Uma parcela significativa das lesões na molécula do DNA é causada por espécies reativas de oxigênio e a sua produção excessiva e/ou o funcionamento deficiente dos sistemas celulares antioxidantes, que neutralizam a sua ação, é conhecido como estresse oxidativo. Os danos em células normais são prontamente detectados por um sistema de defesa e, em conseqüência, uma rede intrínseca de sinalizações é ativada, sendo que uma das vias resulta na ativação dos mecanismos de reparo do DNA. O reparo por excisão de bases (BER) parece ser a via preferencial de reparo de bases oxidadas, mas existem outras vias de reparo implicadas na reversão do dano oxidativo. A doença de Alzheimer (DA), uma patologia causada particularmente por danos oxidativos, acomete atualmente cerca de 25 milhões de pessoas no mundo, sendo o risco aumentado a partir dos 65 anos de idade. Com isso, a necessidade da identificação de fatores de risco, além de fatores protetores relacionados à DA, tornou-se de grande importância. Por outro lado, há também a necessidade de estudos em nível molecular, que possam fornecer informações sobre os mecanismos que levam ao desenvolvimento da doença. Nesse sentido, foi realizado no presente trabalho, um estudo de expressão gênica transcricional pelo método de microarranjos de DNA, bem como uma análise por PCR em tempo real para uma série de genes envolvidos na resposta ao dano oxidativo no DNA (percepção de danos e reparo do dano), além de outros genes relacionados à doença. Adicionalmente, foram também avaliadas as quebras na fita dupla de DNA causadas por bases oxidadas, em linfócitos de pacientes de Alzheimer (grau moderado) e indivíduos sadios, usando-se métodos de detecção de bases oxidadas (8-oxoGuanina). Entre os vinte genes analisados pelo método de PCR quantitativa em tempo real, apenas a APOE mostrou-se induzida, enquanto 19 genes (ADAM17, APEX1, APP, BACE1, OGG1 ATM, ATR, TREX1, FEN1, FANCG, RAD17, DUSP, ERCC1, ERCC3, ERCC6, HUS1, RAD9, RAD1, PRKDC) foram reprimidos transcricionalmente. Essa repressão verificada para a maior parte dos genes estudados indica que várias vias de sinalização celular ligadas a respostas ao estresse oxidativo, incluindo-se as várias vias de reparo do DNA, podem estar envolvidas na condição DA. Adicionalmente, a análise de expressão gênica por microarranjos de cDNA indicou uma série de 41 genes significativamente modulados (q < 0,06) (dentre eles, NOTCH1, MARK3, PAK, SMC1L1) mas para a maioria destes não há relatos na literatura sobre uma possível relação com DA. Por essa razão, o método de microarranjos de cDNA aponta novas vias que possam estar alteradas em DA, o que constitui uma informação importante. Em conjunto, os dados obtidos no presente estudo fornecem uma contribuição relevante, que futuramente poderão contribuir em termos de intervenção terapêutica. / A great amount of DNA molecule lesions is caused by reactive oxygen species and its synthesis in excess and/or misfunctioning of antioxidant cell systems, which neutralize its effects, is known as oxidative stress. Damage in normal cells is readily detected by a defence system and as consequence, a complex signaling pathway is activated, among them DNA repair mechanisms. The base excision repair (BER) seems to be the primary repair pathway in base oxidative damages, however there are other pathways that are involved in their repair. The Alzheimers disease (AD), a pathology caused particularly by oxidative damages, hits 25 million people worldwide, and its prevalence increases every 5 years beyond age 65. Therefore, there is an emerging need of finding risk factors, as well as protective factors related do AD. By the other hand, it is also necessary molecular studies, which could provide precious information about the mechanisms which lead to the disease development. In the present work, it was made a study about transcriptional gene expression by cDNA microarray, as well as Real Time PCR analysis in a series of genes involved in oxidative DNA damage response (sensing and damage repair), and others associated with the disease. In addition, it were also evaluated DNA strand breaks induced by oxidized bases in lymphocytes from Alzheimers patients (moderate level) and healthy individuals, by oxidized bases (8- oxoguanine) detection methods. Among the twenty genes tested by the quantitative Real Time PCR assay, only APOE was induced, as the remaining 19 (ADAM17, APEX1, APP, BACE1, OGG1 ATM, ATR, TREX1, FEN1, FANCG, RAD17, DUSP, ERCC1, ERCC3, ERCC6, HUS1, RAD9, RAD1, PRKDC) were found repressed. This observed inhibition in most of genes studied shows that many cell signaling pathways associated to oxidative stress response, including DNA repair pathways, may be also involved in the AD pathology. Additionally, the gene expression analysis by cDNA microarrays showed transcriptional alterations in 41 genes (q < 0.06) (among them, NOTCH1, MARK3, PAK and SMC1L1), but for most of them, there are no reports in the literature about their possible relationship with AD, what brought us new important information. Together, all the data obtained in the preset study provide a relevant contribution, which, in the future, may help on new therapeutic designs.

