Part one of this volume is a review of the literature on the association between chronic pain and depression. It presents the results of 15 prospective studies, divided into three categories: studies investigating outcomes of chronic pain in patients with depression, studies investigating outcomes of depression in patients with chronic pain, and studies investigating variables associated with chronic pain and depression. The review highlights problems with the measures of depression used in the majority of the studies. The clinical implications are discussed, and suggestions for how future research can overcome methodological limitations are made. Part two presents an empirical study which investigates the influence of history of depression, perceived trustworthiness and gender of the patient; and training level of the clinician on judgements and treatment decisions in patients with chronic pain. The results showed that participants were affected by patient gender and trustworthiness in their pain judgements and management decisions. Implications for reducing bias in training clinicians are discussed. Part three is a critical appraisal of the research process as a whole. It contains some personal reflections on the different stages of research: designing the study, recruiting participants and analysing data. It also reflects further on the research findings.

The interaction between a human's genes and their environment is dynamic, producing phenotypes that are subject to variance among individuals and across time. Metabolic interpretation of phenotypes, including the elucidation of underlying biochemical causes and effects for physiological or pathological processes, allows for the potential discovery of biomarkers and diagnostics which are important in understanding human health and disease. The study of large cohorts has been pursued in hopes of gaining sufficient statistical power to observe subtle biochemical processes relevant to human phenotypes. In order to minimise the effects of analytical variance in metabolic profiling and maximise extractable information, it is necessary to develop a refined analytical approach to large scale metabolic profiling that allows for efficient and high quality collection of data, facilitating analysis on a scale appropriate for molecular epidemiology applications. The analytical methods used for the multidimensional separation and detection of metabolic content from complex biofluids must be made fit for this purpose, deriving data with unprecedented reproducibility for direct comparison of metabolic profiles across thousands of individuals. Furthermore, computational methods must be established for collating this data into a form that is suitable for analysis and interpretation without compromising the quality achieved in the raw data. These developments together constitute a pipeline for large scale analysis, the components of which are explored and refined herein with a common thread of improving laboratory efficiency and measurement precision. Complimentary chromatographic methods are developed and implemented in the separation of human urine samples, and further mated to separation and detection by mass spectrometry to provide information rich metabolic maps. This system is optimised to derive precision from sustained analysis, with emphasis on minimisation of sample batching thereby allowing the development of metabolite collation tools that leverage the chromatographic reproducibility. Finally, the challenge of metabolite identification in molecular profiling is conceptually addressed in a manner that does not preclude the further reinvention of the analytical approaches established within this thesis. In summary, the thesis offers a novel and practical analytical pipeline suitable for achieving high quality population phenotyping and metabolome wide association studies.

Persistent infection with hepatitis B virus (HBV) is a global health burden accounting for more than a 1 million deaths worldwide. Much work has focused on the failure of the adaptive immune response in persistent HBV infection. In contrast innate responses remain poorly characterised. Accumulating evidence indicates the involvement of NK cells in the control of viral infections and shaping of adaptive immunity. Here we investigated the antiviral and immunoregulatory potential of NK cells in chronic Hepatitis B virus infection (CHB). The combination of immunosuppressive cytokines seen in CHB suppresses the non-cytolytic antiviral function of NK cells, whilst maintaining TRAIL mediated killing, which has been associated with hepatocyte apoptosis. Our findings further implicate NK cells in the down-regulation of antiviral T cells, which are profoundly diminished and prone to apoptosis in CHB. Our data show that in vitro depletion of NK cells results in recovery of HBV-specific CD8 T cells, an effect that was not observed for control virus responses. When NK cells were depleted or not in contact with T cells, the degree of caspase activation in HBV-specific T cells was decreased, supporting a direct and contact dependent NK cell effect on virus-specific CD8 T cell survival involving induction of apoptosis. NK cells, the predominant population of TRAIL expressing cells in the HBV infected liver, were found in intimate contact with T cells within the liver sinusoidal spaces. The observed upregulation of the death receptor, TRAIL-R2 on CD8 T cells, likely imposed by the intrahepatic milieu in HBV infection may sensitise them to apoptosis. Our findings illustrate an important and novel mechanism of immune dysregulation contributing to viral persistence in humans. The potential to selectively deviate NK cells from pathogenic roles, whilst augmenting antiviral functions could improve HBV control and enhance the design of future therapeutic strategies.

