This study was undertaken to increase understanding of recovery from chronic pain in the absence of medical intervention. Chronic pain is a debilitating condition that inflicts significant human suffering and costs the economy of the Western world £billions each year. Despite advances in modern medicine pain remains poorly understood and difficult to treat. Applying a heuristic methodology, an in-depth exploration was conducted into the author’s personal experience of recovery and participants (N=8) who had recovered from chronic pain were interviewed. The results indicate: • Chronic pain is a multi-dimensional phenomenon that serves a purpose and has personal meaning. • Pain serves as a non-verbal communication whose meaning can be revealed through tuning in to the felt sense of the experience. • Medical approaches were ineffective and often exacerbated pain. • Recovery occurred after all medically prescribed interventions had been exhausted and participants hit rock-bottom. This triggered a radical epistemological shift from the commonly held medical perspective into one that considers the physical, psychological, social, and spiritual aspects of the experience. • Chronic pain is difficult to define within the realms of medical pathology and might be alternatively viewed as a healthy response to an unhealthy social system and world which are inextricably linked to the body. • Adult chronic pain was associated with physical pain during childhood as well as repressed childhood trauma. • Key factors in recovery were engaging in a deep personal exploration that involved: (a) remembering and acknowledging childhood adversity, (b) reflecting on the current circumstances of life, (c) challenging previously held views of pain that were based on a medical understanding, (d) Confronting pain and the fear of pain, and (e) making significant life changes.

Chronic fatigue syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a debilitating disorder of unknown aetiology and is characterised by severe disabling fatigue in the absence of an alternative diagnosis. Historically, there has been a tendency to draw psychological explanations for the origin of fatigue. However, this model is at odds with patient descriptions of their fatigue, with many citing difficulty in maintaining muscle activity due to perceived lack of energy and discomfort. In vivo studies have revealed profound and sustained intracellular acidosis following a standardised exercise protocol, suggestive of underlying bio-energetic abnormality and pointing towards an over-utilisation of the lactate dehydrogenase pathway. Similarly, a recent in vitro pilot investigation reported aberrantly low intracellular pH in CFS/ME patient myoblast samples when compared to healthy controls. Remarkably, intracellular pH in CFS/ME myoblasts was normalised to control level following treatment with pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) , suggesting bio-energetic dysfunction in CFS/ME may be modifiable and therefore treatable. In this thesis, in vitro approaches were used to investigate possible mechanisms leading to muscle dysfunction and the fatigue phenotype exhibited in CFS/ME. Validation work was performed to assess the capacity of a novel pH responsive nanosensor system to measure intracellular pH in CFS/ME patient myoblast cells. The work was unable to reliably detect any acidosis in CFS/ME cells, or any difference between CFS/ME and control cells. In addition, DCA did not modify intracellular pH in either CFS/ME or control cells. The fluorescent pH responsive dye 2’7’-bis (2-carboxyethyl)-5 (6) carboxyfluorescein (BCECF) was used to measure intracellular pH at rest, following electrical pulse stimulation (EPS) and after treatment with DCA in myoblast and differentiated myotube cells. Intracellular pH did not differ between CFS/ME patient and control cells at rest or post-EPS. In addition, treatment with DCA did not modify pH in either CFS/ME patient or control cells. Glycolytic function was assessed via a combination of extracellular flux analysis (XF) and through the measurement of cellular L-lactate concentration. XF analysis revealed extracellular acidification rate (ECAR) measurements for all glycolytic ii parameters to be comparable in CFS/ME patient muscle samples when compared to controls. Additionally, DCA did not alter ECAR in either group. L-lactate concentration was elevated at rest of post-EPS in CFS/ME cells compared to controls. DCA did not modify L-lactate concentration in either sample group. Mitochondrial function was assessed via extracellular flux analysis. Bio-energetic function was investigated by manipulating glucose substrate availability in the assay medium. Basal oxygen consumption rate (OCR) was reduced in CFS/ME myoblasts under hypoglycaemic conditions compared to control cells, however this was not observed in CFS/ME myotubes. ATP-linked OCR was reduced in CFS/ME myoblasts under hyperglycaemic conditions compared to control cells but was not observed in CFS/ME myotube cells. There was no difference between CFS/ME and control cells for any of the other mitochondrial parameters tested. A direct real-time electrochemical approach was used to monitor superoxide (O2.- ) generation in CFS/ME cells following ethanol stimulation and lactic acidification of the assay medium. O2.- generation was not elevated in CFS/ME cells compared to controls following ethanol stimulation or lactic acidification. The in vitro muscle culture approaches reported in this thesis have enabled the investigation of the biochemical basis of muscle cell dysfunction in patients with CFS/ME. It is possible to conclude there to be no evidence of impaired muscle function in CFS/ME patients. Additionally, there was no impairment found in PDK enzyme function. Therefore, it can be determined that bioenergetic function is normal in CFS/ME patients and cannot be attributed to the excessive peripheral muscle fatigue phenotype frequently exhibited.

