The dystonias are a common group of movement disorders, which are mechanistically poorly understood. Rapid-onset dystonia-parkinsonism (RDP) is a hereditary movement disorder without neurodegeneration resulting from mutations in the ATP1A3 gene encoding the α3-isoform of the Na+/K+ ATPase. RDP patients are generally asymptomatic until a stressful life event triggers disease onset. Basal Ganglia dysfunction has previously been implicated in RDP aaetiology. To investigate the neuronal pathology in RDP we have generated patient-specific induced pluripotent stem cell (iPS) lines and derived mature midbrain dopaminergic neurons. Using functional live- cell imaging, we have shown that RDP neurons show higher intracellular Na+ levels at rest and smaller Na+ signals in response to stimulation by glutamate or KCl. Consistent with this, resting membrane potential in RDP neurons was chronically elevated and cells failed to return to resting potential after stressful depolarisation. Interestingly, spontaneous spiking activity in the absence of stress was normal. Apart from abnormalities in sodium homeostasis, there was reduced uptake of calcium from the cytosol by mitochondria in RDP neurons probably due to a reversal of the mitochondrial sodium/calcium exchanger in response to elevated intracellular calcium levels. Furthermore mitochondrial pathology in RDP included depolarised mitochondrial membrane potential, reduced oxidative phosphorylation and increase ROS production. Stressful depolarisation induced a sharp increase in ROS production. There was a compensatory increase in Krebs cycle activity to counteract energy depletion due to reduced oxidative phosphorylation and marked upregulation in levels of glutathione, an endogenous antioxidant. This high level of compensation may explain the absence of neurodegeneration in RDP. Based on our findings, we suggest antioxidant therapy as a potential treatment to improve neuronal function in RDP patients.

The aim of this thesis is to apply a particular category of machine learning and pattern recognition algorithms, namely the kernel methods, to both functional and anatomical magnetic resonance images (MRI). This work specifically focused on supervised learning methods. Both methodological and practical aspects are described in this thesis. Kernel methods have the computational advantage for high dimensional data, therefore they are idea for imaging data. The procedures can be broadly divided into two components: the construction of the kernels and the actual kernel algorithms themselves. Pre-processed functional or anatomical images can be computed into a linear kernel or a non-linear kernel. We introduce both kernel regression and kernel classification algorithms in two main categories: probabilistic methods and non-probabilistic methods. For practical applications, kernel classification methods were applied to decode the cognitive or sensory states of the subject from the fMRI signal and were also applied to discriminate patients with neurological diseases from normal people using anatomical MRI. Kernel regression methods were used to predict the regressors in the design of fMRI experiments, and clinical ratings from the anatomical scans.

One of the fundaments of associative learning theories is that surprising events drive learning by signalling the need to update one’s beliefs. It has long been suggested that plasticity of connection strengths between neurons underlies the learning of predictive associations: Neural units encoding associated entities change their connectivity to encode the learned associative strength. Surprisingly, previous imaging studies have focused on correlations between regional brain activity and variables of learning models, but neglected how these variables changes in interregional connectivity. Dynamic Causal Models (DCMs) of neuronal populations and their effective connectivity form a novel technique to investigate such learning dependent changes in connection strengths. In the work presented here, I embedded computational learning models into DCMs to investigate how computational processes are reflected by changes in connectivity. These novel models were then used to explain fMRI data from three associative learning studies. The first study integrated a Rescorla-Wagner model into a DCM using an incidental learning paradigm where auditory cues predicted the presence/absence of visual stimuli. Results showed that even for behaviourally irrelevant probabilistic associations, prediction errors drove the consolidation of connection strengths between the auditory and visual areas. In the second study I combined a Bayesian observer model and a nonlinear DCM, using an fMRI paradigm where auditory cues differentially predicted visual stimuli, to investigate how predictions about sensory stimuli influence motor responses. Here, the degree of striatal prediction error activity controlled the plasticity of visuo-motor connections. In a third study, I used a nonlinear DCM and data from a fear learning study to demonstrate that prediction error activity in the amygdala exerts a modulatory influence on visuo-striatal connections. Though postulated by many models and theories about learning, to our knowledge the work presented in this thesis constitutes the first direct report that prediction errors can modulate connection strength.

