G protein coupled receptor agonists can be described by two parameters; affinity (ability to bind a receptor) and efficacy (ability to activate a receptor once bound). While affinity is now an accessible parameter through binding studies, agonist efficacies have historically been difficult to determine. This is due to the significant system dependence of agonist responses and is particularly true of traditional maximal response and potency measurements. The Black and Leff operational model determines agonist affinity and efficacy estimations directly from functional data. Its measure of efficacy (τ) offers improvement over maximal response determinations but remains a system dependent parameter. More recently, the Slack and Hall operational model has been described. This model uses receptor constitutive activity to estimate the system contribution to agonist responses to derive system independent measures of intrinsic agonist efficacy. This thesis explores the use of operational models in the analysis of agonist functional responses at the dopamine D2 receptor. This receptor is an important therapeutic target in the treatment of schizophrenia and Parkinson’s disease, and has well documented receptor constitutive activity. Maximal responses and potencies in two representative functional assays, CRE-SPAP reporter gene and receptor internalisation, were determined for a panel of ligands at the D2L receptor. In the receptor internalisation assay, agonist maximal responses and potencies were decreased, consistent with an assay of reduced receptor reserve. Applying the Black and Leff operational model to the responses of four representative agonists, in the absence and presence of the irreversible antagonist phenoxybenzamine, yielded affinity and efficacy estimations consistent with the known pharmacology of the compounds. However, experiments did not provide evidence for the receptor constitutive activity necessary to apply the Slack and Hall operational model. As such, site-directed mutagenesis was performed to generate a constitutively active D2L receptor. T6.34R mutation increased agonist potencies and relative maximal responses consistent with a receptor more likely to adopt the active state; however, potencies of the ergo-derived compounds were selectively decreased. Mutagenesis of three key binding site serine residues examined the mode of agonist binding in both wild type (WT) and T6.34R backgrounds. Each serine mutation had differential effects on the responses of structurally distinct agonists, suggesting agonists engage specific serine residues; while S5.42 and S5.46 were required for catechol agonist responses, S5.46 was essential for the responses of the ergo-derived compounds, bromocriptine and dihydroergocristine. It was unknown whether these specific modes of binding would generate functionally similar active conformations. Generally, the differences between the effects on CRE-SPAP reporter gene and receptor internalisation assay responses were sufficiently explained by differences in receptor reserve. However, there were differential effects of serine mutations on WT and T6.34R D2L receptors, particularly the opposing effect of S5.42A on quinpirole potencies in the CRE-SPAP reporter gene assay. This suggests that WT and T6.34R D2L receptors adopt different conformations. Finally, we have described a method to determine agonist affinity and efficacy from [35S]-GTPγS binding assays using operational models, which does not rely on irreversible antagonist treatments. Buffer Na+ substitution revealed consistent constitutive activation of the D2S receptor, but not of the D2L or D2L T6.34R receptors. Agonist responses at the D2S receptor, in the presence and absence of buffer Na+, were analysed with the Black and Leff and Slack and Hall operational models respectively, generating agonist affinity and efficacy estimations. A novel operational model, written specifically for the analysis of [35S]-GTPγS binding assays, was also examined. This model provided similar affinity derivations to the Black and Leff operational model but provided a τ value that may have reduced system dependency. Although there may be compromises in the associated experimental conditions and which receptors can be investigated, the Slack and Hall operational model can be applied to constitutively active systems to provide system independent measures of agonist intrinsic efficacy.

