Trace elements are involved in many key metabolic pathways. Literature review demonstrated that the trace element content in liver metastases is lower than that in surrounding livers and that in livers of normal individuals. The significance of trace elements in the diagnosis, prognosis, and treatment of patients undergoing surgical resection for colorectal liver metastases (CLM) is not known. Samples obtained from patients who underwent liver resection for colorectal liver metastases were used for this research. Iron, copper and zinc content were measured by X-ray fluorescence (XRF) and were confirmed to be lower in colorectal liver metastases than normal liver. Measuring zinc levels in the tissues could differentiate CLM and normal liver with a sensitivity and specificity of 100% and 93.1% respectively. There was a positive correlation between zinc content but not copper content and microvessel density (MVD), a surrogate marker of angiogenesis. However, tumour copper content was found to be a very good predictive factor of survival in patients undergoing liver resection for CLM (area under receiver operating characteristics curve - AUROC 0.919). Micro-XRF revealed that the zinc was preferentially found in normal liver cells (hepatocytes) followed by tumour cells and found least in fibrous stroma. ZnT1, the main zinc efflux transporter, was downregulated in CLM compared with normal liver (5.2 fold, P = 0.002 respectively). Tissue culture experiments using the cell line HT-29 demonstrated no correlation between zinc in culture medium and the main zinc transporters (ZnT1 and ZIP1). However, zinc chelation inhibited genetic markers of apoptosis and angiogenesis and was lethal to the HT-29 cell line. Zinc did not have any effect on cell viability but had a protective effect against short periods of zinc chelation. This thesis has demonstrated unique and biologically important relationships between zinc levels and colorectal metastases which require further biochemical and genetic investigation.

The high demand on the upper extremity during manual wheelchair use contributes to a high prevalence of shoulder pathology in people with spinal cord injury. The overall purpose of this thesis was to investigate shoulder muscle recruitment patterns and wheelchair kinetics in able-bodied participants over a range of daily activities and mobility tasks requiring manual wheelchair propulsion. With a complete understanding of the muscle recruitment patterns, physiotherapists and wheelchair users can improve rehabilitation protocols and wheelchair propulsion performance to prevent shoulder pathology and maintain comfort during locomotion. Motor unit recruitment patterns were examined first during isometric and isotonic contractions to determine if spectral properties from EMG and MMG could be related to the different motor units in biceps brachii by using wavelet techniques coupled with principle component analysis. The results indicated that motor unit recruitment patterns can be indicated by the spectral properties of the EMG and MMG signals. EMG activity of 7 shoulder muscles was recorded with surface electrodes on 15 able-bodied participants over a range of manual wheelchair propulsion activities. Wavelet and principle component analysis was used to simultaneously decompose the signals into time and frequency domain. There are three main conclusions that can be drawn: 1) Uphill and faster speed (1.6m/s) propulsion required higher activity levels in the shoulder muscles and greater resultant joint force than did slow speed propulsion on the ergometer (0.9m/s), thus potentially resulting in shoulder pathology. 2) Prolonged wheelchair propulsion and greater muscle activity may result in fatigue and play a factor in the development of shoulder pain and pathology over time. 3) The instructed semicircular pattern has a positive effect on shoulder muscle recruitment patterns. Further investigations need to focus on a systematic integrated data collection and analysis of kinematic, kinetic, and electromyography (EMG) data from people with spinal cord injuries.

The Bioartificial Liver Devices (BAL) could provide treatment for acute liver failure by supporting patients awaiting transplantation or aid the process of liver regeneration. For use within a clinical setting, a number of regulatory criteria must be met, including controlling DNA, endotoxins and particulates. The aim of this thesis was to begin the development of a system to control plasma quality returning to the patient. Methods for DNA, endotoxin and particulates detection were established with human plasma to measure sensitivity for use with the BAL system. DNA detection by QPCR using Alu repeats were validated for use as an analytical method to demonstration DNA removal, achieving a Limit of Quantification (LoQ) of 0.1ng/ml DNA. Endotoxin analysis utilised a fluorescent derivative of the widely used LAL assay to increase sensitivity, enabling 2EU/ml to be detectable. Particulates down to 1μm were measured using laser light obscuration. Initially the removal of particulates from alginate as a starting material (alginate prior to encapsulation) was shown, using filtration by depth charge filter, sand bed filtration and gas solid cyclonic filtration. Encapsulated bead integrity, cell function and growth were compromised with all techniques of filtering alginate in solution, including depth charged and sand bed filtration. Conversely, gas solid cyclonic filtration maintained bead integrity, cell growth and function. Testing potential DNA levels in the large scale BAL system required the development of a scaled down model of the BAL treatment phase, replicating the large scale BAL system with cell number to plasma volume ratio. This provided an indication of the DNA challenge a removal system at a large scale would need to contend with, predicted to be 68ng/ml for a full scale BAL. A scaled down filtration model was then established to model the DNA removal capability of different 3M® Cuno® DNA depth charged filters. This established a requirement for a predicted surface area of 1300cm2 to achieve complete DNA removal. The volumetric capacity of the filters were calculated using established filter blockage models, in order to scale the capacity to the full BAL system size. Finally, the chosen depth charge filter was tested at a large scale with the extracorporeal BAL system, spiking human plasma with DNA and endotoxin, whilst measuring endotoxin and DNA removal over 8 hours of treatment.

