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Delirium, or “acute confusion” is a common and serious acute neuropsychiatric syndrome mainly affecting older people. It is associated with multiple adverse outcomes, including an increased risk of developing dementia and increased mortality. The underlying mechanisms of delirium are poorly understood, and there are currently no specific treatments. This thesis investigated the roles of the hypothalamic-pituitary adrenal axis and inflammation in the pathophysiology of delirium, persistent delirium and cognitive decline following delirium. It investigated whether levels of cortisol in blood and cerebrospinal fluid (CSF) are elevated in delirium, with elevated pro-inflammatory and reduced anti-inflammatory cytokines. It also investigated whether there is loss of cortisol diurnal rhythm (in saliva) with elevated afternoon cortisol levels. The thesis investigated whether any hypercortisolaemia was sustained during the year after delirium, and whether this was associated with deterioration in cognition during the year after hip fracture. Finally, it also tested whether there are high levels of a marker of central nervous system damage (S100B) and of a dementia marker (tau) in CSF in delirium. A prospective observational cohort study was conducted in N=108 patients aged over 60 who had sustained a hip fracture, in whom 40% developed delirium. Participants gave informed consent or if they lacked capacity to give informed consent, this was given by their next of kin. Participants were assessed regularly for delirium, according to DSM IV criteria, during the two weeks after hip fracture. A sample of CSF was collected during the spinal anaesthetic performed for the operation to repair their fracture. Samples of blood and saliva were collected during the two weeks after the hip fracture operation. Participants were visited three, six and twelve months after their hip fracture for further delirium assessment, and a cognitive test battery was completed. Further samples of blood and saliva were collected at these visits. The study found evidence of high levels of cortisol and of S100B in CSF in those with active delirium, but there were no differences in levels of tau or cytokines in CSF. Those with delirium had elevated serum cortisol during the perioperative period, and elevated afternoon salivary cortisol, suggesting flattening of cortisol diurnal rhythm with failure to reach the normal diurnal nadir. After adjusting for confounders in a multivariate logistic regression analysis, serum cortisol was still predictive of delirium, but salivary cortisol AM:PM ratio had a trend towards significance. Those who had persistent delirium features in the months after hip fracture had significantly higher serum cortisol three months after hip fracture. There was a change in serum inflammatory profile in those with delirium, with a shift towards a pro-inflammatory state. Testing the study hypotheses surrounding cognition after delirium was very challenging, due to patient attrition and other factors. Some participants showed a trajectory of cognitive improvement, which was probably due to resolution of delirium during the year after hip fracture. Those with resolved delirium had deficits in verbal and visual memory. This study has improved understanding of the mechanisms of delirium, suggested further avenues for research and identified possible new therapeutic targets.

<p>This thesis describes the current status and novel aspects of the analysis of the molecules of life, i.e. peptides and proteins, using capillary electrophoresis (CE) coupled to mass spectrometry (MS) via (sheathless) electrospray ionization (ESI). Early reports of sheathless CE-ESI-MS were plagued by limited lifetimes of the electrospray emitter. In this thesis, two new approaches, the Black Dust and the Black Jack methods, utilizing polymer-embedded graphite instead of noble metals are presented. These emitters have shown improved long-term stability and proven excellent for sheathless electrospray operation. Failure of an emitter is often caused by electrochemical reactions occurring at the emitter-liquid interface. The electrochemical properties of the graphite coated emitters were therefore evaluated by classical electrochemical methods, such as cyclic voltammetry and chronoamperometry. The graphite coated emitters showed excellent electrochemical stability and properties compared to noble metal and polymer configurations.</p><p>Analyte-wall interactions have long been known to cause problems in the CE analysis of biomolecules. This can be circumvented by internal modification of the capillary walls. Additionally, it is of outermost importance to have a stable and sufficiently high electroosmotic flow (EOF) to sustain the electrospray, when using a sheathless approach. New monomer and polymer coatings are presented for rapid and high-efficient CE-ESI-MS separations of peptides and proteins.</p><p>Furthermore, the use of CE-ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) shows great potential for rapid proteomic probing of human cerebrospinal fluid. The results are comparable with more established techniques, such as liquid chromatography and two-dimensional gel electrophoresis coupled to MS. However, the CE-ESI-FTICRMS analysis has significantly lower sample consumption and faster analysis time compared to the other techniques. The applications and use of CE-ESI-MS is expected to have a bright future with continued growth as current trends of multidimensional hyphenation and microfabricated devices are further developed and explored.</p>

