high surface to volume ratio and hence enhanced performance. This work was focussed to develop and characterise scaffolds for small intestinal tissue engineering using a new nanocomposite polymer of polycaprolactone and silsesquixane, developed in our laboratory. An in vitro study was also performed to test the scaffolds for cell viability and proliferation using rat’s intestinal epithelial cells. Our results have shown that biodegradable polycaprolactone-silsesquioxane nanocomposite can be fabricated in desired scaffold morphology using simple techniques like particulate leaching, and that it supports intestinal cell growth and proliferation. Future studies incorporating these scaffolds for in vivo use in animal models need to be carried out in order to investigate further about their ability to withstand natural forces within the abdomen, and whether they support cell growth based on principles of cell migration, before a more definitive and continuous cell supply is available in form of stem cells cued specifically to intestinal lineage.