Introduction and methods Asymmetric dimethylarginine (ADMA) is a potent endogenous competitive inhibitor of nitric oxide synthases, which has attracted considerable attention as a marker and mediator of atherosclerotic disease and as a potential mediator of multiple organ failure in critical illness due to endothelial dysfunction. However, data regarding basic aspects of its biology such as biological variation and its response to acute inflammation are lacking. Moreover, significant methodological variability has been a barrier to collating the burgeoning data available. Therefore, this thesis describes the development and validation of a reliable assay for measurement of ADMA and related compounds in plasma, urine and other biological fluids based on isocratic reverse phase high performance liquid chromatography (HPLC). This method was used to determine the biological variation of ADMA in human plasma, and its response to acute inflammation using a model of elective knee arthroplasty. Further HPLC methods for measurement of dimethylamine (DMA), the main metabolite of ADMA, and nitrate were developed and used to determine excretion of these compounds in acute inflammation to complement the observed changes in plasma ADMA concentration. Results Complete chromatographic separation of arginine, homoarginine, monomethyl-arginine, ADMA and its structural isomer SDMA was achieved, permitting their accurate quantification using a novel, non-endogenous, internal standard. The intra-individual biological variation of ADMA was found to be low at 7.4%, imposing a tight imprecision goal for analytical methods. Plasma ADMA concentration decreases rapidly during the acute inflammatory response, with a median decrease of around 30%, and a significant change already evident as little as 12 hours following the onset of inflammation. No similar change was seen in the concentration of the closely related compound SDMA. No significant increase in the urine excretion of DMA was noted during the early phase of the response, with a significant increase seen 5 days following the insult by which point the plasma ADMA concentration had returned to baseline levels. A small, but significant, decrease in nitrate excretion during the inflammatory response was seen, mirroring the observed changes in plasma ADMA. Conclusion The low biological variation of ADMA suggests physiological regulation. The rapid and significant decrease in plasma concentration during inflammation does not appear due to increased catabolism, but rather is more likely to represent increased cellular partitioning. This may be associated with an impairment in NOS activity. It is unclear whether this is of pathological significance, or represents a physiological response to regulate NO production in inflammation. Further study is warranted in relevant models, particularly with attention to intracellular concentrations.

This submission for PhD is based around a series of thematic and original research studies, which have been published during the past seven years (2001 to 2007), focused upon arm crank ergometry exercise testing. A number of topical issues have been considered, including: 1) the development of fundamental exercise protocols; 2) the assessment of oxygen uptake (VO2) kinetics; 3) the assessment of external power production during sprint arm crank ergometry, and 4) the design and implementation of novel equipment configurations and research designs. During preliminary testing we demonstrated that the selection of an imposed crank rate was an important consideration. In another study we also demonstrated that it was feasible to use either step or ramp incremental exercise tests for the purpose of establishing peak physiological responses during arm crank ergometry. Two further studies considered the measurement of external peak and mean power production during sprint arm crank ergometry. The first of these studies compared measurement techniques. A follow-up publication considered the reproducibility of uncorrected peak and mean values of power output. In the final publication an interdisciplinary approach was employed. This study examined how crank configuration (asynchronous vs. synchronous) affected the magnitude and pattern of muscle activity, and torque production at two work rates. Our findings demonstrated the complexity of arm crank ergometry as the activity of muscles of the arms, shoulders and legs became progressively involved as external work rate increased from 50 to 100 W. Based on parameters associated with the pattern of torque profile, it was speculated that the asynchronous crank configuration was more efficient for the group of able-bodied participants.

Mesenchymal stem cells (MSC) are a tissue-resident stromal cell population that are able to regulate immune responses, in particular the capacity for endothelial cells (EC) to support leukocyte recruitment. In this thesis we examined the ability of MSC from different sources (bone marrow, Wharton’s jelly and trabecular bone) to regulate neutrophil recruitment to inflamed EC and how these responses are altered upon adipogenic differentiation of MSC. Using two flow based adhesion models with varying degrees of proximity between MSC and EC, we observed that all MSC populations suppressed neutrophil recruitment. IL-6 and TGFβ were identified as common bioactive agents found in all co-cultures. Upon differentiation, MSC exhibited a diminished capacity to suppress neutrophil, but not peripheral blood lymphocyte, recruitment. Loss of suppression by MSC-derived adipocytes was reversed by neutralising IL-6. Adipose tissue-derived mature adipocytes and culture differentiated pre-adipocytes did not recapitulate the effects of MSC-derived adipocytes. These data suggest that crosstalk between tissue-resident MSC and EC, dampens the endothelial response to cytokines and limits the aberrant infiltration of circulating leukocytes during inflammation. Upon adipogenic differentiation, MSC lose this regulatory capacity. This could impact on the beneficial effects of MSC in chronically inflamed sites where aberrant infiltration of leukocyte is a main driver of the disease.

