The prediction and measurement of surgical outcome is difficult. Current methods of perioperative risk prediction do not perform particularly well on an individual basis with guidelines suggesting a stepwise approach to perioperative risk assessment. Part of this stepwise approach is an assessment of functional capacity. Cardiopulmonary exercise testing has a body of evidence to support its use as a measurement of functional capacity and predictor of perioperative risk. In addition grip strength as assessed by handgrip dynamometry has been shown to be predictive of surgical outcome. This thesis examines the development and testing of a standardised forearm handgrip exercise model to predict mortality and morbidity in orthopaedic surgical patients. This thesis investigates the development of two standardised forearm handgrip exercise models, one using an intermittent (cyclical) exercise protocol and the other using a static (isometric) protocol. Having established reliable methods of using each as a preoperative test, the metabolic output i.e. the measurable venous products of metabolism; lactate, SO2, PO2, PCO2, pH and tissue oxygenation were compared. The comparison showed that the isometric exercise model was the stronger stimulus for anaerobic respiration. Each exercise model was also compared to an anaerobic threshold as measured by cardiopulmonary exercise testing in the same individuals. The isometric model showed a consistent and statistically significant relationship with the anaerobic threshold as measured by cardiopulmonary exercise testing but not the cyclical model.Finally the isometric forearm exercise model was prospectively tested in a pilot study of 21 orthopaedic patients undergoing joint replacement surgery for its ability to predict surgical outcome. The maximal voluntary contraction from handgrip dynamometry was predictive of complications and length of stay and although not statistically significant there was a clear trend for those with fewer complications and shorter lengths of hospital stay to produce more lactate during isometric forearm exercise testing.

Quantum dots (QDs) are semiconductor nanocrystals (<100 nm), which are emerging as a novel class of multifunctional fluorescent probes for many potential biological and medical applications. In comparison to conventional organic fluorescent probes (organic dyes), QDs have substantial advantages, such as, bright fluorescence, narrow emission, broadband excitation, photostability and extended half-life. Imaging with diagnostic assessment, plays an important part in clinical settings for determining disease (cancer) progression and therapy. However, current imaging techniques have certain limitations, and they include insufficient sensitivity to detect low numbers of cancer cells at primary or metastatic sites and appropriate probes to detect specific cancer cell surface markers. To address these limitations, studies were conducted (1) to develop an aqueous synthesis of a series of near infrared (NIR) QDs, incorporating cadmium (Cd), tellurium (Te), cobalt (Co) and mercury (Hg) in its core (2) to minimize its potential toxicities, by developing coating strategies with a novel coating nanomaterial, mercaptopolyhedral oligomeric silsequioxane (MPOSS) while maintaining strong emission, stability and biocompatibility, (3) to apply conjugated NIR QDs as probes in targeting and detecting immunogenic apoptosis in cancer cells in vitro and mapping biodistribution in vivo. The inclusion of mercury (Hg (ClO4)2) and cobalt (Co) to the QD core resulted in NIR emission at 800 nm with paramagnetic properties. Characterization by transmission electron microscopy (TEM) confirmed size of the QDs. Detoxification of QDs was demonstrated, by toxicity studies, using two different vital stains, Alamar Blue and Neutral Red on human umbilical vein endothelial cells (HUVECs), human breast cancer cells (MCF-7), colorectal cancer cells (SW620) and prostate cancer cells (PC3). A short synthetic peptide to calreticulin (CRT) was chemically synthesised and antibodies generated against the peptide (Anti-CRT) with specificity to the native CRT protein (a cancer cell immunogenic apoptosis marker). The presence of functional groups on the coatings of QDs provided an additional advantage for conjugation to Anti-CRT for targeting, and carbon nanotubes (CNT) for thermal strategy. QDs conjugated to Anti-CRT showed specificity to cancer cells in vitro undergoing apoptosis when exposed to the following: 1) Doxorubicin (an anticancer drug), 2) cadmium and 3) QD-CNT (photothermal effect). Characterization by Fourier Transform Infrared Spectrophotometry (FTIR) confirmed conjugation of QDs to Anti-CRT. Confocal microscopy images further confirmed targeted and non-targeted QDs in vitro, and NIR sensitive camera for in vivo imaging. These studies and findings demonstrate the feasibility of applying these engineered nanocrystals for clinical diagnostics, drug delivery and therapy.

