Ischemia reperfusion injury (IRI) following liver transplantation and major liver surgery results in a systemic inflammatory response syndrome (SIRS) with damage to the liver and remote organs. Physical methods of reducing liver IRI such as direct ischemic pre-conditioning (IPC) where a brief period of IRI is applied to the liver have been shown to reduce the adverse effects of liver IRI. However, direct liver IPC has been shown to impair liver regeneration in experimental models. Remote ischemic preconditioning (RIPC) is a novel strategy for reducing IRI through a brief period of IPC to a remote organ and this has been shown to reduce IRI to the heart and other organs. The purpose of the experiments described in this thesis is to investigate the effect and mechanism of RIPC in reducing liver IRI in an animal model and in patients. A rabbit model of total hepatic ischemia was established to study early warm liver IRI and in this model 25 minute of total portal inflow occlusion resulted in severe liver IRI at 2 hours of reperfusion. In the main experimental study rabbits were divided into four equal groups to study the effect of RIPC on early phase (2 hours) warm IRI following total portal inflow occlusion. RIPC before IRI reduced the adverse local and systemic effects of IRI, reduced acidosis and nitric oxide was shown to be an important mediator of protection. In a proof of concept study 16 patients undergoing major liver resection surgery were randomised into two groups, control and RIPC. In the RIPC group, post resection, there was a reduction in liver IRI.

Studies of Sacral Nerve Stimulation (SNS) have demonstrated significant symptom improvement in Faecal Incontinence (FI); however, mechanisms of action remain poorly understood. Various authors have examined anorectal physiological parameters with SNS; and apart from an observed increase in squeeze pressures, findings were mostly inconsistent. It is currently believed that effects are mediated through neuromodulation. Identification of the involved neuronal pathways and the associated changes at the level of the target organ can further inform the process of patient selection for this costly treatment. The aim of this thesis was to examine potential SNS mechanisms by studying its effects on the sphincteric and suprasphincteric properties utilising physiological and structural tests. A total of 30 patients (29 female, median age 49 years) with intractable FI undergoing temporary SNS were recruited into four different studies designed to examine associated physiological and structural changes. The study of rectal properties revealed no change in rectal compliance following stimulation. However, rectal pressures associated with urge perception and maximally tolerated distension were significantly increased; predominantly in clinical responders. Anal squeeze pressures were significantly increased after stimulation in both responders and nonresponders. However, an increase in resting pressure was only noted in responders. Furthermore, Recto-Anal Inhibitory Reflex (RAIR) recovery time was significantly shorter after stimulation. An acute ON/OFF alteration of stimulation did not result in an acute change in anal pressures or RAIR parameters. Magnetic Resonance Proctography revealed a trend of reduced duration of rectal emptying after stimulation. Furthermore; it has suggested that more efficient contrast evacuation occurs after SNS. Mechanisms of SNS are most probably complex and multi-factorial. The observed changes in rectal sensory thresholds, RAIR recovery time and rectal evacuation in this study suggest that SNS influences the anorectal autonomic function and that it has an afferent-mediated mechanism.