The discovery of new treatments for chronic pain relies on the detection of pre-clinical targets and the progression to successful clinical trials. In order to improve this transition reliable translational models must be identified, based on mechanisms that underlie the symptoms of chronic pain. This thesis aimed to validate the use of 3 potential translational models: topical capsaicin, ultraviolet irradiation (UVB) and UVB rekindling. Furthermore, using a mechanism based approach to treatment, the modulation of capsaicin induced sensitisation was explored in animals and humans. In order to characterise the models in rats, in vivo electrophysiological recordings were made from single unit dorsal horn wide dynamic range neurones. Evoked responses to thermal, mechanical and electrical stimulation were quantified. To complement the animal studies, full QST profiling was undertaken on healthy human volunteers. Assessments of the pain thresholds were made, as well as numerical ratings to sub and supra threshold stimuli, in order to best compare these results with rodent data. All of the models tested evoked similar sensory changes across species, and the symptoms induced in each of the models were used to infer the peripheral and central components. Sensory changes evoked by capsaicin included mechanical hypersensitivity accompanied by a facilitation of responses in the Aδ fibre range. These are reflective of both a peripheral and central sensitisation. Furthermore, these changes were prevented by pre-treatment with the adenosine receptor 1 (A1R) agonist, CPA. UVB appeared to be a strictly peripheral model, resulting in no secondary changes or receptive field expansion. On the other hand, the UVB rekindling model showed clear signs of engaging both peripheral and central mechanisms, including thermal allodynia, secondary brush hypersensitivity and a facilitation of Aβ fibre responses. Overall, we confirmed that similar short-term sensory consequences, that may mimic certain pathophysiologies, could be engaged and quantified in rats and human volunteers in response to topical capsaicin, UVB irradiation and UVB rekindling. The UVB rekindling model induced signs of the engagement of a number of clinically relevant phenomena, such as peripheral inflammation/ sensitisation driving central modifications. As such this model will be useful in investigating mechanisms of inflammatory pain and assessing analgesic efficacy of novel medications.

Natural and synthetic nanoparticles (NP) are microscopic particles, which are characterized by their small size (< 0.1 μm). It is now well established that exposure to NPs can represent a serious risk to human health and the environment. To establish the modes of action of NP toxicity, this project utilizes the nematode Caenorhabditis elegans to conduct baseline studies to screen the toxicological effects (on life-history traits, fitness, and metabolism) of metal oxide based synthetic (30 nm ZnONPs), and naturally occurring (Carbon Black M120 and NIST 1648a) NPs. The results indicate that: 1) The Nanosight NTA technique is a suitable tool to evaluate particle aggregation in biological test media. 2) All tested particles exert a shared toxic response that is manifested by a decrease in reproductive potential. The toxic effects were dose responsive to ZnONPs exposure, but not to NIST and CB. 3) The DCFH-DA assay provided in vitro evidence of the oxidative potential of particles, as the intracellular total ROS levels were altered. 4) The whole genome, qPCR analyses, and microscopy provided evidence that the majority of transcripts involved in stress response pathways (e.g. sod family; cat-2,-3; hsp-16.1) did not alter or were only marginally affected by the particles. Nevertheless, the most profound effects were the down regulation of the ribosomal RNA transcript (rrn-3.1) with increasing NIST concentrations, and the induction of cep-1 gene (p53 human orthologous) with ZnONPs. 5) Finally, spectroscopic strategies identified the importance of metallochelators in the protection from ZnONP induced toxicity. Overall, the results of this study suggest that the use of sensitive nematode mutants combined with genomic tools represents a powerful approach to assess the physicochemical toxicity of different types of particles.

New methods of fetal anomaly detection will offer increasingly detailed information to prospective parents but are likely to lead to more frequent discovery of anomalies with an ambiguous prognosis for the future child. It is important to consider the ethical implications of such ambiguity prior to introducing new tests. An examination of the bioethics literature reveals 'promoting autonomy' as the predominant justification for fetal anomaly detection. Two questions arise: does the fetal anomaly detection programme as it stands promote autonomy, and is 'promoting autonomy' an appropriate ethical principle in this context? To answer these questions, an empirical bioethics approach using a reflexive balancing methodology is employed. This examines qualitative data from interviews of prospective parents who had had to decide, on the basis of such an ambiguous prognosis, whether or not to continue their pregnancy. On the basis of different accounts of autonomy, it is argued that the fetal anomaly detection programme does not promote autonomy when decisions are based on an uncertain prognosis. Moreover, 'promoting autonomy' is not, on its own, an appropriate aim in this setting, as the participants did not use their choice as a means of self-expression, the decisions were made by two people and were heavily swayed by considerations for the future child, and under conditions of uncertainty it is not possible to attain the level of rational decision-making required by most accounts of autonomy. Furthermore, the impacts of the decision reduce the benefit of any potential opportunity for self-expression through these choices. The data indicated that a welfare approach might be more appropriate, and it was concluded that, whilst a welfare approach did not entirely encompass all of the nuances of the participants' experiences, it was the better fit, giving some useful indications for an ethical framework for an expanded offer of testing.