A systematic review of the efficacy of a specific perioperative haemodynamic management strategy was performed to explore the balance between therapeutic benefit and adverse effects. Whilst mortality and length of hospital stay were reduced in the intervention group, pooling of morbidity data for between-group comparisons was limited by the heterogeneity of morbidity reporting between different studies. Classification, criteria and summation of morbidity outcome variables were inconsistent between studies, precluding analyses of pooled data for many types of morbidity. A similar pattern was observed in a second systematic review of randomised controlled trials of perioperative interventions published in high impact surgical journals. The Post-operative Morbidity Survey (POMS), a previously published method of describing short-term postoperative morbidity, lacked validation. The POMS was prospectively collected in 439 patients undergoing elective major surgery in a UK teaching hospital. The prevalence and pattern of morbidity was described and compared with data from a similar study using the POMS in a US institution. The type and severity of surgery was reflected in the frequency and pattern of POMS defined postoperative morbidity. In the UK institution, many patients remained in hospital without morbidity as defined by the POMS, in contrast to the US institution, where very few patients remained in hospital in the absence of POMS defined morbidity. The POMS may have utility as a tool for recording bed occupancy and for modelling bed utilization. Inter-rater reliability was adequate and a priori hypotheses that the POMS would discriminate between patients with known measures of morbidity risk, and predict length of stay were generally supported through observation of data trends. The POMS was a valid descriptor of short-term post-operative morbidity in major surgical patients.

The aim of this thesis is to improve our understanding of muscarinic and purinergic neurotransmission within the urinary bladder both within the detrusor muscle and the urothelium as both sites are therapeutic targets. The M2 receptor is the most populous muscarinic receptor in the detrusor muscle however its role is unclear, as detrusor contractility has been demonstrated to be mediated principally by the M3 receptor. The role of the M2 muscarinic receptor in guinea pig and human detrusor contractility was examined through organ bath experiments. Significant M2 modulation of contractility in patients with neuropathic overactivity and overactive bladder symptoms was demonstrated through inhibition of agonist dose response curves and electrical field stimulation with the selective M2 inhibitor methoctramine. Furthermore cAMP elevation through the adenylate cyclase activator forskolin produced an identical and non-additive inhibition to that achieved through methoctramine suggesting that cAMP inhibition is an important mechanism of M2 activation in the detrusor. Detrusor contractility was further assessed through an isolated cell technique and this demonstrated further evidence of M2 mediated contraction of the detrusor indicating that the site of action of M2 agonism is directly within the detrusor cells. Ussing chamber experiments to examine the effect of the exogenous addition of neurotransmitters on the electrical properties of the urothelium were carried out. These demonstrated that cholinergic agonists had no effect but ATP resulted in an increased negativity of the basolateral surface of the urothelium only when added to the luminal but not the basolateral surface. As ATP release from the urothelium has been found to be associated with inflammation and the sensory nerves are adjacent to the basolateral surface, we believe this represents a sensory mechanism whereby a luminal inflammatory signal is transduced electrically across the urothelium to activate the sensory nerves.

Introduction: Short bowel syndrome is characterised by a severe reduction in the amount of functional intestine available as an absorptive surface. Attempts to lengthen the intestine by interposition of artificial tubular scaffolds juxtaposed between healthy tissues have shown limited success. Transplantation is limited due to organ shortage. The most promising solution may be implantation of tissue-engineered small intestine using natural scaffold. Materials and Results: Using a completely novel approach, up to 30cm lengths of ileum with the attached vasculature were harvested from porcine donors. Separate intestinal and vascular loops were identified and de-cellularised using individually tailored detergent-enzymatic protocols. The resulting scaffold was compared to native tissue in terms of retention of cellular and nuclear remnants, as well as structural and functional proteins. Its biocompatibility was assessed by subcutaneous implantation of 1cm2 pieces into rat recipients. The remodeling fate of grafts was determined by time related changes in the ratio of sub-populations of residual macrophages. Its mechanical strength and ease of handling was evaluated by performing a left-sided nephrectomy in an unrelated pig model, followed by end-to-end anastomosis of the de-cellularised scaffolds’ mesenteric vasculature to the appropriate renal artery and vein. In the last stage, porcine organoid units were isolated and their yield estimated for future in vitro studies. Conclusions: It is possible to simultaneously de-cellularise two different tissues of varying cellular configuration and composition effectively and efficiently over a relatively short period of time. The two key features of the de-cellularised scaffold are that 1) the scaffold has in place the necessary architectural topography of small intestine (including mucosal villi) and molecular cues for optimum re-cellularisation and 2) the attached vascular tree provides an ideal conduit for re-cellularisation using either vascular committed endothelial or progenitor cells. Ultimately, this scaffold can be used for constructing long segments of bio-engineered intestine with the possibility of immediate blood supply and re-vascularisation.