This thesis describes the current status and novel aspects of the analysis of the molecules of life, i.e. peptides and proteins, using capillary electrophoresis (CE) coupled to mass spectrometry (MS) via (sheathless) electrospray ionization (ESI). Early reports of sheathless CE-ESI-MS were plagued by limited lifetimes of the electrospray emitter. In this thesis, two new approaches, the Black Dust and the Black Jack methods, utilizing polymer-embedded graphite instead of noble metals are presented. These emitters have shown improved long-term stability and proven excellent for sheathless electrospray operation. Failure of an emitter is often caused by electrochemical reactions occurring at the emitter-liquid interface. The electrochemical properties of the graphite coated emitters were therefore evaluated by classical electrochemical methods, such as cyclic voltammetry and chronoamperometry. The graphite coated emitters showed excellent electrochemical stability and properties compared to noble metal and polymer configurations. Analyte-wall interactions have long been known to cause problems in the CE analysis of biomolecules. This can be circumvented by internal modification of the capillary walls. Additionally, it is of outermost importance to have a stable and sufficiently high electroosmotic flow (EOF) to sustain the electrospray, when using a sheathless approach. New monomer and polymer coatings are presented for rapid and high-efficient CE-ESI-MS separations of peptides and proteins. Furthermore, the use of CE-ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) shows great potential for rapid proteomic probing of human cerebrospinal fluid. The results are comparable with more established techniques, such as liquid chromatography and two-dimensional gel electrophoresis coupled to MS. However, the CE-ESI-FTICRMS analysis has significantly lower sample consumption and faster analysis time compared to the other techniques. The applications and use of CE-ESI-MS is expected to have a bright future with continued growth as current trends of multidimensional hyphenation and microfabricated devices are further developed and explored.

The pre-symptomatic stage of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) occurs several decades before the clinical onset. Changes in the lysosomal network, i.e. the autophagosomal, endosomal and lysosomal vesicular system, are among the first alterations observed. There are currently no treatments to slow or cure neurodegenerative diseases, and there is a great need for discovery of treatment targets in cellular pathways where pathology pre-dates the neuronal death. It is also crucial to be able to diagnose neurodegenerative diseases earlier, both to enable early intervention treatment and aid in selecting clinical trial populations before the patient has widespread pathology. This thesis aims at investigating the potential of lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease. A targeted search for lysosomal network proteins was performed in cerebrospinal fluid (CSF) from AD patients, and seven proteins: early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), lysozyme, microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were elevated. The levels of EEA1, LAMP-1, LAMP-2, LC3, lysozyme and Rab3 were also measured in CSF from parkinsonian syndrome patients: PD, clinically diagnosed 4-repeat tauopathy, pathologically confirmed corticobasal degeneration (CBD) and pathologically confirmed progressive supranuclear palsy (PSP) patients. LAMP-1 and LAMP-2 were decreased in PD. LC3 and lysozyme levels were increased in 4-repeat tauopathy patients. EEA1 was decreased and lysozyme increased in PSP, and LAMP-1, LAMP-2, LC3 and lysozyme were increased in CBD. The lysosomal network proteins had different CSF protein profiles in all the parkinsonian syndromes, as well as in AD. It should be emphasized that only a select few of the lysosomal network proteins were observed to be changed, rather than a general change in lysosomal network proteins, which implicates the involvement of these seven proteins in specific pathological processes. The most interesting candidates, LAMP-2 and lysozyme, were selected for further study for their involvement in the pathology of AD. Lysozyme was found to co-localise with Aβ plaques in AD patients and overexpression prolonged survival and improved the activity in a Drosophila model of AD. Lysozyme was found to alter the aggregation pathway of Aβ1-42, to counteract the formation of toxic Aβ species and to protect from Aβ1-42 induced cell toxicity. Aβ1-42 in turn was found to increase the expression of lysozyme in both neuronal and glial cells. These data suggest that lysozyme levels rise in AD as a compensatory response which is protective against Aβ associated toxicity. LAMP-2 mRNA and protein were found increased in brain areas relevant for AD pathology and various cellular models showed complex involvement of LAMP-2 in Aβ related pathology, with extensive crosstalk between LAMP-2 and Aβ. Exposure to oligomeric Aβ1-42 caused an upregulation of LAMP-2 and in turn, overexpression of LAMP-2 caused a reduction in secreted levels of Aβ1-42, as well as changing the generation pattern of Aβ and affecting clearance and secretion of Aβ1-42. These data indicate that the increased levels of LAMP-2 in AD could be an attempt to regulate Aβ generation and secretion. In summary, this thesis reports that utilising lysosomal network proteins as biomarkers and novel therapeutic targets for neurodegenerative diseases holds great promise.

