Introduction Acute viral encephalitis is a severe form of brain inflammation due to sporadic infection, typically with herpes simplex virus, or epidemic/pandemic infections. Epidemiological data are particularly important for pandemic viruses. Although new reporting approaches are often considered, no real-time clinical data collection tool has been developed. These data are dependent on diagnosis of individual cases. However, the aspects of management that result in delays and missed diagnoses are not clear and it is not known if interventions can improve sample collection and diagnosis. Whilst the importance of cytokines and associated mediators is increasingly recognised, signatures associated with specific aetiologies have not been established. Also, it is not known whether these mediators correlate with clinical severity and outcome, or their impact on blood-brain barrier permeability. Methods I undertook a national surveillance study through neurology networks, and investigated alternative notification approaches. I undertook a multicentre cross-sectional study of clinical investigation, studied viral load and assessed the impact of a lumbar puncture pack. I used bead array to assess mediator profiles and assessed the albumin ratio and viral load, in samples from a Health Protection Agency study. I examined profiles with respect to aetiology, disease severity and outcome and compared this with histopathology tissue and a blood-brain barrier model. Results In the context of a pandemic influenza virus, existing mechanisms identified limited cases, and a smartphone application was developed to collect real-time data. Delays in lumbar puncture and sub-optimal sample collection were identified, in association with a lower viral load. A lumbar puncture pack improved sample collection. Mediator profiles differed between those with an infectious versus immune-mediated aetiology, and those of unknown aetiology best reflected infectious; particularly myeloperoxidase, in part relating to neutrophils in cerebrospinal fluid and parenchyma. The interleukin1 antagonists, IL1RA and IL10, were associated with coma and outcome; and IL10 with reduced blood-brain barrier permeability. Adhesion molecules may counteract this, in both clinical samples and the model. Conclusions Current limitations of detection may be augmented with novel real-time technologies. Diagnosis is limited by delayed and sub-optimal sample collection, which can be improved with a simple pack. Mediators profiles may assist in the distinction of infectious from immune-mediated encephalitis, and cytokines that act against IL1 correlated with clinical severity and outcome. This may be more closely associated with outcome than viral load, although this may reflect sample timing. These findings should direct future research to develop approaches for improved diagnostics and adjunctive therapies.

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Since identification that mutations in NOTCH3 are responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) in the early 1990s, there has been extensive characterisation of the clinical and radiological features of the disease. However therapeutic interventions remain elusive, partly due to a limited understanding of the vascular pathophysiology and how it leads to the development of strokes, cognitive decline and disability. The apparent rarity and heterogenous natural history of CADASIL potentially make conducting any longitudinal or therapeutic trials difficult. The role of disease biomarkers is therefore of some interest. This thesis focuses on vascular function in CADASIL and how it may relate to clinical and radiological markers of disease. Establishing the prevalence of CADASIL in the West of Scotland was important to assess the impact of the disease, and how feasible a trial would be. A mutation prevalence of 10.7 per 100,000 was demonstrated, suggesting significant under diagnosis of the disease across much of Scotland. Cerebral hypoperfusion is thought to be important in CADASIL, and it has been shown that vascular abnormalities precede the development of brain pathology in mouse models. Investigation of vascular function in patients, both in the brain and systemically, requires less invasive measures. Arterial spin labelling magnetic resonance imaging (MRI) and transcranial Doppler ultrasound (TCD) can both be used to obtain non-invasive and quantifiable indices of vascular function. Monitoring patients with MRI whilst they receive different concentrations of inspired oxygen and carbon dioxide can provide information on brain function, and I reviewed the practicalities of this technique in order to guide the design of the studies in this thesis. 