Introduction Patients with symptomatic carotid artery disease have increased risk of cognitive impairment. Multiple mechanisms of cognitive impairment have been proposed. However, the contribution of each mechanism to cognitive impairment and the interrelation between these mechanisms remain unclear. Lesional and non-lesional damage of brain tissues may lead to cognitive impairment in patients with symptomatic carotid artery disease. The research presented aims to use whole brain statistical brain mapping and quantitative neuroimaging techniques to understand vascular cognitive impairment (VCI) in a group of patients with symptomatic carotid artery disease. Methods Studies presented in this thesis were based on available multimodal MRI and cognitive performance data from a study in patients with symptomatic carotid artery disease. Totally 108 subjects had FLAIR and diffusion MRI scans and 62 of them had resting-state fMRI scans in a 3T MRI scanner. All participants (n=108) underwent cognitive assessments using Addrenbrooke’s cognitive examination-revised (ACE-R). Voxel-based lesion symptom mapping (VLSM) technique was used to identify the contribution of ischaemic lesion presence to cognitive performance. Tract-based spatial statistics (TBSS) approach was used to investigate the association between microscopic damage of white matter and cognitive performance. Independent component analysis (ICA) and seed-based analysis approaches were used to investigate functional disconnection of large-scale cognitive networks and its contribution on cognitive impairment. The association between cognitive performance and ischaemic lesions volumes and microscopic damage of brain tissues were also assessed. Results The presence of chronic subcortical ischaemic lesions located in strategic white matter tracts including anterior thalamic radiation, forceps minor and forceps major contributed to global cognitive impairment in patients with symptomatic carotid artery disease. Ischaemic lesion volumes and microscopic damage of brain tissues including strategic brain areas, strategic white matter tracts, normal appearing white matter and white matter tract skeleton were related to cognitive impairment. Functional disconnection of large-scale cognitive networks including default mode network (DMN), dorsal attention network (DAN), left fronto-parietal network and salience network was found in patients with probable vascular mild cognitive impairment (VaMCI). Functional disconnection beyond the large-scale cognitive networks, showing as decreased functional connectivity between bilateral hippocampi, bilateral dorsal lateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) with the rest of the brain, was also found in patients with probable VaMCI. Also, the thesis presented preliminary results of the role of lesional and non-lesion brain damage in fluency performance in patients with symptomatic carotid artery disease. Conclusions This thesis provides further evidence for the role and to the nature of disconnection in VCI and proposes several disconnection related imaging biomarkers.

This thesis focuses on the use of the Magnetic Resonance Imaging (MRI) white matter lesion (WML) central vein as a biomarker for multiple sclerosis (MS). MS remains a clinical diagnosis, with reliance on MRI to support the diagnosis. Misinterpretation of the MRI can lead to misdiagnoses of diseases that mimic MS. With the increase in disease modifying treatments, accurate and timely diagnosis is needed now more than ever. Using T2* weighted imaging at 3 Tesla (T) MRI, I explored different aspects of WML central veins in patients with relapsing-remitting (RRMS), primary progressive MS (PPMS), and ischaemic small vessel disease (SVD) including: (1) the effect of using different T2* weighted sequences; (2) how T2* and susceptibility weighted imaging (SWI) and fused imaging techniques such as fluid attenuated inversion recovery (FLAIR)-SWI affected the proportion of WML central veins and; (3) determining if WML central veins were as prevalent in patients with PPMS. Further objectives included: (4) attempting to determine if vascular risk factors altered the proportion of WML central veins in patients with MS and; (5) using statistical modelling to calculate a simple diagnostic rule using WML central veins to differentiate MS from SVD. The proportion of WMLs with central veins differed significantly between patients with MS and SVD. Variations of the T2* sequence altered the proportion of WMLs with central veins, but the difference between MS and SVD remained statistically significant. T2* and SWI allowed a higher proportion of WMLs with central veins to be detected, with T2* being just as accurate as FLAIR-SWI in allowing the diagnosis of MS or SVD. Patients with PPMS and RRMS have a similarly high proportion of WMLs with central veins. High sensitivity and specificity for the diagnosis of MS versus SVD can be achieved by identifying a subset of WMLs with central veins. WML central veins could be used as an MRI biomarker using T2* imaging at 3T to differentiate cases of diagnostic uncertainty with RRMS, PPMS and SVD. Application of this imaging technique to patients with diagnostic uncertainty in prospective studies needs to be studied along with refining a clinical diagnostic rule.