Ejaculatory dysfunction after spinal cord injury (SCI) is common with more than 90% of SCI men unable to produce an ejaculate. If the ejaculate is obtained by vibro or electro ejaculation the motility, morphology and forward progression are all subnormal. The exact cause of deterioration of sperms is not known although a number of factors can lead to poor quality semen. A randomized control trial was designed to evaluate if repeated ejaculation with a Ferticare® vibrator can improve the sperm quality in chronic SCI men. All had a spinal cord lesion above thoracic level 10 with a minimum duration of 6 months. The subjects who vibroejaculated (VE) with a Ferticare® vibrator were randomised into the study or control arms. In the study arm VE was applied weekly for 3 months. In the control arm VE was given only once at the beginning and end of 3 months. The semen analysis was performed by two observers according to World Health Organization (WHO) criteria. A paired Student t test was used for statistical analysis. Forty two of 79 subjects (53%) vibro-ejaculated successfully. Thirty four were randomized into study (n=18) and control (n=16) arms. No serious adverse events were encountered. Only morphology and forward progression on WHO criteria demonstrated significant improvement. There was no statistical improvement in either volume, count or motility. It is concluded that repeated ejaculation can improve some parameters of sperm in the semen of SCI men. Hence, patients with a SCI above the level of T10 can improve their sperm quality by applying VE weekly for at least 3 months. It is hoped that larger scale multi-centre studies will be undertaken to confirm the effectiveness of repeated ejaculations.

Objective: A randomized clinical trial seeking evidence as to whether cardioplegic arrest (CA) or intermittent cross-clamp fibrillation (ICCF) method of myocardial protection technique would have any effect on the post-operative neuropsychological outcome in patients undergoing elective coronary artery bypass grafting (CABG) surgery. Methods: One hundred and ninety-five patients were randomized to either CA or ICCF as the method of myocardial protection technique. Cerebral microemboli (ME) during surgery were recorded by transcranial Doppler monitor over the right middle cerebral artery. Evidence of cerebral impairment was obtained by comparing the patients’ performance in a neuropsychological test battery (9 tests) administered 6-8 weeks post-operatively with their pre-operative scores. Results: The groups proved well balanced in pre-operative variables. During cardiopulmonary bypass (CPB) the median number and range of microemboli was 110 (1-1306) in the CA group compared to 105 (9-1757) for the ICCF group (p<0.567). One hundred and seventy-seven patients completed all the neuropsychological tests. The difference between the two groups did not reach significance (p=0.326, 2 tailed t test). Conclusion: Given the similarity of effect in CA and ICCF techniques on the generation of ME and neuropsychological outcomes during CABG surgery, this investigation suggests that other than the myocardial protection technique analysed, there are other multiple causes that need to be studied.

Signals from adipose tissue, such as interleukin-6 (IL-6) and asymmetric dimethyl arginine (ADMA), an endogenous nitric oxide inhibitor, explain the link between obesity and metabolic diseases. Previously published human studies have used omental adipose tissue to study visceral depots, based on the assumption that all visceral adipose tissues are similar. This study, for the first time, assessed the release of five adipokines (adiponectin, leptin, IL-6, MCP-1 and RANTES) from the subcutaneous and two omental depots. Components of the cyclooxygenase (COX) and the nitric oxide (NO) pathways, that regulate cytokine release in other tissues, were also investigated for their putative role(s) in mediating adipokine release. RANTES release was greatest from the gastric fat pad. However, significantly higher circulating RANTES levels suggest that adipose tissue is unlikely to be the main source of RANTES release. Inhibition of the COX pathway, especially COX-2, reduced IL-6 release from subcutaneous adipose tissue. Prostacyclin synthase (PGI2S) activity was higher in the omental tissue and its protein expression was elevated in the stromavascular fraction from this depot. PGI2S activity appears to mainly reside in the non-adipocyte cells and is more coupled to IL-6 production in adipose tissue. Serum insulin and CRP levels, and systolic blood pressure, directly associated with subcutaneous tissue ADMA content, while BMI correlated with omental ADMA release. ADMA release was higher from the omental depot. However, while DDAH2 expression was higher compared to DDAH1 in adipose tissue, there was no depot specific difference in the expression of either isoform. In conclusion, this study showed adipose depot specific differences of RANTES release, a novel adipokine, from a hitherto poorly studied depot, the gastric fat pad. Characteristics of the omental adipose tissues differed depending on location and paracrine factors that may mediate the adipokine release. These regulatory pathways included components of the COX and NO pathways.