22 CADASIL patients were recruited to a longitudinal study. Testing included peripheral vascular assessment, assessment of disability, neurological dysfunction, mood and cognition. A CO2 reactivity challenge during both TCD and arterial spin labelling MRI, and detailed MRI sequences were obtained. I was able to demonstrate that vasoreactivity was associated with the number of lacunes and brain atrophy, as were carotid intima-media thickness, vessel stiffness, and age. Patients with greater disability, higher depressive symptoms and poorer processing speed showed a tendency to worse cerebral vasoreactivity but numbers were small. This observation suggests vasoreactivity may have potential as a therapeutic target, or a biomarker. I then wished to establish if arterial spin labelling MRI was useful for assessing change in cerebral blood flow in CADASIL patients. Cortical grey matter showed the highest blood flow, mean (SD), 55 (10) ml/100g/min and blood flow was significantly lower within hyperintensities (19 (4) ml/100g/min; p <0.001). Over one year, blood flow in both grey matter (mean -7 (10) %; p = 0.028) and deep white matter (-8 (13) %; p = 0.036) declined significantly. Cerebrovascular reactivity did not change over one year. I then investigated whether baseline vascular markers were able to predict change in radiological or neuropsychological measures of disease. Changes in brain volume, lacunes, microbleeds and normalised subcortical hyperintensity volume (increase of 0.8%) were shown over one year. Baseline vascular parameters were not able to predict these changes, or those in neuropsychological testing. NOTCH3 is found throughout the body and a systemic vasculopathy has been seen particularly affecting resistance vessels. Gluteal biopsies were obtained from 20 CADASIL patients, and ex vivo myography investigated the response to vasoactive agents. Evidence of impairment in both vasodilation and vasoconstriction was shown. The addition of antioxidants improved endothelium-dependent relaxation, indicating a role for oxidative stress in CADASIL pathology. Myography measures were not related to in vivo measures in the sub-group of patients who had taken part in both studies. The small vessels affected in CADASIL are unable to be imaged by conventional MR imaging so I aimed to establish which vessels might be responsible for lacunes with use of a microangiographic template overlaid onto brain images registered to a standard brain template. This showed most lacunes are small and associated with tertiary arterioles. On the basis of this thesis, it is concluded that vascular dysfunction plays an important role in the pathophysiology of CADASIL, and further assessment of vascular measures in longitudinal studies is needed. Arterial spin labelling MRI should be used as it is a reliable, non-invasive modality that can measure change over one year. Furthermore conventional cardiovascular risk factor prevention should be undertaken in CADASIL patients to delay the deleterious effects of the disease.

According to the European Brain Council, the annual total cost of brain disorders such as Huntington’s disease (HD) and Parkinson’s disease (PD) in Europe is approximately €386 billion. In order to develop therapies for neurodegenerative diseases, model systems that accurately reproduce the complex circuitry of the adult brain are needed. Neuron circuits developed in vitro could be used for studying pathogenesis of disorders and for high-throughput screening of potential therapies. In vitro models may offer the potential for highly reproducible and controllable cell circuitry, mimicking to some extent the complex neural connectivity required for function. Nano- and micro-scale substrates could be fabricated using techniques such as electrospinning, lithography and microfluidics to direct neurite orientation in order to build in vitro models that mimic in vivo circuitry. Poly-lactic acid (PLA) nano-fibres and polydimethylsiloxane (PDMS) micro-groove constructs were either pre-coated with poly-D-lysine (PDL) and laminin (LN) or pre-aligned astrocytes to study attachment and orientation of striatal neurites. Neurites were more responsive to substrates made up of combined topography and chemical cues; PDL and LN coated PDMS micro-grooves yielded the best neurite alignment. Excitability of striatal and cortical neurons was verified via an electrophysiology technique of patch clamp. PDMS microfluidic devices fabricated via lithographic techniques, were developed for co-culturing basal ganglia (BG) cells to model BG in vivo circuitry. Cell populations in the microfluidic device displayed electrical activity monitored using a calcium imaging technique. Connectivity was determined by eliminating activity of one cell population using tetrodotoxin (TTX) and studying response of remaining cell populations. Micro-contact printing was further explored as a technique for patterning BG cell circuitry instead of microfluidic devices. The microfluidic-based functional and complex model developed herein provides platform technology that can be useful for pharmaceutical and regenerative therapy and evaluation, therefore massively reducing costs currently associated with neurodegenerative diseases.