The human skin is a complex ecosystem which supports a diverse population of bacteria. Comparative genomic analyses are increasingly being used to explore the functional potential of this bacterial population . The ubiquity of Staphylococcus on human skin means this genus represents the most well-studied of the microbial skin residents, however most analysis has focussed on the significant clinical pathogenic species S. epidermidis and S. aureus. To investigate the biology of S. hominis, the second most frequent Staphylococcus species isolated from human skin after S. epidermidis, seven isolates were sequenced using Illumina and PacBio technologies. An intraspecies comparative genomic analysis was performed with these and several publically available S. hominis genomes to identify core and accessory genes. The complement of encoded cell wall-anchored proteins was studied using bioinformatics to describe the range of surface-attached proteins and revealed a unique species set. Investigation also revealed the presence of S. hominis genes described as virulence factors in S. aureus and S. epidermidis. This further highlights non-pathogenic staphylococci as a reservoir of genes, which can be exchanged with pathogenic S. aureus, and the potential for recruitment of these genes into virulence pathways. Interspecies comparative analysis of twenty Staphylococcus species, based on clusters of orthologous genes, confirmed the designation of staphylococcal species groups previously established by DNA-DNA hybridisation and single gene analysis methods. The bioinformatic algorithm randomForest was used to identify drivers forming species groups based on the orthologous gene cluster analysis leading to a subset of orthologous clusters defined as being contributory. This interspecies analysis also revealed diversity between the staphylococcal species groups with respect to their response mechanisms for antimicrobial peptide (AMP) resistance. Specifically, the presence or absence of the BraRS two-component system (TCS) was identified to be one of the important drivers differentiating a nine species member group that included S. aureus, S. hominis and S. epidermidis. Experimental evolution in the presence of the lantibiotic nisin was used to dissect differences in the global response of the BraRS-positive species S. hominis and S. aureus, from the BraRS-negative species S. saprophyticus, . Identified SNPs from the resistance evolution revealed complex relationships between the regulons of staphylococcal TCSs and identified that YurK should be investigated for a potential role in AMP resistance of S. aureus and S. hominis.

Chronic pain is a debilitating symptom of a wide range of conditions. These conditions are both highly prevalent and create adverse consequences for individuals and society. Whilst understanding of chronic pain conditions has improved, in a number of cases the mechanisms of chronic pain are not fully understood and no cure is available. It is appreciated that chronic pain is not only unpleasant in itself, but can also lead to a reorganisation of the nervous system resulting in further suffering. These factors present a justification for further investigation into the mechanisms and effects of chronic pain to enable progress towards more effective treatments. Neuroimaging techniques have helped our understanding the mechanisms and effects of chronic pain. Techniques have been developed to examine the structure, chemistry and activity of the brain. This thesis describes investigations that used neuroimaging to examine the effects of chronic pain on the human brain. A distinction has been drawn between chronic widespread pain (CWP) and chronic localised pain (CLP). Historically, the latter was seen as a condition of the peripheral components of the pain system. More recently, however, an understanding has been gained that central mechanisms may also be a factor in these conditions. The purpose of my investigations was to examine differences and similarities in the effects of two CWP and CLP conditions on the human brain. Fibromyalgia (FM) and Knee Osteoarthritis (OA) were chosen as representatives of these classes of condition. The effects on neurochemistry, brain structure and coordinated brain activity in these conditions were compared using magnetic resonance spectroscopy (MRS), voxel-based morphometry (VBM) and resting state functional connectivity (rs-FC). Using MRS I observed a reduction in N-Acetylaspartic acid (NAA) in the thalamus of OA patients when compared to FM. Using VBM I observed that grey matter volume (GMV) was reduced in the left brainstem and posterior cingulate cortex in FM patients when compared to OA. GMV was reduced in the left precentral, middle frontal and supramarginal gyri in OA when compared to FM. Using rs-FC I observed an increase in functional connectivity in the default mode network of FM patients when compared to OA. I observed increased functional connectivity within the default mode network (DMN) in both pain conditions compared to healthy controls. I also observed increased functional connectivity between the precuneus and regions in both the DMN and executive attention networks. Consideration is given to these findings in the context of previous relevant research. The implications of the results are related the patients’ own experience of their condition and links to clinical measures are also discussed. The findings provide further evidence for the neural basis of elements of patients’ experience of their condition and further understanding of the differences between the wider presentations of these conditions. The findings are drawn together to demonstrate where the effects of CWP and CLP overlap and where their effects contrast. Consideration is given to the mechanisms at work in these conditions that suggest differing effects on the components of the pain system and also to demonstrate where prolonged abnormal peripheral input may be a factor driving adaptation in CLP.