Background: The recent unprecedented interest in islet allotransplantation has been tempered by the observation that sustained freedom from exogenous insulin is rarely achieved and the rate of insulin independence drops to 10% at 5 years follow-up. One critical determinant of successful islet allografting is preservation injury to the pancreas. The two-layer method (TLM) of pancreas preservation was developed to mitigate the deleterious effect of cold ischaemia, but the mechanism is unclear and its clinical efficacy is controversial. Hypothesis, Aims and Objectives: There is no clinical or experimental evidence for benefit of the two-layer method as currently employed in pancreatic islet transplantation. A potentially beneficial effect on pancreas preservation by improvement in graft ATP production is possible by modification of the two-layer method. The principal aim was to develop optimal protocols for pancreas preservation in islet transplantation by clarifying the mechanism of TLM. A second objective was to develop a dynamic model for the study of mitochondrial function during organ preservation. Methods: 1. Perfluorocarbon (PFC) content of porcine pancreases preserved in TLM and in University of Wisconsin (UW) solution for 24 hours was compared. Pancreatic samples were analysed using Varian INOVA 9.4T MR scannerspectrometer. External PFC standard was introduced for quantification. Four consecutive transverse images of 4mm thickness were obtained using a spin-echo sequence. 19F MRS was performed with the same parameters except with more averages. MR data was confirmed by headspace chromatography. 2. Real-time changes in pancreas bioenergetics were studied with 31P MRS for rat pancreases preserved at 4°C - 6°C in five different groups: chilled Marshall‟s, static TLM, continuous TLM with oxygen perfused at 0.5L/h, and static or continuous TLM both the latter following 30 min warm ischaemia. 31P spectra were analyzed for phospho-mono-esters, inorganic phosphate (Pi) and α-, β- and γ-nucleotide triphosphate. Results: 1. PFC standard was readily detected in 19F MR images. There was no signal from porcine pancreas in 19F MR images following either UW or TLM storage. 19F MR spectra typical of PFC were not obtained from either UW-or TLM-preserved pancreas with non-localized 19F MRS. Mean concentration of PFC in TLM pancreas measured by head space chromatography was not significantly different from background concentration in UW pancreas. 2. Intergroup rates of change of [γ-ATP]/[Pi] and [β-ATP]/[Pi] throughout preservation period were significantly different. For continuous TLM there was an increase relative to baseline but decrease for both static TLM and Marshall‟s with respect to continuous TLM. Rate of decrease was similar for the Marshall's and static TLM groups. [γ-ATP]/[Pi] and [β-ATP]/[Pi] increased with WI continuous TLM but decreased for WI static TLM. Conclusions: There is no evidence of penetration of perfluorocarbon into pancreas tissues investigated either by MR or chromatography in organs preserved at hypothermia. 31P-MRS is an effective tool for non-invasive assessment of pancreas bioenergetics. Continuous TLM preserves cellular bioenergetics and is superior to current non-PFC based solutions for pancreas preservation.

Dysplasia arising in Barrett’s oesophagus (BO) confers risk of progression to oesophageal adenocarcinoma (OAC), but it is variable and confounded by sampling error and diagnostic inter-observer variability. There is a need for biomarkers to accurately define risk and guide surveillance and treatment strategies. Oesophagectomy is the preferred treatment of high grade dysplasia (HGD), although this has been challenged by the emergence of endoscopic therapy. This thesis has shown image cytometric DNA ploidy analysis (ICDA) is accurate when compared to flow cytometry. In a multi centre biomarker validation study, ICDA predicted cancer risk in non dysplastic BO. ICDA also predicted relapse following photodynamic therapy (PDT). Nucleotyping (NT) is an imaging technique that evaluates textural changes of nuclei, which correlate with chromatin content and organisation. In this thesis NT was demonstrated to yield additional diagnostic information over ICDA alone, and the combination was highly accurate for dysplasia classification. In an experiment evaluating replication licensing factors in OAC, those representing the S-G2-M phases of the cell cycle correlated with aneuploidy. Polo like kinase 1 upregulation had the strongest correlation, the first time this has been shown in BO. Elastic scattering spectroscopy detected DNA ploidy abnormalities with equal accuracy using a standard or near infra-red enhanced spectrometer. In a prospective in vivo surveillance study, fewer biopsies were taken without a change in the diagnostic yield for dysplasia. A field carcinogenesis effect was also demonstrated, independent of both dysplasia and DNA ploidy abnormalities. Finally, in a randomised control trial of PDT for HGD, patients with BO less than 7cm had significantly higher efficacy and better tolerability with ALA than photofrin. Radiofrequency ablation was shown to be a safe and effective rescue therapy for patients that failed PDT. In summary these studies advance our understanding of biomedical optics for the risk stratification and treatment of Barrett’s oesophagus.