The secretory pathway Ca\(^{2+}\)-ATPase (SPCA) provides the Golgi apparatus with a luminal Ca\(^{2+}\) store, which is used to modulate the activity of Ca\(^{2+}\)-dependent enzymes involved in controlling the secretory pathway and post-translational modification of proteins. This Ca\(^{2+}\) store controlled by SPCA is also believed to be agonist-releasable. Regucalcin (RGN), (also known as senescence marker protein 30 (SMP30)) is believed to be a Ca\(^{2+}\)-binding protein expressed in an age-dependent manner, whereby its protein levels decrease in a number of organs as aging progresses. It has been suggested to be able to affect the activities of the sarco/endo-plasmic reticulum Ca\(^{2+}\)-ATPase (SERCA), as well as other Ca\(^{2+}\)-dependent enzymes. On the other hand, RGN’s ability to bind Ca\(^{2+}\) has been argued against and this protein has been shown to modulate the activities of enzymes not involved in Ca\(^{2+}\) homeostasis, as well as have intrinsic enzymatic activity in itself as a gluconolactonase. It was of interest in the present studies to examine how SPCA can be regulated and how RGN can contribute to the regulation of Ca\(^{2+}\) homeostasis within intact cells. As a result, the following novel attributes of SPCA and RGN were shown: (1) SPCA expression and activity are sensitive to glucose homeostasis in rat vascular smooth muscle cells (VSMCs); (2) hSPCA1d activity can be inhibited without altering hSERCA2b activity by using bis-phenol and 2-APB; (3) TFP and TBBPA affect both hSPCA1d and hSERCA2b activities to the same degrees, while cADPR and NAADP have no effect on hSPCA1d (and possibly hSERCA2b in the case of cADPR) activity; (4) RGN mRNA expression can be found in some specialised cell types of the male rat reproductive system; and (5) human RGN over-expression can influence Ca\(^{2+}\) mobilisation stimulated by 1\(\mu\)M thapsigargin and 10\(\mu\)M histamine, possibly via alteration of SERCA expression. The present study has also made available a custom-made recombinant form of rat RGN and anti-RGN polyclonal antibodies. It can be strongly suggested that SPCA plays a role in diabetes because of its sensitivity to glucose concentrations, coupled with its involvement in the secretory pathway and ability to influence vasopressin response in VSMCs. Bis-phenol has now been quantitatively shown to be a potent and specific inhibitor of SPCA, giving it great potential to be used as a pharmacological tool for future research on this Ca\(^{2+}\)-ATPase. For RGN, it appears promising that this cytosolic protein has a role to play in male fertility, while its role in Ca\(^{2+}\) homeostasis is likely to involve modulating ER Ca\(^{2+}\) storage capacity and strength of Ca\(^{2+}\)-mediated hormone response.

Symptoms of gastro-oesophageal reflux disease are equally common in both sexes and at all ages. However, complications of gastro-oesophageal reflux disease such as reflux oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma, although more common in men, increase sharply in older women, suggestive of a protective effect of female sex hormones in menstruating women. Oestrogen has anti-inflammatory properties, improves healing in oral and skin wounds and may therefore reduce the severity of reflux-induced oesophageal mucosal injury, consequently protecting women from developing severe reflux oesophagitis. Long-term oestrogen treatment with hormone replacement therapy seems to be additionally associated with a reduction in the risk of oesophageal cancer. Moreover, there are gender-specific genotypic differences in the response of oesophageal mucosa to chronic acid reflux suggestive of multiple factors that may play a role in explaining the male predominance of oesophageal adenocarcinoma. Finally, oestrogen has no association with the severity of acid reflux once adjustment is made for the influence of increasing body mass index in women undergoing oesophageal pH monitoring. The gender difference in the prevalence of gastro-oesophageal reflux disease and its complications may thus be related to the effect of female sex hormones, particularly oestrogen and its 'protective' effect in pre-menopausal women.

The cardiovascular system (CVS) has emerged as an important target of corticosteroid hormones. Mineralocorticoid receptor antagonists provide cardiovascular protection and are now routinely used in disorders such as primary hyperaldosteronism, resistant hypertension and congestive heart failure (CHF) but the underlying molecular mechanisms of corticosteroid hormone action remain unclear. We have characterised corticosteroid hormone action and metabolism by 11β- hydroxysteroid-dehydrogenases (11β-HSDs) in isolated adult rat cardiomyocytes (CM) and cardiac fibroblasts (cFb). We have detected 11β-HSD1 expression and activity in CM and cFb where it facilitates glucocorticoid hormone action, whereas 11β-HSD2 was absent. We have shown differential gene regulation by aldosterone (Aldo) and corticosterone in CM and identified novel Aldo target genes which may provide insights into the molecular mechanisms of Aldo action. We have also studied the role of corticosteroids in essential hypertension and the effect of spironolactone (Spiro) upon their secretion and metabolism in patients with chronic kidney disease. We have shown that mineralocorticoids but not glucocorticoids are involved in elevated blood pressure in essential hypertension and that Spiro treatment results in compensatory activation of the renin-angiotensinaldosterone system (RAAS), whereas glucocorticoid secretion and metabolism remain unchanged. In summary, these data provide novel molecular and clinical insights into corticosteroid hormone action in the CVS.