The interaction between angiotensin II (Ang II), a smooth muscle constrictor peptide and nitric oxide (NO) a vasodilator, as well as the role of oxidative stress (OS), have been investigated in human and chronic partial bladder outlet obstructed (PBOO) rabbit corpus cavernosal tissue. The PBOO rabbit model is characterised by an increase in corpus cavernosal collagen deposition and a marked reduction and impaired relaxation of corpus cavernosal smooth muscle (CCSM) cells, making it a useful model for erectile dysfunction (ED). Immunohistochemical analysis identified Ang II peptide distribution in human corpus cavernosal tissue, while organ bath studies determined the Ang II/NO interaction. OS was determined using apocynin and diphenylene iodonium chloride (DPI), inhibitors of NAD(P)H oxidase, which inhibit superoxide production and superoxide dismutase (SOD, the enzyme that accelerates the breakdown of superoxide). Human penile Ang II was distributed in the arteriolar endothelium, the endothelium lining sinusoids and CCSM cells. The peptide caused a dose dependent contraction of CCSM strips that was inhibited by losartan (AT1 receptor antagonist) and apocynin. In contrast, CCSM relaxation induced by either sodium nitroprusside (SNP, an NO donor) or electrical field stimulation (EFS) was potentiated by losartan. The Ang II contractile response was enhanced in CCSM strips taken from PBOO rabbits and inhibited by losartan, DPI and SOD. CCSM relaxation induced by SNP/EFS was impaired in this model and improved by vardenafil (PDE5 inhibitor) and losartan. Taken together, these findings suggest that Ang II and NO interact to modulate human and rabbit penile smooth muscle tone. Moreover, the Ang II response involves the production of superoxide and the development of OS. The increase in Ang II-mediated CCSM contraction following PBOO is likely to be a pathological consequence of the condition. Importantly, AT1 receptor inhibition may be a therapeutic target for the treatment of ED associated with PBOO.

Semiconductor nanoparticles, often referred to as quantum dots (QDs), have attracted considerable interest due to their unique photophysical properties such as high photostability and fluorescence quantum yields. In the biomedical arena, QDs are being studied both for their diagnostic and therapeutic applications, in particular the possibility of using QDs in photodynamic therapy, which is based on the destruction of tissue with light through photosensitised formation of reactive oxygen species (ROS). In this thesis, the ability QDs to induce the formation of ROS through Type I photoinduced electron transfer and Type II energy transfer mechanisms was investigated. Firstly, the effectiveness of quantum dot-photosensitiser complexes for photosensitised production of ROS was investigated. A sulfonated phthalocyanine was found to form stable complexes with water soluble pegylated quantum dots. Efficient Förster resonance energy transfer (FRET) between the quantum dot and phthalocyanine was demonstrated to generate singlet oxygen by the Type II mechanism with a quantum yield of up to 0.15. Secondly, the potential of QDs alone to produce ROS in aqueous solutions and cellular environments was studied. In aqueous solution, the production of superoxide radical anions by photoinduced electron transfer to molecular oxygen was demonstrated with a quantum yield of 0.005. The yield could be significantly enhanced via a Type I mechanism in the presence of the electron transfer agent, NADH, which was demonstrated by oxygen consumption measurements, electron paramagnetic resonance (EPR) spin trapping and cytochrome c reduction. Production of hydroxyl radicals was shown using a fluorescence probe. In the cellular studies, QD uptake could be significantly enhanced by conjugation with the cell penetrating Tat peptide, which enabled studies of phototoxic effects induced by QD photosensitised ROS production. QDs can also be used as fluorescent imaging probes in vivo for a variety of biological applications; however their fluorescence properties in tissue have not been widely investigated. The uptake of intravenously administered CdSeTe/ZnS QDs in a range of organs including liver, was investigated. By comparing a range of fluorescence quantitation methods, including fluorescence microscopy and chemical extraction, it was found that these QDs were highly resistant to degradation under physiological conditions, demonstrating their effectiveness as imaging probes in vivo.

