Metabolites are a potentially useful source of detecting and identifying disease specific biomarkers. This thesis investigates the possibility of using metabolomics applications to detect Alzheimer’s disease associated metabolite peaks in patients and to detect longitudinal changes of the disease. Serum samples and clinical data were collected from 60 healthy controls and 60 Alzheimer’s disease patients (60 at baseline and 60 at 12 month follow-up). The metabolic fingerprinting of serum samples using the FT-IR lacked discriminatory power to discriminate Alzheimer’s disease and non-disease samples due to the similar magnitude of biological and analytical variation. The metabolic profiling of serum samples using the GC-ToF-MS did not reveal any significantly altered metabolite peaks between the Alzheimer’s disease and non-disease groups. Metabolic profiling of serum samples using the UPLC-LTQ/Orbitrap-MS operated in the positive ionisation mode did not reveal any significantly altered metabolite peaks between the disease and non-disease groups. Up to twelve metabolite peaks were significantly altered in the Alzheimer’s disease baseline and follow-up samples, indicating a potential association with disease progression. Metabolic profiling of serum samples using the UPLC-LTQ/Orbitrap-MS operated in the negative ionisation mode did not reveal any significantly altered metabolite peaks between Alzheimer’s disease and non-disease groups. Three metabolite peaks were significantly altered in the Alzheimer’s disease baseline and follow-up samples, indicating a potential association with disease progression. Metabolic profiling of serum samples with the UPLC-LTQ/Orbitrap-MS may potentially be used to detect disease and disease progression associated metabolite peaks. The metabolite peaks require identification followed by a validation experiment.

Part one is a systematic review of the literature relating to perceived stigma and support amongst family caregivers of people with dementia. Numerous studies have suggested stigma is associated with negative outcomes in various mental and physical health difficulties. Older people with dementia and their family care givers could be especially vulnerable to experiencing stigma and this could have a negative impact on their engagement with services. Out of an initial literature search resulting in 101 articles, 15 articles met the inclusion criteria for the present review and their methodological quality was assessed. The 15 articles were critically analysed and four main themes from the articles were extracted. The themes were sociocultural aspects, services use and diagnosis, stigma and aging and managing stigma. A proposed model of perceived stigma that explained the potential origins of stigma, the difficulties it may bring and possible links to service engagement was developed from the results of this review. The clinical implications of this and possible further research areas was discussed. Part two is an empirical paper consisting of two aims. The first aim investigated levels and types of hope experienced by older people with and without dementia. The second aim focused on potential relationships between stigma, hope and quality of life in older . people with dementia. Many studies have indicated that hope is positively associated with quality of life in chronic health conditions and there are two contrasting theories of I " hope, multi-dimensional hope and goal directed hope. Preliminary studies suggested that older people with dementia may predominantly experience multi-dimensional hope and that stigma may be a barrier towards hope. A group of 60 older people without dementia completed two different questionnaires on hope and a group of 40 older people with dementia completed two additional questionnaires on their experiences of stigma and their quality of life. The results supported the hypothesis that older people with dementia experienced higher levels of multi-dimensional hope but stigma was not a statistically significant barrier towards this. Implications for services, including the development of psychosocial interventions and possible further research were discussed. Part three are the appendixes including a reflective statement on the designing, planning and implementation of the research.

This thesis is divided into 3 parts:Part 1 is a systematic literature review entitled ‘Living Positively with Dementia’. The notion that people living with dementia can have positive experiences in spite of - or even because of - their illness remains widely disputed. In light of this controversy and the potential conceptual and clinical importance of documenting the subjective experience of dementia in all its variations, this review examines the current research evidence relating to positive lived experience in dementia. The review uncovered 63 studies containing evidence of positive experiences reported by people living with dementia. The methodological quality of the studies is evaluated along with consideration of how researchers have managed such reports. The findings of the review suggest that people with dementia are capable of experiencing positive emotions, and furthermore that dementia can uncover strengths in people, such as hope; faith; resilience; wisdom; altruism; humor; optimism; perseverance; and courage.Part 2 is an empirical study which investigates one of the positive constructs highlighted by the review - hope. The paper examines the subjective experience of hope in older people with early-stage dementia and explores factors that facilitate or hinder hopefulness in this population. Ten participants were each interviewed and interpretative phenomenological analysis was used to identify themes within their accounts. The study considers how hopefulness in dementia compares with existing definitions of hope and the existing literature on age; dependency; and coping, in order to provide a thematic framework for understanding how hope may support individuals with dementia to maintain their quality of life, and to consider the scope for hope-fostering strategies.Part 3 consists of the appendixes relating to the research, including a reflective statement on the process of carrying out the research. The statement includes reflection on all aspects of research from planning to carrying out the research project.