Multiple receptor tyrosine kinases (RTK) have been shown to be over-expressed in the malignant Hodgkin Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). However, the activation status of many of these RTKs has not been studied. Furthermore, the contribution of aberrant RTK activation to the pathogenesis of HL is currently unknown. In chapter three, I have shown using a human phospho-receptor tyrosine kinase array that HL cells are characterised by the activation of multiple RTK. I have confirmed the over-expression and activation in HRS cells of two of these RTK, MET and RON and provided preliminary evidence that MET is negatively regulated by LRIG1 in these cells. In chapter four, I have shown for the first time that DDR1 is over-expressed in primary HRS cells. Furthermore, I have shown that in many cases, DDR1-expressing HRS cells are intimately associated with collagen, the ligand for DDR1. However, knockdown of DDR1 in a HL cell line in which DDR1 appeared to be constitutively phosphorylated revealed no detectable change in phenotype and few transcriptional changes. While exploring possible reasons for this, I identified that HL cells express multiple DDR1 isoforms including several novel transcripts. Finally, in chapter five, I have shown that HL cells are sensitive to the RTK inhibitor, dasatinib. Furthermore, consistent with the aberrant activation of multiple RTKs in HL cells, I observed that these cells were also sensitive to lestaurtinib and dovitinib, two next generation multiple-target RTK inhibitors.

This study investigated the myoelectric effects of muscle fatigue in Longissimus Thoracis, Iliocostalis Lumborum pars Thoracis and lumbar Multifidus during standardised muscle fatigue tests and in a marine environment. These muscles contribute significantly to the extensor moment of the lumbar spine and are an important postural stabiliser. Fatigue of these muscles is considered to contribute to the aetiology of low back pain; rehabilitation approaches that improve muscle endurance capacity would benefit from a better understanding of how these muscles fatigue A feasibility study methodology was developed and a case series study undertaken to establish the utility of standardised fatigue testing before and after a marine high-speed transit that is considered to cause muscle fatigue. Surface EMG data were collected in combination with heart rate and motion data of the vessel and study participants. Standardised fatigue testing was utilised before and after the task to determine the sensitivity of the test; the test was not shown to have clinical utility in this case. Methodology was developed, using high-density surface electromyography (sEMG) and high-density surface electrodes, in order to analyse data from multiple locations over each muscle of interest. Software was developed that enabled specific channels and segments of data to be analysed. A post-mortem anatomical study, in an older population, was completed and this provided position and angle data from bony landmarks for the accurate positioning of high-density surface electrodes and subsequent interpretation of high-density sEMG signal data. A pilot validation study, utilising magnetic resonance images (MRI), compared results of the anatomical study with a MRI series from an older population and a further series in a younger population. The results of these studies partly concur, add to previous study results and provide data on sEMG electrode placement that will aid further sEMG studies.

This work is motivated by an attempt to explain the origins of syringomyelia. This serious disease is characterized by the appearance of cavities, called syrinxes, in the central canal of the spinal cord resulting in partial or complete paralysis. The causes of syringomyelia are unknown but pressure propagation is probably implicated. Pressure pulses, caused by coughing, sneezing and similar activities, propagate up the cerebrospinal fluid (CSF) in the spinal subarachnoid space. Williams (1990) suggested a possible cause of syrinx formation is that when such pressure pulses encounter a partial or total blockage a large pressure rise would be generated in the spinal central canal. Our theoretical model modeling was undertaken to investigate Williams’ hypothesis. Essentially, we model the spinal system as a channel separated into two parts by a flexible diaphragm representing the spinal cord. The upper part (A) represents the subarachnoid space while the lower part (B), which has a much smaller cross-sectional area, represents the central canal. A theory for pressure wave propagation in such a two-chamber system is described. It has been used to study the wave characteristics of the two-chamber system. In this way it has been found that the leading edge of a pressure pulse/wall bulge tends to steepen into a shock-like wave or elastic jump. When such a pressure pulse is incident on a blockage in the subarachnoid space, a large pressure rise is generated in its vicinity. We showed that this pressure rise could be momentary or permanent depending on whether the pressure pulse bulge is positive or negative. This provides a possible mechanism for the formation of the syrinxes. An experimental rig has also been built in order to qualitatively confirm some theoretical results. The experimental wave speed agreed with the wave speed defined in the theoretical model. We were also able to confirm that the reflection of a pressure pulse from a blockage placed in the upper part A (subarachnoid space) leads to high pressure in the vicinity of the blockage.