Chronic pain is a debilitating condition that affects approximately 20% of adults, yet effective treatments remain sparse. This is partly due to an inadequate representation of clinical pain in animal models utilised during drug development. The UVB and heat re kindling (UVB/HR) model is a promising model of chronic inflammatory pain, however the presence of central sensitisation is contentious. The first part of this project utilised centrally acting, MK-801, in electromyographic (EMG) recordings to objectively validate the induction of central sensitisation in the rat UVB/HR model, providing an essential step toward future utility in pre-c1inical trials. The UVB/HR model was utilised in subsequent studies as a model of inflammatory pain. It is well documented that nociception can be modulated supraspinally, with previous studies suggesting a differing contribution from the cerebellum, depending on the cerebellar lobe activated. However, these studies involved either visceral (rather than somatic) nociception or electrical activation, where confounds such as current spread are important limitations. Previous studies also did not consider the modular organization of the cerebellum . In terminally anaesthetized rats, chemical activation (with DLH) of the cerebellar cortex of module A in the posterior and anterior lobe produced a pronociceptive effect. This was demonstrated by a decrease in EMG threshold to mechanical stimulation. Activation of lateral vermal lobule VIII also produced robust pronociception in naive rats but had no effect in UVB/HR rats. This is possibly due to other supraspinal sites playing a more central role in pain states and masking effects from the cerebellum. EMG recordings involve a polysynaptic withdrawal reflex pathway and thus the site of spinal nociceptive modulation following cerebellar cortical activation was unclear. To try and address this issue the effect of cerebellar stimulation on monosynaptic H reflex amplitude was examined. No detectable effect was found, suggesting the changes in EMG withdrawal responses were unlikely due to alteration in motor neurons alone. Finally, extracellular recordings from spinal dorsal horn neurons were obtained to determine whether cerebellar activation alters processing of noxious sensory inputs. Results from this part of the study were inconclusive, potentially due to the heterogeneity of the cell population, where it is possible that descending control from the cerebellum targets a particular subset of neurons in the dorsal horn that were not recorded in our study. Overall the novel findings reported in this thesis suggest that various modules of the cerebellum can be pharmacologically manipulated to alter nociceptive reflex responses in naive animals, but may have a lesser effect in lasting pain states.

The descending noradrenergic system forms part of the body's endogenous analgesic system, however there are conflicting views as to the exact role the system plays during the development of the neuropathic pain phenotype. The experiments described in this thesis aim to explore the longitudinal changes in descending noradrenergic control following nerve injury and the potential utility of selective noradrenaline re-uptake inhibitors in the treatment of neuropathic pain. All experiments were performed on adult male Wistar rats using a combination of behavioural, immunohistochemical and electromyographical techniques. Using a method chronic intrathecal catheterisation, it was found that administration of an uz adrenoceptor (AR) antagonist at day 3 following nerve injury revealed the early onset of allodynia and hyperalgesia. However, as the phenotype progressed, uz-AR antagonism had no effect over allodynia however could still modulate heat hyperalgesia. There was also a reduced noradrenergic innervation to the lumbar dorsal horn in nerve injured rats. Combined, this suggested a progressive failure of the noradrenergic system to prevent the onset of ipsilateral behavioural hypersensitivity. Interestingly, uz-AR antagonism also unmasked contralateral allodynia and hyperalgesia and was associated with an increase in lumbar dorsal horn c-fos expression. Changes in the descending noradrenergic modulation of bilateral heat hypersensitivity mediated by AD- versus C-heat nociceptors was then explored in anaesthetised rats. These experiments further suggested a functional deficit in noradrenergic control, opposed to uz -AR desensitisation, which contributes to the onset of neuropathic ipsilateral hypersensitivity. Further investigations involved pharmacologically restoring descending noradrenergic tone and observing the effects on the development of the neuropathic pain phenotype. It was shown that chronic intrathecal dosing with reboxetine (a selective noradrenaline re-uptake inhibitor) from the time of nerve injury could suppress the onset of allodynia. In addition, the effects of intrathecal versus systemic administration of reboxetine for alleviating spontaneous and evoked neuropathic pain behaviours were also examined, alongside a discussion of the potential clinical implications.