The Amyloid Cascade Hypothesis states that fibrillation of the amyloid beta (Aβ) peptide is the primary cause of Alzheimer’s pathology. The trigger for the fibrillation is a subject of much debate, although it is clear, oxidative stress is a key feature of Alzheimer’s aetiology. This thesis explores a possible role of oxidation of Aβ, in particular the effect of histidine and methionine side-chain oxidation, on Aβ fibril growth rates. Within chapters 2 and 3 of this thesis is a discussion of various approaches to chemical synthesis of 2-oxo-histidine with a view to the incorporation of the oxidised amino acids into Aβ peptide using Fmoc approaches. Chapter 2 describes attempted chemical transformation of (protected) L-histidine into L-oxohistidine. Dimethyldioxirane oxidised Boc-His-OMe yielded products containing isopropylidene groups, while oxidation using a Cu(II)/ascorbate generated 2-oxo-histidine but gave very low yields. Within chapter 3, a successful synthesis of protected 2-oxo-histidine is described, via the known imidazolin-2-one-4-carboxylic. Chapter 4 analyses Aβ(1-40) fibrillation kinetics by treating the intact peptide with various oxidants. Contrary to previous reports, hydrogen peroxide alone did not slow fibrillation rates. Cu(II)/Cu(I)- catalysed oxidation increased the likelihood of amorphous aggregation over fibrillation. This thesis shows oxidation of Aβ has a profound influence on fibril growth and that incorporation of a stable oxidised histidine into Aβ is a realisable goal.

The N-terminally pyroglutamyl-modified β-amyloid (Aβ) peptides are found in abundance in the pathological Alzheimer disease (AD) brain deposits. Such modification not only increases the hydrophobic properties of a given molecule, but also plays a protecting role against proteolytic degradation. This project involved the study of the human type I and type II pyroglutamyl peptidases and their involvement in Aβ processing in AD. Human PcpI has been successfully overexpressed in Escherichia coli strain and purified to homogeneity. The protein displayed significant instability in vitro. To overcome this problem a number of methods were employed such as screening for an optimal protein expression system and buffer composition, site-directed mutagenesis and chemical modification of selected surface residues. This resulted in the selection of the HEPPS buffer system as providing the most stabilising conditions for human PcpI. Improvement in the protein stability enabled initial crystallisation experiments and the identification of favourable conditions for crystal production. Further optimization of this process is needed in order to obtain good quality crystals which are required for structural study. The study on human PcpII involved an extensive screening for optimal expression conditions in bacterial, baculovirus/insect and mammalian systems. The truncated PcpII isoform PcpII/S62-H1024, which lacks the N-terminal transmembrane domain, was successfully expressed and secreted from the HEK 293T cell line using three different pOPIN-based constructs. Moreover, homology modelling of human PcpII catalytic domain was performed, which helped to gain an insight into the three-dimensional structure of the protein and its mode of substrate binding. Lastly, immunohistochemical staining of the human AD brain tissue sections was performed to compare the level and distribution of PcpI and PcpII enzymes between diseased and control cases. The results confirmed that the neurodegenerative conditions lead to the increased synthesis of both enzymes in the cortical AD tissues. Additionally PcpI was shown to be able to participate in the degradation of pGlu-modified Aβ peptides.

There has been accumulating scientific evidence, over the last three decades, of the role of inflammatory processes in the development of Alzheimer's disease (AD). Population based studies suggest that plasma levels of inflammatory markers are raised in peripheral blood of people with AD. People on long term use of non-steroidal anti-inflammatory drugs have a lower prevalence of AD. Moreover, both animal and human histopathology studies have reported localization of inflammation in brain areas primarily affected by AD pathology. Areas of increased inflammation can be visualized in vivo by Positron Emission Tomography (PET) scans using the PK11195 ligand that binds with the benzodiazepine receptor sites of activated microglial cells. Cognitive decline in AD has been shown to correlate with levels of microglial activation using PK11195 PET scans. People with amnestic mild cognitive impairment (MCI) are known to be at high risk of developing AD.We aimed to investigate the association between peripheral and central markers of inflammation and cognitive decline in a group of people with amnestic MCI.MCI subjects (n=70) underwent cognitive testing, IL-6 and CRP in peripheral blood were measured and repeated after 1 year. A sub group (n=15) was followed up for another year and central brain microglial activation was measured by PET using PK11195 along with cognitive and peripheral inflammatory marker measurement. The mean CRP and IL-6 levels of the cohort increased over one year but the rise was only significant for CRP. No association was detected between inflammatory markers levels and cognition as measured by a battery of cognitive instruments. Group comparisons of the PET cohort with healthy controls (n=5) showed increased PK11195 binding (mean binding potential) in frontal lobe, temporal lobe, parietal lobe, putamen, occipital lobes and significantly increased binding in posterior cingulate gyrus. This study, to our knowledge, is unique in studying makers of inflammation in amnestic MCI participants both in peripheral blood and brain. The results of this study, in the light of current literature, add to the importance of recognition of inflammatory processes in people at risk of developing AD. The results suggest that CRP levels rise significantly over time and are detectable in peripheral blood by using practically simple laboratory techniques. The results also suggest that activated microglia in amnestic MCI patients can be visualized in vivo by using PK11195 PET scans and show higher levels of activation as compared to healthy controls. These finding could be useful in identifying people with malactivated (pro-inflammatory) microglia as potential targets for prevention/early treatment strategies. Further studies with larger samples sizes and long term follow-up are needed to investigate whether these peripheral and central inflammatory markers could shed light on the aetiology of AD and be useful in monitoring disease progression.