Polyurethanes are an attractive class of material for bioprosthesis development due to the ability to manipulate their elasticity and strength. However, their use as long term biological implants is hampered by biodegradation. A novel polyurethane has been developed which incorporates nano-engineered polyhedral oligomeric silsesquioxane within poly(carbonate-urea) urethane to improve the biostability of the latter. Previous investigators have found this material to be cytocompatible and to have low thrombogenicity. The medium and long term clinical results of currently available prosthetic small calibre vascular bypass grafts are poor, due to neo-intimal hyperplasia associated with their non-compliant properties. The investigation reported here commences with the benchtop manufacture of compliant small calibre grafts using an original extrusion- phase inversion technique. The reproducibility of the technique as well as the effect on the pore structure of different coagulation conditions is demonstrated. Fundamental mechanical characterisation of the grafts produced is then presented, by way of tensillometry to demonstrate the viscous and elastic properties of the material. These are made more relevant to the clinical setting with functional mechanical characterisation of the grafts, showing graft compliance in a biomimetic flow circuit along with viscoelastic hysteresis, along with burst pressure testing. An examination of burst pressure testing methodology is also shown, in the light of the various non-standardised strategies reported in the graft-testing literature. Mechanical characterisation shows the short-term safety for use, but durability studies in the biological haemodynamic environment serve to assess longer term fatigability as well as confirming biostability. This has been reported using a stringent ovine carotid interposition model which remained patent over the full investigation period representing at least 45 million pulsatile cycles. Physico-chemical analysis; integrity of the structure, microstructure and ultrastructure; preservation of mechanical properties and immunohistological analysis were used to examine the grafts after implantation to show their healing properties and biostability.

Background: Sinusoidal obstruction syndrome (SOS) occurs in 50-70% of patients receiving oxaliplatin for hepatic metastasis. Patients suffering from SOS preoperatively are at increased risk of developing postresectional liver failure. To date, there is no ideal therapy to prevent SOS. Moreover, SOS delays liver regeneration following liver resections instigating the possible role of cell therapy. It is essential however to understand the dynamics of the transplanted cells. First, we studied the effect of a flavonoid (monoHER) on prevention of SOS. Second, we studied the role of a dual labeling technique for non-invasive tracking of stem cells in-vivo. Methods: A monocrotaline (MCT) induced SOS model was used in rats, with/without monoHER pretreatment. We studied hepatocellular damage and matrix metalloproteinase (MMP) expression. The potential inhibition of oxaliplatin-induced cytotoxicity by monoHER was tested in-vitro. In the second experiment, liver damage was induced in mice by acetaminophen. Green fluorescence protein (GFP) positive mouse embryonic stem cells (ESCs) were stained with a near infrared dye before transplantation. The distribution of the cells was monitored real-time. Immunohistochemistry was used to identify expression of GFP and albumin. Results: MonoHER ameliorated the increase in portal pressure after MCT and prevented hepatocellular damage. The liver damage score was lower in the monoHER group and was associated with less inflammation. Livers of MCT-treated rats had higher expression of MMP-9 when compared to monoHER group. MonoHER had no effect on in-vitro proliferation of colorectal cancer cells. In the second experiment, labeled ESCs were easily tracked by non-invasive technique. Within 24-hr of transplantation, homing of almost 90% cells was confirmed in the liver. Constitutively expressed GFP was used to study the cell distribution. Conclusions: MonoHER prevented MCT induced portal hypertension and hepatic injury in rats. Dual labeling is an effective method for longitudinal monitoring of distribution, survival and engraftment of transplanted cells.