Recent scientific discoveries have confirmed a pivotal role for the NaV 1.7 voltage-gated sodium channel in human familial gain-of-function and loss-of-function pain syndromes. NaV 1.7 is comprised of four hexameric transmembrane domains encoded by SCN9A, a gene preferentially expressed in dorsal root and sympathetic ganglion neurons. Gain-of-function lesions in SCN9A lead to the development of primary erythromelalgia (PEM). To date, fourteen PEM mutations have been identified which all map to the first three domains of NaV 1.7. I have identified four SCN9A mutations, two of which map to the fourth domain of NaV 1.7 and have used a combination of molecular biology and electrophysiology tools to investigate the biophysical properties of the mutated channels. The results provide insights into the function of NaV 1.7 and are useful in a wider clinical context for offering a confident genetic diagnosis of pain channelopathies. Recessive loss-of-function mutations in SCN9A cause congenital insensitivity to pain in humans. It is known that global deletion of NaV 1.7 in mice is lethal whilst peripheral nociceptor-specific ablation of SCN9A (i.e. knockout in NaV 1.8-positive cells) leads to viable animals with a loss of acute and inflammatory pain but leaving neuropathic pain-sensing and noxious cold-sensing modalities intact. I investigated the effects of ablating NaV 1.7 in all sensory and sympathetic neurons by crossing the Wnt-1 Cre mouse line with the floxed SCN9A colony and performed behavioural characterization. The findings of an abolished neuropathic pain phenotype in the NaV 1.7 X Wnt-1-Cre (NaV1.7Wnt-1) knockout mice demonstrate an important role of Nav1.7 in non-nociceptive neurons which contributes to our understanding of development of pathological chronic pain conditions such as erythromelalgia. Finally, I have designed and generated a targeting construct to flox HSNII, a gene which when mutated causes the pain insensitivity disorder Human Sensory Neuropathy Type II. A mouse model generated from this construct may help to explain the function of the largely uncharacterized HSNII gene, which is critical to normal neuronal development and function.

Failure of surgical or endo-vascular treatment in critical limb ischemia (CLI) often leads to amputation of leg. Erythropoietin (EPO) has tissue-protective properties, which can be utilized for managing CLI. However EPO also causes haemopoiesis that precludes its use in CLI. Non-haematopoietic EPO derivatives including ARA 290 act specifically on EPO’s tissue-protective receptor (EPOR-CD131) and avoid the haematopoetic side-effects. The aims of this study were to demonstrate the expression of EPOR-CD131 in CLI; develop a model system to simulate myotube ischaemia in vitro; demonstrate the anti-apoptotic and anti-inflammatory potential of EPO and ARA- 290 in vitro; and assess the proangiogenic properties of EPO and ARA 290 in vitro. Samples were obtained from gastrocnemius muscle of patients undergoing leg amputation for CLI (n=12). Controls were obtained from patients undergoing heart surgery (n=12). EPOR-CD131 expression was demonstrated by immunohistochemistry and western blot. An in vitro model of myotube ischemia was developed and myotubes were subjected to simulated-ischemia after pre-treatment with EPO or ARA 290. Apoptosis was measured by nuclear staining, cleaved caspase-3 and LDH-release assays. Inflammatory cytokines were measured by ELISA. Angiogenic potential of EPO and ARA-290 was assessed in HMEC-1 by proliferation, migration and capillary-like tube formation assays. There was clear expression and colocalization of EPOR-CD131. The expression was upregulated in CLI (p < 0.01). ARA-290 and EPO significantly decreased the number of apoptotic nuclei, cleaved caspase-3, LDH and Interleukin-6 release in myotubes exposed to simulated ischemia (p<0.01). However, only EPO significantly increased proliferation, migration and capillary-like tube formation of HMEC-1 (p < 0.05). This is the first study demonstrating expression of EPO receptors within skeletal muscle and their elevated expression in CLI. Using a model for simulated ischaemia of myotubes, it was shown that EPO and ARA 290 decrease inflammation and apoptosis of ischemic myotubes. The use of EPO derivatives to selectively enhance the tissue protective activity of EPO may provide a novel therapeutic avenue for CLI.