Neuronal cell loss and damage in the central nervous system are characteristics of debilitating brain related-degenerative disorders such as Parkinson’s disease. The last four decades of research have focused on the promise of cell replacement therapy to replace lost cells, repair the damage and provide functional recovery in affected neural circuits. A thorough understanding of the signals implicated in the development of neurons will greatly facilitate the use of cell replacement therapy. This project aimed to investigate the possibility of using nicotinamide, the amide form of vitamin B3, to promote the development of mature neuronal subtypes from mouse embryonic stem cells, and whether these could form a dopaminergic phenotype, to progress research in stem cell-derived therapies for Parkinson’s disease. Treatment of mouse embryonic stem cell monolayer cultures (46C Sox1GFP reporter cell line) with nicotinamide at the early onset of development not only increased the efficiency of neuronal generation but also enriched the ratio of purified neurons to non-neuronal cells. Nicotinamide acted at the initial stages of differentiation to promote accelerated neural lineage entry by embryonic stem cells in adherent monolayer cultures. The pluripotent stem cell and neural progenitor cell populations could be reduced by treating cells with nicotinamide, which also facilitated accelerated neuronal differentiation. Nicotinamide selectively enhanced the production of catecholaminergic, serotonergic and GABAergic neurons and, moreover, accelerated neuronal maturation. A reduction in the proportion of proliferating cells in nicotinamide-treated cultures was demonstrated– that is, nicotinamide enhanced cell-cycle exit, thereby promoting neuronal differentiation. The potential of nicotinamide was introduced to a novel, small-molecule-based strategy using pluripotent stem cell sources. Nicotinamide was shown to function synergistically with signalling molecules known to enhance a dopaminergic phenotype, to direct differentiating cells to adopt a dopaminergic cell fate. Thus, novel findings suggest that nicotinamide is a key signalling factor in brain development, and is required in a definable dosage range and times for the normal formation of dopamine neurons. This study supports previous evidence that vitamins and their metabolites play a fundamental role in neuronal development.

Background: Theories of motivational conflict are key in understanding alcohol misuse. Research suggests that approach and avoidance motivation are two distinct systems and that level of alcohol consumption depends on which system is most activated at one time. One factor thought to influence this balance is the role of implicit processes. This study aimed to investigate the effects of implicitly priming one motivational system (i.e. approach / avoidance) on the opposing system in regard to alcohol-related motivation in heavy drinkers. Methods: Heavy drinkers were recruited from a non-clinical community sample to complete a protocol of stimulus response compatibility and visual probe tasks designed to measure implicit motivation by recording reaction times to alcohol cues. Participants were assigned to one of three groups and attempts were made to manipulate implicit motivation using a masked priming paradigm. Measures of explicit attitudes towards alcohol were also administered. Results: No significant effects of priming were found. The overall sample showed attentional avoidance for alcohol cues presented at 50 ms duration but not at 500 ms. On the SRC task, participants were slower to avoid alcohol cues than neutral cues. Positive correlations were found between attentional bias for alcohol cues presented for 500 ms on the visual probe task and craving and consumption as measured by the Alcohol Approach Avoidance Questionnaire (AAAQ) and the Alcohol Use Disorders Identification Test (AUDIT) respectively. Significance: Implicit priming of alcohol-related motivational states had no influence on indices of alcohol approach and avoidance motivation or on attentional bias. As an overall sample, heavy drinkers showed automatic attentional avoidance of alcohol cues presented at short durations (50 ms). This is the first study to find avoidance of alcohol cues presented at this duration in heavy drinkers.

Background: There are multiple conflicting theories of depression and clients are frequently given contradictory explanations of their difficulties. Evidence that brings together biological, psychological and social factors of depression would be particularly useful in addressing this. The current study investigates the neural correlates of self-referential processing following psychological intervention for depression. This provides neurological evidence of how a central feature of psychological models may change following therapy. Methodology: Fourteen participants, who had received psychological intervention for depression, underwent functional magnetic resonance imaging scans whilst completing three types of cognitive task: a self-referential processing task, an other-referential processing task, and a graphical task. Participants’ neural activation during self-referential processing was compared to that of ten depressed participants and twelve control participants, which had been collected for a previous study. Results: When positive and negative self-referential processing were considered together, there was no normalisation of neural activation in the post-therapy group, despite normalisation on the BDI II. When positive and negative self-referential processing were considered separately there were fewer areas of significant neural activation during negative self-referential processing in the post-therapy group than in the depressed group. Indicating that neural activation in the post-therapy group normalised. In contrast, during positive self-referential processing, a lack of difference between the control group and the depressed group precluded the possibility of normalisation. Conclusions: The findings provide further support for the importance of the self in models of depression. In presenting neurological evidence in relation to psychological models and psychological therapy, they help bring together biological and psycho-social models of depression. It is possible that the ongoing patterns of atypical activation during self-referential processing represent a vulnerability to future episodes of depression. Possible explanations for the valence-specific findings are discussed and these are highlighted as interesting future research questions. Limitations of the research methodology are discussed and possible directions for future research are outlined.

Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) on the brain is a revolutionary method that provides in-vivo access to tissue macrostructure non-invasively (Basser et al., 1994). Recently, DW-MRI has been shown to have great potential in characterising brain microstructure, such as diameter and size distribution of neuronal fibres, features that were available so far only postmortem or through animal studies (Zhang et al., 2011). Using a process known as Tractography the existence of brain connections can be estimated using a set of DW images (Basser et al., 2000). The main aim of this thesis is to develop efficient methods for studying Tractography within a Bayesian framework. In order to characterise the white matter in the brain we focus on the widely used partial volume model (Behrens et al., 2003). We describe methods that are both time and computationally efficient for estimating the parameters of the partial volume model, before reparametrising the model, so that parameter estimation is viable in some special cases. The partial volume model allows for multiple fibre orientations so we develop methodology to choose between the number of white matter fibres in a voxel. We then take into account the uncertainty in the number of fibre orientations and provide a Fully Probabilistic Tractography method as an alternative to existing Tractography algorithms. Finally we look into the Global Tractography model (Jbabdi et al., 2007) and develop efficient methods for inferring connections between brain regions by investigating methods based on Thermodynamic Integration.

This thesis focuses on the care of people with dementia in long-term care settings. It considers both the experiences of staff working with people with dementia in such settings as well as the perceptions and satisfaction of family members of people with dementia who reside in long-term care. It is hoped that these papers will contribute to understanding how staff working with people with dementia in long-term care settings can be supported to improve the quality of care provision to this population. Chapter 1 is a systematic literature review investigating the staff factors which contribute to family satisfaction with ongoing care provision for their relatives with dementia who reside in long-term care. Empirical evidence from 14 articles was critically evaluated in order to identify relevant staff factors which contribute to family satisfaction. Relevant staff factors in three broad areas were found to contribute to family satisfaction with care provision for their relatives with dementia. Each area is explored and consideration is given to the implications for future research and clinical practice. Chapter 2 is an empirical study exploring the lived experience of healthcare assistants working with clients with dementia in residential care homes. Interpretative Phenomenological Analysis was used to analyse the data derived from semi-structured interviews with eight healthcare assistants. Three superordinate themes and nine subordinate themes emerged following the data analysis. Each theme is explored and consideration is given to the implications for future research and clinical practice. Chapter 3 is a reflective account of the researcher’s personal experiences of working with people with dementia. It explores the change in the researcher’s perceptions and attitude towards working with people with dementia as their knowledge and experience has increased over time and the impact that this has had on their outlook regarding working with people with dementia in the future.

Background Stroke is the foremost medical condition responsible for acquired disability and dependency. The initial psychological and physical deficits should arguably be identified early to allow interventions to be put in place if potential long-term sequelae are to be minimised. Physical impairments within stroke cohorts have been extensively researched and reported. We have access to numerous scales that describe general physical functioning during daily activities and its effects on independence and quality of life. Deficits in movement are easily identified and attributes of physical movements can be associated with such obvious measurement terms as strength, speed, co-ordination and task completion. As these attributes can be graded, it is simple to compare patients over time, within stroke cohorts and against the general population: controlled studies are straightforward, even if natural biological variation demands large samples. However, though physical impacts on a patient may be easily observed and measured, corresponding deficits in cognition and mood are less easily detected and quantified. Psychological problems are common within stroke populations, and exert both short and long term effects throughout all stages of rehabilitation. Despite our awareness of the potentially critical effects of psychological factors on patient outcome, there is a dearth of high quality research in this area. Although many cognitive and mood assessments are available, including some that were developed specifically for use in stroke, these are neither regularly administered nor have been convincingly shown to be accurate and reliable in identifying specific deficits. Thus, it is understandable that research available to us that describes prevalence and effects of cognitive and mood problems post-stroke is sparse in comparison to our knowledge of physical deficits. Methodology In order to begin to understand the effects that cognition and mood have on patient outcome post-stroke, a way of identifying prominent issues is required. Knowing that their effects can have both short and long term impact, reliable screening beginning early after stroke onset could offer opportunities to improve patient outcome through early implementation of interventions. Before an appropriate screening tool can be selected, evidence is required to support its feasibility and accuracy within acute stroke cohorts. In this thesis, I investigate cognitive and mood-screening assessments in stroke, across a series of linked projects. I carried out a review of the current research and I surveyed stroke units to identify which