Background: Central Nervous System (CNS) inflammatory syndromes represent a spectrum of diseases caused by infiltration of inflammatory cells into the tissue of the brain and spinal cord. They comprise a group of diseases that can be acute or chronic, including optic neuritis, transverse myelitis, acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO) and its spectrum disorders (NMOSD), clinically isolated syndrome (CIS) and multiple sclerosis (MS). MS is the most common one while NMO/NMOSD is relatively rare although the exact population-based epidemiological parameters are lacking. The distinction of MS and NMO/NMOSD in adult patients and of MS and ADEM in paediatric patients is important for considerations of prognosis and treatment. This thesis aims to differentiate NMO/NMOSD from MS using epidemiology analyses and quantitative magnetic resonance imaging at 7 Tesla. The hypothesis of the thesis is that NMO/NMOSD can be differentiated from MS by disease incidence, prevalence, severity, and the quantitative characteristics of the lesion and non-lesion white matter of the brain. Methods: The thesis firstly employed a comparative epidemiological study to estimate incidence and prevalence of MS and NMO/NMOSD, and to estimate risks for associated comorbidities and mortality between NMO/NMOSD and MS. The thesis then applied two quantitative techniques to compare the white matter lesions and non-lesion white matter (so-called normal-appearing white matter [NAWM]) of both NMO/NMOSD and MS. Results: The results are presented in seven chapters. Chapter 1 provides a general review of CNS inflammatory syndromes. Chapter 2 shows the estimated incidence and prevalence of CNS inflammatory diseases in 2012 using the Clinical Practice Research Datalink (CPRD). MS is estimated to be the most prevalent and the NMO/NMOSD the rarest. Chapter 3 shows the case-controlled analytic results on the impact of the burden of comorbidity in MS. The burden is cumulative after MS diagnosis and impacts on patient survival compared to non-MS patients. With an interest of new-onset epilepsy in the course of MS compared to non-MS controls, chapter 4 shows that MS patients have a higher risk of having the first medical recorded epilepsy at and after MS diagnosis. Chapter 5 shows the comorbidity burden in NMO/NMOSD, and estimates the risk for mortality between NMO/NMOSD and age- and gender-matched MS populations. Chapter 6 compares in vivo white matter lesions and NAWM of the brain between adult patients with NMO/NMOSD or MS and healthy subjects using quantitative T1 relaxation time and magnetisation transfer ratio (MTR) magnetic resonance imaging at 7 Tesla. The results show that myelin content of white matter lesions is relatively better-preserved in NMO/NMOSD than that in MS; and they also show some changes in the NAWM of NMO/NMOSD, despite the fact that the metrics of the histogram of both sequences are not different to those in MS. Conclusions: In the UK, NMO/NMOSD is rare with a higher disease severity compared to MS, which is the most prevalent inflammatory disease. In vivo, using T1 relaxation time and MTR imaging at 7 Tesla, the white matter lesions of the brain have more preserved myelin in patients with NMO/NMOSD than in those with MS. There is at least a small proportion of intracranial NAWM having subtle changes in patients with NMO/NMOSD that is detectable, but the changes could not be differentiated from MS in the current study. This thesis helps to better understand the epidemiology of NMO/NMOSD and MS, and MRI detectable changes of lesions and NAWM in NMO/NMOSD.