The endocannabinoid system (ECS) comprises the endocannabinoids (ECs), including anandamide (AEA) and 2-arachidonoyl glycerol (2AG), which interact with the G protein-coupled type-1 and type-2 cannabinoid receptors(CB1 and CB2 respectively). The ECS is thought to have a role in a number of central processes including neuroinflammation, neurogenesis, neuroprotection, learning and memory. Due to its influence on a diverse number of processes, it has been suggested that modifying the ECS may be therapeutically beneficial in Alzheimer's disease (AD). AD is an age-related neurodegenerative disorder characterised by the presence of extracellular amyloid beta (Ab) plaques and intracellular neurofibrillary tangles (NFTs) resulting in impairments in learning in memory. The aim of this thesis was to determine the status of the brain ECS in the APPswe/PS18E9 mouse model of AD and wild type littermates at 4, 6 and 8 months of age and the performance of these animals in a behavioural test battery. The results of this study indicated that APPswe/PS18E9 animals were hyperactive compared to their wildtype counterparts at all ages and that they also displayed deficits in behavioural flexibility. EC levels increased with age in both wild type and APPswe/PS18E9 mice. Cannabinoid receptor coupling was increased in the frontal cortex and striatum of APPswe/PS18E9 mice relative to wildtype. This study concluded that the status of the brain ECS is altered in AD. Modifications to the performance of the ECS were made in the form of chronic administration of a CB1 receptor antagonist (SR141716A1rimonabant) and a CB2 receptor agonist (JWH133). Chronic administration of SR141716A was able to reverse some learning impairments in APPswe/PS18E9 animals. In contrast, chronic administration of JWH133 resulted in impaired memory extinction in both wildtype and APPswe/PS18E9 mice. The results support the potential benefit of modulating the endocannabinoid system in the treatment of memory impairment in AD.

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system and the formation of protein inclusions in surviving neurones. The mechanisms underlying neurodegeneration and neuroprotection in the nervous system remain elusive. Ubiquitin is one of the hallmarks of neuropathological inclusions in the majority of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, dysfunction of the ubiquitin proteasome system has been implicated in disease cause and/or progression. This thesis investigates a unique conditional genetic mouse model of neurodegeneration caused by conditional genetic 26S proteasomal depletion in mouse forebrain neurones. We have identified potential proteins targeted for ubiquitination in brain using bio-affinity chromatography of zinc finger protein ZNF216 coupled with mass spectrometry. This lead to the identification of several potential ubiquitinated proteins involved in gene expression and regulation. We have also investigated the global brain proteome in response to 26S proteasomal depletion in neurones using two-dimensional fluorescence difference in-gel electrophoresis coupled to mass spectrometry for protein identification. Several differentially expressed proteins were identified in the 26S proteasome-depleted cortex. Astrocytic intermediate filament proteins glial acid fibrillary protein and vimentin, as well as the antioxidant peroxiredxoin-6, were upregulated. Mitochondrial fumarate hydratase and stathmin-1, involved in the tricarboxylic acid cycle and cytoskeletal microtubule dynamics respectively, were downregulated. These proteins have been validated by biochemical and immunohistochemical approaches. Further analysis of oxidative stress revealed increased lipid and protein oxidation that may be involved in the neurodegeneration associated with 26S proteasomal depletion. However, we also show increased phospholipase A2 activity associated with peroxiredoxin-6 expression that may have additional roles in neurodegenerative and/or neuroprotective functions. Interestingly, the levels of reactive oxygen species were inversely correlated with the upregulation of peroxiredoxin-6. We suggest that peroxiredoxin-6 may play an important role in the brain in the protection against oxidative stress and our studies may improve our physiological and pathological understanding of neurodegenerative disease.