Ischaemic priapism is a pathological condition characterised by a prolonged painful penile erection. Corporal blood aspirates show a combination of hypoxia, acidosis and glucopenia. Initial treatment includes ice packs, corporal aspiration and subsequent washout with room temperature fluids. The effect of ischaemia on cavernosal smooth function was examined. In vitro guinea-pig cavernosal smooth muscle strip experiments showed that simulated ischaemia caused a significant and marked reduction in phenylephrine-induced (PE) contraction (plateau PE30 response 35±19%, plateau PE60 response 29±16% of control). The degree of depression was similar to that seen in nerve-contraction although there appeared to be some metabolic reserve as shown by early preservation of the peak PE response (peak PE30 response 83±31%, peak PE60 response 36±35% of control). Nerve-contraction did not recover upon reperfusion whereas agonist-contractures demonstrated complete recovery. Experiments recording the effect of the elements of ischaemia showed this depression to be secondary to combined hypoxia and substrate depletion (absence of superfusate glucose and Na pyruvate). Isolated muscle cells showed a significant reduction in agonist-induced calcium transients during similar interventions. Simulated ischaemia markedly reduced nerve- and abolished agonist-relaxation. These detrimental effects were completely reversible upon reperfusion. Nerve-relaxation recovered whereas nerve-contraction did not. This effect was again secondary to the combination of hypoxia and substrate depletion. This suggests that relaxatory nerves are more resistant to ischaemic damage, a finding which would contribute to the pathogenesis of ischaemic priapism and the contractile failure observed in this condition. Intracellular acidification caused a significant and reversible increase in nerve-mediated contraction. Intracellular acidification also augmented PE contractures at 30 min. (peak PE30 response 120±12%, plateau PE30 response 117±9%). Intracellular acidification induced a significant and reversible increase in PE-induced calcium transients in isolated cells. This augmentation of function was via an oxygen-dependent mechanism. Reduction in superfusate temperature significantly suppressed nerve-contraction. This was not due to reduced recruitment of nerve fibres at low temperature. The time-course of phasic nervecontractions and agonist-contractures was prolonged, slowing responses significantly. Nerverelaxation was significantly ameliorated at low temperatures. The phasic relaxation was also prolonged with the return to precontracted tension following EFS-mediated relaxation slowed to a greater degree than the initial relaxatory response. No change in magnitude of agonist-induced relaxation was observed. Overall reduced temperature interventions affect contraction to a greater degree than relaxatory mechanisms. These effects were not due to changes in the viscoelastic properties of the tissue at low temperature. Prolonged ischaemia is detrimental to contractile function before relaxatory responses. Substrate depletion is a late finding in ischaemic priapism, with undetectable blood glucose after 6-12 hours of priapism. Depletion of the energy substrates glucose and Na pyruvate, combined with hypoxia, is central to the contractile failure seen in ischaemic priapism. This depression is irreversible on contractile nerves at an earlier stage when compared to relaxatory nerves and the smooth muscle itself, propagating the ischaemic priapic state. Reversal of these conditions should form part of any treatment regimen for patients who have priapism. Low temperature interventions do not improve CSM function with nerve-mediated function significantly reduced at low temperature as well as slowing CSM contractile responses. It may be beneficial to use oxygenated washout fluids at body temperature which contain energy substrates such as glucose and Na pyruvate to treat ischaemic priapism.