Objective─ Intracerebral hemorrhage (ICH) is accompanied by a pronounced inflammatory response that mediates brain damage but is also essential for the tissue reparative process. Here we assessed the effect of CORM-3, a water-soluble carbon monoxide-releasing molecule possessing anti-inflammatory properties, on inflammation and brain injury after ICH. Design─ In vivo, in vitro and ex vivo laboratory study. Setting─ Research laboratory. Subjects─ Male Sprague-Dawley rats, 250-350g. Interventions and Measurements─ A model of collagenase injection (2 μl) in brain was established to induce ICH. CORM-3 (4 or 8 mg/kg) was administered i.v. at different times as follows: a) 5 min prior to collagenase, b) 3 hours after collagenase and c) 3 days after collagenase challenge. Saline was used as a negative control. Brain damage, brain water content and behavioural assessment were evaluated. The inflammatory response was determined at set intervals after ICH by counting peripheral neutrophils and lymphocytes, neutrophils and activated microglia/macrophages in the ICH area, brain water content and measuring plasma TNF-α levels. BV2 microglia and DI-TNC1 astrocytes were exposed to triton (1%) or CORM-3 (10-100 μM) and cytotoxicity (LDH assay) measured at 24 hours. Main Results─ Challenge with collagenase to induce ICH caused marked brain damage and modified the levels of inflammatory markers. Pre-treatment with CORM- 3 significantly prevented injury, modulated inflammation and reduced plasma TNF-α. CORM-3 given 3 hours after collagenase significantly increased brain injury and TNF-α production. In contrast, CORM-3 given 3 days after collagenase afforded partial protection, modulated inflammation and decreased TNF-α starting from the day of application. No dose-dependent effects were observed. Conclusions─ CORM-3 promotes neuroprotection or neurotoxicity after ICH depending on the time of administration. Beneficial effects are achieved when CORM-3 is given either before or 3 days after ICH, namely, as a prophylactic agent or during the post-acute inflammatory phase.

Introduction: The greatest challenge facing the success of orthopaedic implants is improving their fixation to bone to enhance their longevity. Bone marrow stromal cells (BMSC), are a population of plastic-adherent cells derived from the bone marrow. The main hypothesis of this thesis is that viable BMSC can be applied to implants using a fibrin glue-spray system; and increase bone formation adjacent to the implants and improve bone-implant contact. Methods: The experiments were undertaken in a large animal model. Four scenarios were tested 1) The ability of BMSC to improve implant fixation using models of total hip replacement, massive endoprosthetic replacement and bone defect around pins. 2) The effect of varying cell dosages of BMSC in their ability to produce new bone and improve bone implant contact. 3) The effect of differentiating the BMSC along the osteogenic pathway in their ability to produce new bone and improve bone implant contact. 4) The effect of using semi-permeable barriers around BMSC sprayed on implants to prevent cell migration Results: 1) BMSC sprayed on the surface of implants resulted in increased bone formation in the total hip replacement, massive endoprosthetic replacement and bone defect around pin models. 2) Bone formation was higher with osteogenic 10x106 BMSC (112.67 ± 30.75 mm2) compared to osteogenic 2x106 BMSC (76.84 ± 2.25 mm2). No significant difference was noted in bone formation between undifferentiated 1x105 BMSC (30.76 ± 9.43%) and undifferentiated 10x106 BMSC (28.27 ± 14.64%). 3) Osteogenic differentiated 10x106 BMSC (112.67 ± 30.75 mm2) produced more bone than undifferentiated 10x106 BMSC (58.22 ± 17.22 mm2). 4) Using semipermeable barriers resulted in significantly increased bone formation when undifferentiated 1x105 BMSC (61.32 ± 6.94% vs 30.76 ± 9.43%) or undifferentiated 10x106 BMSC (57.46 ± 4.39% vs 28.27 ± 14.64%) was used. This difference was not noted when osteogenic differentiated 10x106 BMSC was used. The experiments confirm that viable BMSC can be successfully isolated from bone marrow aspiration, differentiated along the osteogenic pathway and sprayed on the surface of various orthopaedic implants to improve bone-implant contact. Conclusion: This technique of using BMSC may be an ideal alternative to improve osseointegration of implants in challenging clinical scenarios with deficient bone stock.