This thesis investigates auditory and speech processing in Specific Language Impairment (SLI) and dyslexia. One influential theory of SLI and dyslexia postulates that both SLI and dyslexia stem from similar underlying sensory deficit that impacts speech perception and phonological development leading to oral language and literacy deficits. Previous studies, however, have shown that these underlying sensory deficits exist in only a subgroup of language impaired individuals, and the exact nature of these deficits is still largely unknown. The present thesis investigates three aspects of auditory-phonetic interface: 1) The weighting of acoustic cues to phonetic voicing contrast 2) the preattentive and attentive discrimination of speech and non-linguistic stimuli and 3) the formation of auditory memory traces for speech and non-linguistic stimuli in young adults with SLI and dyslexia. This thesis focuses on looking at both individial and group-level data of auditory and speech processing and their relationship with higher-level language measures. The groups of people with SLI and dyslexia who participated were aged between 14 and 25 and their performance was compared to a group of controls matched on chronological age, IQ, gender and handedness. Investigations revealed a complex pattern of behaviour. The results showed that individuals with SLI or dyslexia are not poor at discriminating sounds (whether speech or non-speech). However, in all experiments, there was more variation and more outliers in the SLI group indicating that auditory deficits may occur in a small subgroup of the SLI population. Moreover, investigations of the exact nature of the input-processing deficit revealed that some individuals with SLI have less categorical representations for speech sounds and that they weight the acoustic cues to phonemic identity differently from controls and dyslexics.

Introduction: Muscarinic receptors have been identified both in the suburothelium and urothelium of the human bladder. It has been proposed that increased release of Acetylcholine (ACh) from the urothelial and suburothelial nerves may act on suburothelial and detrusor muscarinic receptors, resulting in detrusor overactivity (DO). Neuropeptide Y (NPY) is a cholinergic co-transmitter. Botulinum neurotoxin type A (BoNT/A) is known to act by blocking the release of ACh. We compared expression of M1, M2, M3 receptors, NPY and SNAP-25 in patients with DO, controls and following successful treatment with BoNT/A. Methods: Flexible cystoscopy bladder biopsies were obtained from 36 patients with DO at baseline, four and sixteen weeks after successful BoNT/A treatment, together with 9 asymptomatic controls. Specimens were immunostained using specific antibodies to the above mentioned antigens. Immunoreactivity (IR) were quantified with image analysis. Results: Reduced levels of M1 IR were noted in DO patients compared to controls. Following BoNT/A, there were increases and „normalisation‟ of M1 IR with similar changes in the urothelium. Significant similar post-BoNT/A increases were seen in M2 IR. Decreased M3 IR was observed at baseline DO compared to controls, with significant increases only in the urothelium following BoNT/A. SNAP-25 IR showed no changes. NPY IR increased in DO, with a decreasing trend following BoNT/A. Inverse IR correlations were found with frequency and urgency. Conclusions: Reduced levels of suburothelial muscarinic receptors in DO are in accordance with previous RT-PCR findings, showing reduced mRNA levels in overactive bladders. NPY IR is increased and may illustrate upregulated cholinergic transmission with DO, similar to ATP. SNAP-25 IR demonstrates the presence of BoNT/A sensitive neurones within the suburothelium. Post-BoNT/A changes in suburothelial muscarinic receptors and NPY appear compensatory to the reduced release of ACh, supporting a neuroplastic effect of BoNT/A on bladder afferent pathways as part of its mechanism of action.