Liver Ischaemia Reperfusion Injury (IRI) leads to production of reactive oxygen species and cytokines, which affects hepatocellular function following liver resection and transplantation. This thesis examines 2 hypotheses: 1) The role of intravenous glycine in amelioration of liver IRI in a in vivo animal model of partial lobar liver IRI. 2) Does prophylactically administered N-acetylcysteine prevent liver IRI in patients undergoing elective liver resection. Materials and Methods 1) A rabbit model of hepatic lobar IRI was used to evaluate glycine. 3 groups (n=6) Sham group (laparotomy alone), ischaemia reperfusion (I/R) group (1 hour ischaemia and 6 hours of reperfusion), and glycine I/R group (IV glycine 5 mg/kg prior to the I/R protocol) were used. Portal blood flow, bile flow and bile was analysed by H1NMR spectroscopy. Hepatic microcirculation, intracellular tissue oxygenation, serum TNFα, IL-8, ALT, AST were measured at 1, 2, 4 and 6 hours following reperfusion. 2) A randomised double blind clinical trial was conducted to assess the effect of NAC on liver IRI following liver resections. The main outcomes were: morbidity and mortality, ICAM-1 expression in liver tissue, liver function tests. Patients were randomised to receive NAC as IV infusion (NACG) or a placebo group (PG) which received 5% dextrose only. Immunohistochemistry for ICAM-1 was carried out on perioperative liver biopsies. Results 1) Glycine normalised the bile flow, reduced phosphatidylcholine shedding, lactate surge, and stimulated bile acid, pyruvate, glucose and acetoacetate release. Glycine improved portal blood flow, hepatic microcirculation by the 2nd hour, and hepatic intracellular tissue oxygenation by the 4th hour of reperfusion. Glycine ameliorated serum TNFα at 1, 2 and 4 hours and serum Il- 8, AST and ALT up to 6 hours post reperfusion as compared to the I/R alone group. 2) Of the 43 patients, 15 received NAC, 16 were randomised to the PG, 12 were excluded due to inoperable tumours. Serum ALT was reduced in NACG (p=0.001), while serum ALP was higher in the NACG (p=0.003). ICAM-1 expression was up-regulated in 6/16 patients in the PG and in 3/15 patients in NACG. ICAM-1 was down-regulated in 1/15 patients in the NACG and none in the PG, the difference was not significant. Conclusions 1) Glycine ameliorated liver IRI, improved bile flow and composition. 2) NAC ameliorated parenchymal liver injury and enhanced liver regeneration in patients undergoing elective liver resection.

Advance in nanotechnology has led to the development of novel fluorescent probes called quantum dots which are being exploited for potential new methods of early cancer detection; spread and therapeutic management. Concerns regarding the release of potentially toxic inorganic core atoms into their surrounding environment and possessing hydrophobic surfaces are hindering the development of quantum dots. In order to abrogate their toxicity and solubilise the nanocrystals in aqueous solution a novel silica nanocomposite (NC) polymer has been used to coat them. Physical and chemical analysis of the coated quantum dots with UV-Visible spectrometry, Photoluminescence, transmission electron microscopy, X-ray microanalysis and diffraction, Atomic force microscope and FTIR Spectrophotometry has enabled us ascertain the characteristics of these unique nanocrystals. The biocompatibility of the nanocomposite coated quantum dots (NCCQD) was assessed by using Alamar blue™ metabolic assay, Pico green assay and by measuring lactate dehydrogenase release on endothelial cell damage. Potential interference of NCCQD with a rat’s normal physiology and systemic tissue distribution were assessed in an in-vivo animal model. Our results demonstrated that the nanocrystals retained their unique optical properties, had a mean hydrodynamic diameter of 10.5 nm, excellent monodispersivity and large absorption spectrum with a narrow emission band at 790nm and were highly photostable after polymer coating. NCCQD were compatible to endothelial cells as viable cells were demonstrated to be present after 14 days of growing cells in cell culture medium exposed to NCCQD at concentrations of 2.25 x 10^{-2}nM. There was no significant disturbance in the physiological parameters on injecting the NCCQD in an in-vivo rat model over a 2 hour period. NCCQD were seen to be deposited in the spleen and thymus as they are reticuloendothelial organs. In conclusion polymer encapsulated CdTe nanocrystals have tremendous potential to be exploited as a medical device in in-vivo imaging.