Scanning ion conductance microscopy (SICM) is a technique for non-contact topographic imaging. In this thesis, a biophysical investigation into Alzheimer's Disease (AD) was carried, with toxic oligomers dosed locally and quantitatively on to single astrocytes using SICM and simultaneously monitoring the response of the target cell. Examination of the effectiveness of antibodies that bind to Abeta or alpha-synuclein (Asyn)peptides depends on the measurement of oligomer-induced abnormal calcium homeostasis in single astrocytes. The method was shown to work at physiological concentrations of oligomers. A series of experiments measuring the reduction in calcium inux in mixtures of antibodies and cerebrospinal fluid (CSF) of AD patients suggested that the binding to co-oligomers composed of Abeta and Asyn may be crucial in the treatment of AD. Furthermore, it may be beneficial to test antibodies before the clinical trial using this assay. The mechanism of this entry of calcium is hypothesised to be the result of the formation of oligomer-induced transient pores in the cell membrane. To verify this hypothesis, a new SICM instrument was built with two nanopipettes; one for dosing and one for detection of the adenosine triphosphate (ATP) release from these pores. A variety of different ATP sensors were made. The best had a sensitivity of 10 micro molar and works as a hexokinase-cofunctioned electrolyte-gated organic field-effect-transistor. However no statistically significant results for ATP release have been obtained in the experiments performed to date. Overall this thesis describes new biophysical methods to study the effect of protein aggregates on live cells and the effectiveness of potential therapies, such as antibodies and nanobodies, to reduce these aggregate induced effects. It can be applied to synthetic aggregates of Abeta or the aggregates present in human CSF.

yes / To investigate cerebrospinal fluid (CSF) dynamics in the aqueduct of Sylvius (AoS) in chronic cerebrospinal venous insufficiency (CCSVI)-positive and -negative healthy individuals using cine phase contrast imaging. Materials and Methods Fifty-one healthy individuals (32 CCSVI-negative and 19 age-matched CCSVI-positive subjects) were examined using Doppler sonography (DS). Diagnosis of CCSVI was established if subjects fulfilled ≥2 venous hemodynamic criteria on DS. CSF flow and velocity measures were quantified using a semiautomated method and compared with clinical and routine 3T MRI outcomes. Results CCSVI was associated with increased CSF pulsatility in the AoS. Net positive CSF flow was 32% greater in the CCSVI-positive group compared with the CCSVI-negative group (P = 0.008). This was accompanied by a 28% increase in the mean aqueductal characteristic signal (ie, the AoS cross-sectional area over the cardiac cycle) in the CCSVI-positive group compared with the CCSVI-negative group (P = 0.021). Conclusion CSF dynamics are altered in CCSVI-positive healthy individuals, as demonstrated by increased pulsatility. This is accompanied by enlargement of the AoS, suggesting that structural changes may be occurring in the brain parenchyma of CCSVI-positive healthy individuals

Background: Normal pressure hydrocephalus (NPH) has been an ongoing and challenging field of research for the past decades because two main issues are still not fully understood: the pathophysiologic mechanisms underlying ventricular enlargement and prediction of outcome after surgery. Purpose: To evaluate changes in diffusion tensor imaging (DTI) derived parameters in patients with suspected normal pressure hydrocephalus before and after withdrawal of cerebrospinal fluid (CSF). Material and Methods: Twenty-four consecutive patients with clinical and radiological suspicion of NPH and 14 agematched control subjects were examined with DTI on a clinical 3T scanner. Patients were examined before and 6–36 h after CSF drainage (interval between scans, 5 days). Fifteen patients were finally included in data analysis. Fractional anisotropy (FA) and mean, parallel, and radial diffusivity (MD, PD, RD) were evaluated using a combination of a ROI-based approach and a whole-brain voxel-by-voxel analysis. Results: Alteration of DTI parameters in patients with suspected NPH is regionally different. Compared to the control group, we found an elevation of FA in the subcortical white matter (SCWM) and corpus callosum, whereas the other diffusion parameters showed an increase throughout the brain in variable extent.We also found a slight normalization of RD in the SCWM in patients after lumbar drainage. Conclusion: Our results show that DWI parameters are regionally dependent and reflect multifactorial (patho-) physiological mechanisms, which need to be interpreted carefully. It seems that improvement of gait is caused by a decrease of interstitial water deposition in the SCWM.