About a third of stroke survivors have some degree of depression. Depression has a significant impact on recovery after stroke, and identification is important so it can be treated. A common symptom of stroke is aphasia, where comprehension or expression of language is significantly impaired. Communication problems following stroke have been shown to be a major predictor of depression after stroke, yet these problems often make assessment of mood using conventional, language-based measures difficult or impossible. Though some non-verbal, self report mood measures exist, their utility is limited and evidence base lacking. The aim of this study was to design, create and validate a non-verbal mood assessment instrument suitable both as a general outcome measure and as a screening measure for depression in stroke patients with aphasia. A series of four judgement experiments were conducted based on 22 photographic sittings, and a series of scales were developed. The resulting prototype instrument Dynamic Visual Analogue Mood Scales (D-VAMS) is a tablet/computer-based instrument consisting of seven bipolar scales comprising images of human faces whose expressions are modulated by sliders. The instrument was then validated in a sample of 46 stroke survivors recruited from online, from stroke clubs and via NHS rehabilitation services. Good construct validity was demonstrated by high correlations between word and face versions of the seven D-VAMS scales (r=.73 to r=.79), however discriminant validity was poor, with substantial cross-correlations between scores for all of the face scales (r=.58 to r=.88). Internal consistency of D-VAMS was very high, with a Cronbach’s α of 0.95. A Principal Components Analysis revealed one factor accounting for 80% of the variance, corresponding to pleasantness or unpleasantness of mood. Excellent criterion validity was evidenced by strong correlations between D-VAMS and Hospital Anxiety and Depression Scale (HADS) depression subscale (HADS-D) scores (r=.73). Excellent test-retest reliability (r=.89), and high sensitivity and specificity against HADS-D cut-offs of 4–7 were also found. The findings suggest that the D-VAMS is a valid, brief measure of pleasantness of mood in a range of 0–100 which is suitable for use as a general outcome measure for stroke survivors with aphasia, and which may serve as an indirect, simplified measure of depression. Though D-VAMS may also be useful as a screening measure for depression following stroke, further validation is needed to examine how it performs in people during the acute stage after stroke. Some supervision may be required for people unfamiliar with using a tablet or PC interface.

The study of spontaneous brain activity using functional Magnetic Resonance Imaging (fMRI) is a relatively young and rapidly developing field born in the mid-nineties. So far, sufficiently solid foundations have been established, mainly in validating the neuronal origin of a significant component of observed low-frequency fluctuations in the 'resting state' fMRI signal. Nevertheless, the field is still facing several major challenges. This thesis first reviews the current state of knowledge and subsequently proceeds to present original research results that are directed towards overcoming these challenges. The first challenge stems from the indirect nature of the fMRI recordings, obscuring the interpretation in terms of the underlying neuronal activity. Two investigations related to this are presented. First, I show that increased head-movement, epiphenomenal to altered states of consciousness, can lead to spurious increases in low-frequency fluctuations in fMRI signal. This may adversely affect inferences on the underlying neurophysiological processes. Second, I demonstrate a direct electrophysiological correlate of increased synchronisation of fMRI activity in areas of the much studied default-mode network. By directly studying electrophysiological correlates of fMRI-based functional connectivity, this study took a pioneering approach to confirming the biological validity of the fMRI functional connectivity concept. Another widely debated question within the field is the optimal method for extracting relevant information from the extreme volumes of neuroimaging data. I present an investigation providing insights and practical recommendations for this question, based on assessing the interdependence information neglected by the commonly used linear correlation for fMRI functional connectivity studies. The results suggest that in typical resting state data, the nonlinear contributions to instantaneous connectivity are negligible. The third major challenge of the field is the integration of the experimental evidence into theoretical models of spontaneous brain activity. In the last part of this thesis, such models are discussed in detail, focusing on the two crucial features of observed spontaneous brain activity: functional connectivity and low-frequency fluctuations. Two specific mechanisms for emergence of the latter are proposed, depending either on the local synchronisation dynamics or the regulatory action of particular neuromodulators. The thesis concludes with discussion of the questions arising from the presented results in the context of the most recent development in the wider field.