cognitive and mood assessments are commonly implemented. I collated and offer synthesis of the published data on accuracy of cognitive assessment instruments. I used these results to inform a diagnostic test accuracy study, examining selected measures that are commonly used in UK practice to screen for cognitive and mood problems. Based on these results, I designed (and was awarded grant funding for) a clinical study to assess test properties of cognitive and mood screening instruments in a rehabilitation setting and to describe potential obstacles affecting patient assessment. Findings There is heterogeneity in the choice of cognitive and mood tests employed across research and clinical practice. There was some overlap in assessment choice within these domains but no clear consensus on a preferred assessment tool. This is in part explained by the substantial number of tests available, it is telling that the most popular assessments accounted for only a fraction of the tool assessments employed. My literature based work also points to a relative lack of published science employing a cognitive or mood assessment tool. My review of diagnostic test accuracy found that properties of cognitive tools commonly used in practice and research (Folstein’s Mini Mental State Examination: MMSE, the Montreal Cognitive Assessment: MoCA, the Addenbrookes’ Cognitive Examination Revised: ACE-R and the Cambridge Cognitive Examination revised: R-CAMCOG) were susceptible to changing populations and purpose of assessment, with test properties differing when screening tools are used in acute and chronic stage of stroke. Depending on the cut-offs that are used to define “screen positive” cases, these tools would have varying ability to identify multi=domain cognitive impairment or dementia. Generally when applying standard (i.e. the traditional cut-off described for test use in an unselected population) cut-offs, sensitivity was good but specificity was low. Specificity could be improved when the cut-offs were altered while maintaining reasonable sensitivity and this suggests that screen positive thresholds may need to be altered to suit a stroke population The need for lowering our standard cut-offs suggests that there may be factors present in typical acute stroke patients which affect assessment accuracy compared to the populations and purpose for which these scales have been developed. Using the MOCA in the acute setting of my clinical study, confirmed that stroke cohorts require altered cut-offs to improve accuracy in cognitive impairment detection. A stroke cognitive assessment that can be derived from a standard neurological examination *the Cog4) has been described. Cross sectional comparison of MoCA and Cog 4 suggest that Cog4 has questionable validity and stroke specific cognitive measures are required since scores derived from other types of measures are not necessarily testing the most appropriate domains for stroke deficits. A lack of published data on cognitive and mood screening in the first days post-stroke suggested that describing the feasibility of assessing stroke patients in an acute setting would be a useful topic for research. My subsequent clinical study incorporated verbal and non-verbal assessments for mood and the MOCA. As well as usual test accuracy outcomes I considered feasibility issues such as proportion of patients suitable for initial approach, acceptance of assessment, prevalence of common stroke related impairments that mandate assistance or cause difficulty in completing assessments, or that preclude assessment altogether. A moderate proportion of patients who were approached declined to take part and several others required external assistance to complete the assessments. Shorter, less cognitively demanding assessments required less assistance and appeared to offer higher accuracy for predicting mood problems at follow-up. These results suggest that delaying cognitive and mood assessments until later during the post-stroke period may reduce the interference from acute stroke deficits. The final piece of work generated from my PhD studies, and that is ongoing, continues the theme of feasibility of cognitive and mood assessments. Cognitive and mood assessments are performed in stroke rehabilitation centres. The rehabilitation setting was chosen, as it will include varying patterns of physical and cognitive impairment. By comparing brief assessments and more lengthy measures of cognition, I hope to identify the most appropriate testing scheme that minimises patient burden. As part of this work I will describe the impact of stroke deficits on assessment and quantification of the patient’s psychological capabilities. Conclusions In conclusion, these studies have demonstrated a lack of guidance and of protocols for cognitive and mood assessment post-stroke. The evident heterogeneity in choices of assessment in research and usual practice indicated a need for evidence based accuracy studies. In conducting these I found that usual measures are susceptible to the population, timing, and cut-off used to define test positive cases, together indicating undesirable sources of variation. Transient stroke-related problems may lead to overestimation of persistent impairments. Although acute screening of cognition and mood would be possible, such screening may not be widely acceptable to patients and would require a high level of assistance from health professionals. Acute screening should only be performed if there are potential benefits that could impact on the patient from identification of cognitive or mood problems at this early stage. With the transient changes in cognition and mood that the majority of stroke survivors experience, screening is best left until later in the patient journey. However, there may still be potential feasibility issues of administration and assessment completion during later stages. Therefore, I suggest that studies that investigate what assessments are feasibly administered to stroke patients in later stages are required. This will inform future trial recruitment for complete data requirements as well as provide clearer picture of stroke survivors’ affected cognitive domains and or mood problems.