No description available.

The integrity of brain white matter is vital for the interneuronal signalling between distinct brain regions required for normal cognitive function. White matter integrity is compromised with ageing and could contribute to age-related cognitive decline. Chronic cerebral hypoperfusion is thought to underlie the development of white matter pathology and cognitive changes, often seen in the elderly. Additionally, the development of regional hypoperfusion and white matter damage are thought to be early events in Alzheimer’s disease (AD) pathogenesis. This thesis set out to test the hypothesis that chronic cerebral hypoperfusion underlies the development of white matter pathology and cognitive decline and also that chronic cerebral hypoperfusion causes the development of Ab pathology in AD. The first aim was to investigate the impact of hypoperfusion on the development of white matter damage and different aspects of cognition in a mouse model of chronic cerebral hypoperfusion. Two studies were undertaken to address this. The first study examined the temporal development of pathology following hypoperfusion induced by bilateral carotid artery stenosis (BCAS) using microcoils Hypoperfusion was induced in wild type (WT) mice and the pathological changes examined at one week, two weeks, one month and two months. Hypoperfused animals developed a diffuse and widespread white matter pathology, present from one week, which occurred predominantly in the myelin component of white matter; this was accompanied by minimal axonal damage. A second study examined the impact of hypoperfusion on different aspects of spatial memory and further investigated pathological changes in the model at one and two months. Behavioural testing revealed a significant impairment in spatial working memory but not episodic memory or spatial reference memory in hypoperfused animals. In the same mice, pathological assessment indicated that there was a significant increase in levels of myelin damage and elevated levels of microglial activation as compared to shams. These results demonstrate that modest reductions in cerebral blood flow are sufficient to cause the development of white matter damage and the development of cognitive deficits. The second aim was to investigate the impact of hypoperfusion on the development of white matter and amyloid pathology in a mouse model (3xTg-AD) of AD. To address this, using 2 different sizes of microcoils (0.18mm and 0.16mm internal diameter) BCAS of varying severities was induced in 3xTg-AD mice and white matter and Ab pathology were assessed at one month. Circle of Willis (CoW) architecture was also compared between WT and 3xTg-AD mice. Overall white matter pathology was not exacerbated in experimental 3xTg-AD mice with BCAS induced by 0.18mm coils. However with a greater level of stenosis (0.16mm coil) ischaemic damage to neuronal perikarya was present in most experimental animals. In addition to ischaemic damage, localised areas of severe white matter pathology were also observed in conjunction with subtle changes to white matter Ab levels. Hypoperfusion did not impact on the development of intraneuronal Ab pathology, other than in the presence of ischaemic damage when levels were reduced. Comparison of CoW architecture between WT and 3xTg-AD mice revealed strain specific differences in the presence and morphology of the posterior communicating artery which may explain the lack of pathology in 3xTg-AD mice as compared to WT following BCAS induced using 0.18mm dia. microcoils. The third aim was to investigate whether white matter protein composition changed with age and also whether ageing conferred increased vulnerability to hypoperfusion. To address this, white matter protein levels were compared between young (3-4 months) and old (12-13 months) 3xTg-AD mice. White matter pathology was compared between sham and hypoperfused animals in the aged cohort. Levels of myelin basic protein and 2', 3'-cyclic nucleotide 3'- phosphodiesterase were found to be significantly increased whilst levels of myelin associated glycoprotein were significantly reduced with ageing. These results suggest that changes in myelin protein composition may contribute to the development of age related white matter pathology. White matter pathology was not exacerbated in aged hypoperfused animals following one month of hypoperfusion as compared to shams. The results presented within the thesis demonstrate that chronic cerebral hypoperfusion precipitates the development of selective white matter damage and impacts on cognition. Also it has been shown that where hypoperfusion is severe enough to cause ischaemic damage to neuronal perikarya and localised areas of severe white matter pathology, alterations in white matter Ab levels can occur. Hypoperfusion does not impact on APP processing or on intraneuronal levels of APP or Ab, other than in the presence of ischaemic damage to neuronal perikarya, when levels are reduced. These findings highlight the importance of early intervention strategies in the treatment of vascular risk factors which can lead to hypoperfusion and the development of white matter damage and a decline in cognitive function in later life. These findings also suggest that repair or prevention of white matter damage may be an appropriate strategy for the attenuation of cognitive decline following onset of hypoperfusion. This thesis also highlights some of the limitations of animal models of human disease.