Chronic Fatigue Syndrome (CFS) is an incapacitating condition characterised by extreme fatigue. In the absence of an objective diagnostic test CFS remains a clinical diagnosis based on a broad spectrum of symptoms, including autonomic dysfunction and cognitive impairment. This has given rise to significant challenges, not least the development of multiple sets of diagnostic criteria that may represent different disease phenotypes. This thesis examines autonomic and cognitive features between subgroups that meet different diagnostic criteria to better understand this possibility. It also examines the overlap between symptoms of CFS and depression, a potential confounder. Methods A subset of data from a larger Medical Research Council funded observational study Understanding the pathogenesis of autonomic dysfunction in CFS and its relationship with cognitive impairment was examined. Patients were screened using the SCID-I assessment tool to exclude major depression prior to the main study. Depressive symptoms were compared to CFS Fukuda criteria. The DePaul Symptom Questionnaire (DSQ) was used to differentiate between diagnostic criteria. COMPASS and COGFAIL questionnaires were administered for self-reported autonomic and cognitive features respectively. The Task Force® Monitor was used for autonomic assessment and a battery of neuropsychological tests administered for objective cognitive assessment. Results Subjective autonomic and cognitive symptoms were significantly greater in CFS subjects compared to controls. There were no statistically significant differences in objective autonomic measures between CFS and controls. There were clinically significant differences between DSQ subgroups on objective autonomic testing. Psychomotor speed was significantly slower in CFS compared to controls. Visuospatial memory, verbal memory and psychomotor speed were significantly different between DSQ subgroups. Conclusion The findings indicate phenotypic differences between DSQ subsets and suggest that elucidating the symptoms seen in CFS, or its disease spectrum, will support research into its underlying pathophysiology and enable more tailored treatment. The absence of significant differences in objective autonomic function between CFS and controls in this cohort contrasts to findings of some other studies and may reflect study exclusion for depression. Together with the overlap between CFS and depressive symptoms, this reinforces the need to better understand the underpinning causality to allow appropriate identification and management.

Barrett's oesophagus is a complication of gastro-oesophageal reflux disease and is the single most important predisposing factor for the development of adenocarcinoma of the oesophagus. New molecular markers are needed for early diagnosis and to monitor disease progression. Telomerase is a ribonuclear protein with reverse transcriptase activity, which uses its own RNA component as a template for the addition of telomeric repeats to the end of chromosomes. Telomerase activity has been studied during the neoplastic progression of Barrett's oesophagus using a TRAP based ELISA technique, which found telomerase to be reactivated early during . disease progression. A non-isotopic method of in situ hybridisation for the detection of the RNA component of telomerase has also been successfully developed. Plasminogen activation is an inducible extracellular proteolytic system involved in the regulation of cellular interactions and invasion. The components of the urokinase-type Plasminogen Activator system have been fully investigated during the progression of Barrett's oesophagus to adenocarcinoma utilising immunohistochemistry and ELISA techniques. Changes in the expression of this invasive phenotype were found to occur late during disease progression in malignant tissues. Two-oesophageal cell-lines have been characterised using molecular biological techniques to detect a range of molecular markers to produce ex vivo models of oesophageal adenocarcinoma and oesophageal squamous cell carcinoma. In order to assess the effects of bile salts and acidity on oesophageal tissues these celllines were then utilised as ex vivo models. Exposure to acidic conditions both alone and with bile salts altered the morphological appearance of the cells and disrupted adhesion molecules in the cellular membrane. Investigations into both telomerase reactivation and the plasminogen activator system have provided new information concerning the nature and timing of molecular changes during the Barrett's metaplasia/dysplasia/adenocarcinoma sequence.

The perception of pain - burning, aching and soreness - acts as a physiological warning that protects us from real or potential injury by employing both behavioural and reflex avoidance responses. Unfortunately, this sensory modality can outlive its usefulness and become chronic and debilitating. Indeed, inflammatory mediators released following tissue trauma can sensitise pain fibres (primary afferent nociceptors) to a diverse range of mechanical, chemical and thermal stimuli. The aim of the present study was to investigate the molecular mechanisms regulating the excitability of primary afferent nociceptors. The present study has demonstrated that cyclooxygenase-1 (COX-1) was constitutively expressed in a subpopulation of putatively defined nociceptors in the rat dorsal root ganglion (DRG), using immunocytochemical techniques. Consistent with these immunocytochemical findings, sharp-electrode recordings revealed that depolarisations evoked by the inflammatory mediator, bradykinin on cultured rat DRG neurons, were significantly attenuated by selective COX-1 inhibition. These findings suggest that activation of bradykinin receptors in nociceptors, may in addition to activating phospholipase C, also release arachidonic acid, which is then specifically metabolised by COX-1 to synthesise pro-inflammatory prostaglandins. There is increasing evidence to suggest that inflammatory mediators can regulate neuronal excitability by phosphorylating ion channels expressed on primary afferent nociceptors. However, the identities of the ion channels underlying these effects have not been fully elucidated. The present studies demonstrated the expression and distribution of the inwardly rectifying potassium channel, Kir2.3 and tetrodotoxin-resistant (TTX-R) sodium channel NaV1.8, in a sub-population of putatively defined nociceptors. Using the whole-cell patch clamp technique, it was demonstrated that the inflammatory mediator, bradykinin could modulate the functions of the Kir and TTX-R sodium currents in cultured rat DRG neurons. Taken together, the results from the present studies have increased our understanding of the molecular mechanisms regulating nociceptor excitability, and as such, may also contribute to the development of more efficacious analgesic therapies.

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