Introduction: This thesis explored Chronic Neuropathic Pain (CNP) patients' experiences of the treatment journey of Spinal Cord Stimulation (SCS) surgery, considering life prior to, and after the surgery. Previous SCS literature has predominantly focused on technology, SCS efficacy, and the role of psychological factors in SCS patient selection and outcomes. Whilst research highlights SCS as an effective treatment for various CNP conditions, it predominantly employs quantitative outcome measures, thereby reducing the depth of information yielded about the experience of SCS surgery and patient satisfaction. There is a dearth of in-depth understanding of the lived experience of the SCS surgery treatment journey. Objectives: The aim of this thesis was to explore participant experiences of the SCS surgery treatment journey considering life prior to and after the surgery. Methods: Ethical and NHS trust approval were obtained. A purposive sample of seven CNP patients who had undergone SCS surgery 2-8 months previously were recruited. Each participant took part in a face-to-face semi-structured interview which was audio recorded. Interviews were transcribed verbatim and analysed using Interpretative Phenomenological Analysis (IPA). Results: Three super-ordinate themes were generated: Diminished control and coping, identity transitions and SCS conflict. The themes were interpreted as being interconnected with each other. To demonstrate the treatment journey, all themes were included in the journal paper and further details of convergences and divergences between participants were included in the extended paper. Discussion: In line with previous research, patients’ expectations of SCS surgery were significant in patient satisfaction with the outcomes, reinforcing the importance of identifying and addressing expectations in pre-surgery preparation. Given SCS is often the last treatment option; the current study found post-SCS participants were going through a process of acceptance of lost identities and of current pain relief and capabilities. Simultaneously, participants were adjusting to living with the stimulator, indicating the significance of offering psychological treatments adjunct to SCS treatment to support participants through these processes. Difficulties in acceptance of identity changes and adjustment to SCS could negatively impact on mood and sense of control which can have adverse effects on pain perception.

Stroke is a major contributor to the global burden of disease. It is the third main cause of death and the largest cause of adult disability in the UK. Stroke is reported to be the second most common cause of disability after dementia in the UK care home population with an estimated 25% of residents living with the consequences of stroke. The aim of this PhD programme of research was to explore the current research evidence for the provision of occupational therapy to stroke survivors living in care homes; investigate current routine occupational therapy practice for this specific stroke population in UK care homes; and to contribute original new knowledge on the health outcomes of sub groups of the care home population with stroke. This study was divided into four distinct projects that were completed alongside a National Institute for Health Research funded phase III multi-centre cluster randomised controlled trial of occupational therapy for care home residents with stroke known as the ‘OTCH study’. The OTCH study evaluated the efficacy of delivering occupational therapy interventions targeted towards increasing and maintaining independent performance of personal self-care activities of daily living and mobility. The PhD student was a member of the OTCH study team with responsibility for delivering the intervention at the Nottingham site. A PhD studentship from the University of Nottingham enabled the development of this complimentary and integrated programme of research. Stage one (reported in chapter two) involved the completion of a Cochrane systematic review and meta-analysis as a means of systematically appraising published randomised controlled trials of occupational therapy interventions for care home residents with stroke to the highest gold standard. Systematic searching identified 1,436 unduplicated records however only 1 study met the inclusion criteria, with another trial ongoing. There was insufficient evidence from the reviewed randomised controlled trial to determine that occupational therapy improves outcomes for care home residents with stroke and therefore further high quality research in this area is needed. Stage two (reported in chapter three) involved a national online survey study to provide contextual demographic data, along with data on the aims, content, funding and provision of occupational therapy services currently being delivered to stroke survivors residing in UK care homes. Out of a total of 138 completed questionnaires, data were analysed from 114 respondents who met the eligibility criteria of providing assessment and treatment to residents in a care home setting. The survey findings confirmed that occupational therapy is being delivered in some care homes; however, interventions for residents with stroke are not routinely delivered by stroke specialist occupational therapists and are not routinely delivered using a systematic, evidence-based approach. Stage three (reported in chapter four) utilised the raw data from the 1,042 participants recruited to the OTCH study to perform subgroup