Prion diseases in mice are characterised by prolonged, clinically silent incubation periods that are distinct in duration and reproducible. Incubation times are influenced by a number of factors with the major genetic determinant being variation within the prion protein gene, Prnp, however, different lines of inbred mice with identical Prnp genotypes show differences in incubation time indicative of a multi-genic effect. A heterogeneous stock of mice was used for fine mapping studies from which emerged several candidate genes including Hectd2, an E3 ubiquitin ligase. A human association study found that this genetic modifier is associated with an increased risk to vCJD and kuru. To characterise Hectd2, a yeast two hybrid screen was performed to search for novel interactors, yielding twenty binding partners. Selected candidates were verified by co-immunoprecipitation in mammalian cells. These included Sh3glb1, which has been linked to autophagy in a Parkinson’s disease model as well as Stambp, which encodes a deubiquitination protein in the CNS, and Stmn2, a gene previously associated with susceptibility to vCJD in a genome wide association study. Pacsin2 and Sept7, also emerged as Hectd2 interactors. These proteins operate in the cytoskeletal network, serving a regulatory role for maintaining neuronal integrity, a pathway not previously recognised in prion pathology. By application of a cell based model of prion propagation, the Scrapie Cell Assay, it has been possible to establish the effect of knocking down candidate genes on cell susceptibility thus providing a direct link with prion pathology. In addition, Hectd2 knockout and transgenic overexpressing mouse models were challenged with three strains of mouse adapted prions to search for an association with incubation time. In conclusion, this has been the first attempt at interactome mapping of a candidate gene associated with incubation time in mouse and susceptibility to human diseases. We have identified novel gene candidates that are implicated in prion pathology, thus providing an insight into the possible pathways that modulate prion disease.

This work focuses on creating MEMS devices to mimic the function of patch pipettes. It is hypothesised that the closer the devices approximate the physical structure of patch pipettes, the more successful they will be in achieving seals and recording from cells. To test this hypothesis, devices were fabricated with a range of physical properties. The parameters varied were aperture diameter, aperture profile, aperture depth, surface roughness and surface chemistry. Devices with apertures of 1.5 <i>µ</i>m and 2.5 <i>µ</i>m were fabricated, with either a flat profile or a protruding nozzle. The surface material was thermal silicon dioxide, which was optionally doped with boron to change the surface chemistry. Devices were also manufactured with 2 <i>µ</i>m diameters. These had a rougher, PECVD silicon dioxide surface, which produced a more rounded aperture profile. Using glass patch-pipettes as a control experiment, attempts were made to form seals using these devices and N2A cells. The results obtained showed that only surface roughness and aperture depth had a significant effect on seal formation. Although it was anticipated (on the basis of the control experiments) that aperture diameter would also play a role, this was not witnessed: attempts are made to explain this discrepancy. It is thus concluded that matching surface roughness and aperture depth to those of glass pipettes is of most importance in the manufacture of planar patch-clamp devices. Although surface chemistry and aperture profile apparently do not affect seal formation, further investigation would be required to determine this for certain.

The N-methyl-D-aspartate (NMDA) type of glutamate receptor has important functions in the processes of neuronal development, forms of neurodegeneration, epilepsy, pain and especially in synaptic plasticity and forms of learning and memory. An NMDA receptor dependent form of synaptic plasticity, long-term potentiation (LTP), has been proposed as a cellular correlate of memory. The cellular and molecular mechanisms that underlie this form of synaptic plasticity are at the present time unclear. The NMDA receptor is part of a macromolecular complex that mediates both the localisation of the receptor and its interactions with signalling molecules that activate the intracellular pathways responsible for the long lasting changes in synaptic efficacy. This thesis details results of experiments investigating long and short-term plasticity in mice lacking signalling molecules associated with NMDA receptor. The <i>in vitro</i> hippocampal slice preparation and extracellular and intracellular electrophysiological recording techniques were used to investigate plasticity in mice lacking the non-receptor tyrosine kinase fyn, focal adhesion kinase (FAK) and the ras guanine nucleotide releasing factor (ras-GRF). Ras-GRF knockout mice displayed normal NMDA receptor dependent LTP in area CA1 of the hippocampus and have been reported to show normal spatial learning in the Morris water maze (which is dependent on hippocampal function). Mice with disrupted FAK function also showed normal LTP in area CA1 of the hippocampus. Fyn tyrosine kinase deficient animals have been reported to show several neurological phenotypes, including impaired LTP induction in area CA1 of the hippocampus. This effect was found to be dependent upon the genetic background of the animal and independent of disruptions to hippocampal morphology. Short-term plasticity (paired pulse facilitation) was reduced in mice lacking fyn tyrosine kinase, an effect which was shown to be independent of age and genetic background. The mechanisms by which fyn mediates these changes were investigated through the use of experiments modulating the probability of neurotransmitter release.