Pain is considered to be one of the most challenging and unmet medical needs. A considerable amount of research effort in both academia.and pharmaceutical industry has, and continues to be, aimed at elucidating the nociceptive and perceptual pain mechanisms in order to develop more effective treatments. Na+ channels are considered to be attractive and well validated analgesic targets. Recent human genetic studies have further implicated both tetrodotoxin (TTX)-sensitive and -resistant Na+ channel isoforms in the mechanisms of pain, arousing fresh interest in them as drug targets. In order to fully understand their role in various mechanisms underpinning nociception, it is essential to determine their contribution to the excitability of nociceptors under physiological as well as paineliciting conditions. This project aims to provide a detailed description of the role of previously validated TTX-sensitive and -resistant Na+ currents in the neurophysiology of mouse nociceptors at near-physiological temperature and different age points. The observed striking temperature and age dependence of Na+ current contribution to nociceptive neurophysiology is presented in chapters 3 and 4 respectively, and is expected to expand the current understanding of the role of Na+ channels in pain. While the current understanding of molecular nociception is based mainly on animal data, recent developments in the field of stem cells and genetics have the enormous potential to expand the knowledge of the mechanisms of human pain and nociception. Specifically, the potential use of human induced pluripotent stem cell-derived nociceptor-like neurons as a research tool in studying 'healthy' nociception, disease modelling and prediction of drug effectiveness has generated a lot of excitement and reinvigorated the concept of personalised medicine in pain research. The data presented in chapter 5 point to iPSe-derived nociceptor-like neurons as an effective avenue in studying the neurophysiology of 'healthy' nociceptors as well as elucidating the molecular mechanisms of pain conditions, in this case primary inherited erythromelalgia.

Aims: Sleep and pain share a complex relationship; pain can disrupt sleep and conversely, poor sleep can exaggerate pain intensity. At present, the exact temporal assoications sunderlying this relationship are not fully understood. Correlational data and preliminary experimental studies suggest that poor sleep augments pain in both acute and chronic situations. This thesis examines the reciprocal interaction between these two processes and the subsequent impact that sleep has on pain, at both the behavioural and brain level. Furthermore, the influence of co-morbid factors such as attention and affective state is considered. Developing an understanding of how sleep affects pain and pain-related attention may have implications for the treatment of chronic pain and its concomitants. Methods: A number of different settings were used to explore the relationship shared by sleep and pain. Epidemiological data was used to explore the association between sleep problems and the later development of musculoskeletal pain. The association between comorbid sleep and pain problems and pain-related complaints were also assessed in adolescent and adult clinical samples. An acute experimental model was used to explore the influence of non-stage-specific sleep disruption on pain and pain-related attention. The aim of this experiment was to explore the effects of sleep disruption on pain sensitivities, central pain modulation and distraction analgesia. Functional magnetic resonance imaging was used to investigate the effect of sleep disturbance on the cerebral signature of pain. Results: Data from a large birth cohort indicated that sleep problems were associated with the later development of musculoskeletal pain. Co-morbid sleep and pain problems were associated with a higher incidence of pain-related and psychological complaints in both adolescent and adult samples. The experimental disturbance of sleep resulted in mechanical and thermal hyperalgesia. Sleep disruption was also found to compromise both conditioned pain modulation and the analgesic benefits of distraction analgesia. Imaging data suggested that sleep disturbance was associated with regional changes in activation in areas previously implicated in pain processing. Neural decoupling was also observed across regions of the cortex and brainstem, the implications of which could be critical for understanding pain processing following sleep disturbance. Conclusions: Results from this thesis suggest that sleep disturbance is an important risk factor in both the development and maintenance of clu'onic pain. The effects of sleep disturbance on both central pain modulation and distraction analgesia are possible pathways by which sleep disturbance facilitates vulnerability for the development of clu'onic pain. Sleep disturbance is reported by two thirds of chronic pain patients and should therefore be identified as a key modifiable target in the treatment of chronic pain. Further explorations of the relationship shared by sleep and pain, in addition to their related concomitants (e.g., mood disturbance) can help establish novel targets which can be manipUlated for diagnostic and therapeutic gain.