Background: Quantum dots (QDs) have emerged as one of the most exciting fluorescent nanoparticles with a potential for diagnostic and therapeutic application in the field of nanomedicine. The aim of this study was to synthesize water soluble QDs; bio-conjugating these QDs with RGD peptides prior to linking the QD-conjugated peptide to cancer cells with the aim to study cytotoxicity and assess its feasibility for in vivo studies. Methods: Water soluble Cadmium Telluride (CdTe) QDs were synthesized by the reaction of cadmium chloride with sodium tellurite in the presence of buffer solution of Mercaptosuccinic acid (MSA) as a capping ligand. Water soluble red emitting QDs thus obtained were characterized using spectrophotometric analysis. These QDs revealed a wide absorption spectrum with an excitonic absorption peak of 380nm and a narrow symmetrical emission spectrum of 630nm. The size and pattern of these QDs were studied using Transmission Electron Microscopy (TEM). These nanocrystals revealed their configuration in the form of isolated crystals or clusters measuring from 5-10nm in diameter. X-ray microanalysis combined with TEM permitted analysis of the elemental configuration of these QDs. These CdTe QDs were subsequently bound to HT 29 colon cancer cells to study the interaction of QDs in vitro. As colon cancer cells over-express integrins, QDs were conjugated with RGD (Lysine) and RGD (Cysteine) peptides for the purpose of active binding with HT29 colon cancer cells. The conjugated QDs were applied to colorectal cancer cells to assess their affinity to cellular adhesion molecules. The toxicity of naked and conjugated QDs was also assessed by analyzing cell survival and cell death after exposure to C2C12 mouse skeletal muscle cells. In vivo experiment using Sprague 5 Dawley (SD) rat established feasibility of biodistribution studies with a small dose of 10μg/ml. Results: These water soluble fluorescent CdTe nanocrystals were synthesized using relatively stable precursors. It was possible to demonstrate binding of these red emitting QDs to the HT29 colon cancer cells in vitro. Significant and stable binding was noted after QDs were conjugated with RGD peptides. Toxicity assay evaluation studies suggested that both nonconjugated and conjugated QDs were nontoxic to C2C12 mouse skeletal muscle cells at a concentration of 50 μg /ml indicating that they are less toxic to normal cells, and are safe to be applied to in vivo models. Further in vivo experimentation in SD rats established feasibility for imaging sentinel lymph nodes following interdigital web space injection of QDs. Conclusions: RGD-conjugated QDs can selectively target HT29 colorectal cancer cells with low toxicity to normal muscle cells offering a potential novel detection strategy for colorectal cancer. This property can be explored for early diagnostic and therapeutic purpose by selectively targeting cancer cells. Further studies are required in an in vivo model to analyze systemic biodistribution and toxicity studies.

Coronary artery bypass surgery is a life saving surgical solution for patients presented with greater than 70% occluded or stenosed arteries. For these patients, autologous vein is the graft of choice. Alarmingly, 5-30% of patients have no suitable veins available due to previous use or diseased vein wall, thus the critical clinical need for nonthrombogenic vascular grafts is underscored. Our group has synthesised and patented a nanocomposite polymer based on polyhedral oligomeric silsesquioxane modified polycarbonate urea-urethane (POSS-PCU) nanocomposite polymer. The polymer was extruded using coagulation technique to match mechanical properties of a native artery. Optimising the interactions with blood on the graft surface is of keen interest. Endothelialisation is a favourable solution for enhancing antithrombogenic properties. The protective effect of the endothelium is recognised to be governed by nitric oxide (NO). A graft designed for in-situ endothelialisation will have an interval, where it will be prone to platelet adhesion before complete endothelialisation. Therefore it is desirable to induce antithrombogenic properties during this initial period and the induction of NO is desirable. In this study, current research on biofunctionalisation of biomaterials to enhance antithrombogenic properties by inducing in-situ endothelialisation and NO release were reviewed. The possibility of biofunctionalisation of POSS-PCU polymer whilst retaining its original polymer chemistry was investigated by using amine functionalised nanoparticles including POSS and fumed silica to anchor bioactive peptides (RGD) and amino acids (larginine). The antithrombogenic properties of the biofunctionalised polymer were demonstrated to be due to NO release and endothelialisation. The biofunctionalised polymer was exposed to whole blood, endothelial progenitor cells and platelets. Overall this study presents a novel method of biofunctionalising vascular bypass grafts to induce endothelialisation as tested in a bioreactor and also a means of exploiting the possibility of adhesion of platelets to induce NO synthesis. Overall I aimed to look into methods of functionalising graft surface to induce nitric oxide synthesis when the graft is implanted and is in contact with blood.

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