Information regarding the performance of tests used to assess the tear film is currently incomplete. This hinders the interpretation of their results, and so obstructs the management of patients with disorders of the tear film. This work determined the repeatability and identified confounding variables of methods that assess tear breakup time and tear meniscus height. It was found that the reproducibility of both these tests were poor when considered over the range of values encountered in clinical practice; however, the situation was improved for lower values. Furthermore, results indicated that the volume of Fluorescein solution instilled prior to the assessment of tear break-up time and the delay after a blink when assessing tear meniscus height should be standardized. Additionally, a new instrument to grade the severity of symptoms of ocular surface irritation was developed and psychometrically tested. This has advantages over its predecessors because it derives measures on a linear interval scale and more satisfactorily accounts for missing data. It is envisaged that this instrument will be used in future clinical trials, and will improve the power of inferences made from their data. Finally, the effectiveness of eyedrops containing Sodium hyaluronate in the treatment of moderate dry eye was investigated. A pharmacokinetic analysis established that their beneficial effects on the tear film last several hours, and a follow-up study found that they reduce symptoms and improve ocular surface health when used for a month

Auditory fatigue is now known as Temporary Threshold Shift (subsequently abbreviated to TTS). It has been defined as the temporary elevation in the absolute threshold of hearing for a given test sound resulting from preceding auditory stimulation by a given stimulus sound (see Hirsh, 1952, page 177). Associated with the phenomenon there are six physical variables which sub-divide themselves into stimulus, test and recovery factors. In IMS study each of the latter factors was systematically Investigated using four groups of six undergraduate subjects. Thresholds were measured using the Bek6sy (1947) technique of threshold measurement. Control experiments were carried out to verify that TTS results from the application of the stimulus tone, to study the mechanisms involved in the Bekesy technique of threshold measurement and the effects of this technique on the measurement of TTS, to investigate any additive effects of TTS and to study the possibility of any measurement errors resulting from the physical test environment. The results of the experiments showed that the stimulus test and recovery factors all produced a consistent duality of results. It was concluded that the unitary definition of TTS is inadequate and that there are two TTS mechanisms. These are referred to as fatigue and temporal stimulation deafness. Fatigue is associated with moderately intense stimulus tones of fairly short duration. It increases linearly with logarithm of the stimulus duration; it is maximal at stimulus frequencies of 1000, 2000 and 3000 cps; it does not vary significantly with stimulus intensities of up to 90 db. but thence increases rapidly to a maximum; it is maximal at a test frequency equal to the stimulus frequency and recovery from it is complete within one minute of the cessation of the stimulus. Temporary stimulation deafness is associated with high intensity stimulus tones of fairly long duration. It increases linearly with the logarithm of the stimulus duration; it is maximal with stimulus frequencies of 4000 to 6000 cps; it increases rapidly as the stimulus intensity is increased; it is maximal at a test frequency an octave above the stimulus frequency and recovery from it takes longer than one minute from the cessation of the stimulus tone. It is hypothesized that fatigue is a neural, possibly bio-chemical, adaptation effect and that temporary stimulation deafness results from structural damage to the organ of Corti. Other work supports this differentiation. The phenomena of fatigue support either a "place" or a "volley" theory of hearing. The phenomena of temporary stimulation deafness are partially explicable in terms of the anatomical characteristics of the ear.