analysis and predictive modelling (including regression modelling and generalised estimating equation (GEE) modelling) with the aim of further investigating the effect of occupational therapy on various subgroups of the participant sample. Subgroup analysis determined that age, time since stroke onset, cognitive status, mood and pain made no difference to the effect of a three month occupational therapy intervention aimed at improving or maintaining independence in basic ADLs (as measured by the Barthel Index (BI)). Predictive modelling found type of care home (residential or nursing) and cognitive status (dementia or normal cognition) to be a far greater predictor of ADL performance and mobility outcome than whether or not the resident had received the occupational therapy intervention. Stage four (reported in chapter five) involved analysis of the content of occupational therapy intervention delivered to the OTCH study participants and their performance in self-care ADLs to account for possible reasons why the trial produced neutral results by (1) exploring the content of the treatment that the intervention arm participants received from the study occupational therapists; and (2) investigating the performance of those participants who had received the allocated occupational therapy intervention, whilst accounting for possible predictor covariates. Binary logistic regression was used to model the relationship between the dependent outcome variable and the explanatory predictor variables. Results of the analyses demonstrated that the therapists did not allocate their time according to those with greater levels of disability and higher levels of need. Residents with dementia received less therapy input than those with mild cognitive impairment or normal cognition. Cognitive status was the strongest predictor of functional outcome. The thesis concludes by highlighting the implications of this new body of research evidence for occupational therapy clinical practice, policy, and future research.

Title: The language of acute pain assessment: a corpus-based Critical Discourse Analysis approach Aim: Through use of real time interactions between healthcare workers and patients in an acute hospital setting this study sets out to investigate how health care workers help or hinder patients to express their pain during the pain assessment process. Background: Pain has long been an issue for investigation and there are a multitude of assessment options available. However, despite using an assessment framework, the ability of patients to use language to express pain has been shown to be more problematic than might be first considered. This study sets out to investigate how both patients and healthcare workers use language in this assessment process. Method: Real time data was recorded in an acute hospital in-patient setting. The use of corpus based critical discourse analysis enabled specific instances of word use and phrases related to pain experience to be identified and analysed. Findings: Two key areas were identified in the analysis of these interactions. The first area related to the traditional aspects of pain assessment relating to terminology used, location and function of pain. The second more important area related to how healthcare professionals presented a certain ‘mentality’ about the assessment process in how they appeared to be patient centred but through the use of brevity of interaction and trivialisation of the issues actually presented an opposite view. Conclusion: The primary conclusion is that although healthcare workers apply pain assessment processes, their use of language can show that they are both patient-centred and have their own motivations and agendas.

The work in this thesis is focused on the study of neuroinflammation with molecular magnetic resonance imaging (MRI) methods. Neuroinflammation is a response of the central nervous system to pathological insult and it is present in many neurological disorders, such as Alzheimer’s disease. Being able to image neuroinflammation non-invasively with MRI techniques would have an important clinical value for diagnosis and assessment of therapy effectiveness. The aim of this work is to develop and validate an MR biomarker of neuroinflammation using MR Spectroscopy (MRS) and chemical exchange saturation transfer imaging (CEST). First, intravenous administration of lipopolysaccharide (LPS) is used as a mild inflammatory stimulus in wild type mice and in a mouse model of Alzheimer’s disease (AD). Elevated levels of the osmolyte myo-inositol, measured with MRS and microglia activation are found in AD mice after LPS administration. Due to the inherent low spatial resolution of MRS, a CEST MRI method is developed next. A myo-inositol CEST protocol is optimised, using Matlab simulations based on the Bloch-McConnell equations for a three pool model, in order to maximize the contrast and to estimate the amount of signal that can be expected in vivo. In vitro and in vivo tests are presented and a fast CEST sequence is developed, while the experimental difficulties and limitations of the technique are discussed. A CEST protocol is finally applied to evaluate the metabolite response to an LPS inflammatory challenge using MRS and histology as validation. A correlation is described between CEST and MRS myo-inositol levels, as well as between CEST and microglia concentration (Iba1 immunostaining), which highlight the potential of CEST as a non-invasive in vivo neuroinflammatory biomarker.