Dentre as várias hipóteses envolvendo a etiologia da doença de Alzheimer (DA), estudos sobre o envolvimento dos elementos traço têm recebido considerável atenção. O objetivo do presente trabalho foi comparar as concentrações de Br, Fe, K, Na, Rb, Se e Zn no encéfalo de idosos com diferentes estados cognitivos bem como entre regiões do encéfalo de casos post mortem, provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral. O desenvolvimento da presente pesquisa foi aprovado pelo Comitê de Ética local. Os casos foram divididos em três grupos levando-se em conta tanto a análise clínica quanto a neuropatológica: (1) grupo DA: indivíduos com sinais clínicos e neuropatológicos de DA (n=13), (2) grupo NN: indivíduos sem sinais clínicos e neuropatológicos de DA (n=11) e (3) grupo RC: indivíduos cognitivamente normais, porém com sinais neuropatológicos característicos de DA (n=4). A análise neuropatológica foi realizada com base em critérios bem aceitos, usando imunoistoquímica. O diagnóstico de demência foi realizado por meio de entrevista com um informante. As concentrações dos elementos foram determinadas no hipocampo e no giro frontal médio utilizando-se o método de análise por ativação com nêutrons. Materiais de referência certificados foram analisados a fim de verificar a qualidade dos resultados analíticos. A análise estatística dos resultados obtidos nas análises do tecido encefálico foi realizada utilizando-se os aplicativos Minitab v15 e SPSS v11. As variáveis foram comparadas entre os grupos utilizando-se o método de análise de variância com medidas repetidas. A análise dos resultados obtidos nos materiais de referência mostrou que a análise por ativação com nêutrons é um método preciso e exato para análise de encéfalos humanos. Os resultados das análises do encéfalo mostraram que há uma diminuição das concentrações de Rb no encéfalo de portadores de DA quando comparadas com as de indivíduos dos grupos NN e RC. Além disso, há uma redução das concentrações de K e uma elevação das concentrações de Na no encéfalo dos indivíduos portadores de DA quando comparados com as de indivíduos NN. Uma elevação nas concentrações de Fe e de Se também foi verificada no grupo DA quando comparado com as do grupo NN. A comparação entre as concentrações dos elementos na região frontal e no hipocampo mostrou que há diferença nas concentrações entre estas duas regiões para vários elementos. As altas concentrações de Rb nos indivíduos RC quando comparadas com as do grupo DA e sua redução no grupo DA quando comparadas com as do grupo NN sugerem um papel do Rb nos mecanismos contra DA. Entretanto, as variações nas concentrações de Rb, assim como as de Na e K podem ser secundárias ao processo patológico. As altas concentrações de Fe nos encéfalos do grupo DA sugerem que este elemento pode ser um fator tóxico na DA e as altas concentrações de Se podem atuar no mecanismo de proteção. No entanto, cabe ainda verificar se a deposição dos elementos é um evento primário ou secundário à doença. As diferenças encontradas entre as concentrações de elementos nas diferentes regiões do encéfalo salientam a importância de realizar-se uma amostragem apropriada para determinação da composição do tecido cerebral / Among the many hypotheses concerning the etiology of Alzheimers disease (AD), studies regarding the involvement of trace elements have gained considerable attention. This study aimed to compare Br, Fe, K, Na, Rb, Se and Zn concentrations in brain of elderly people in different cognitive status and between brain regions in postmortem brains belonging to the Brazilian Aging Brain Study Groups Brain Bank. Protocols were approved by the responsible ethics committee. The cases were divided into three groups, according to clinical and neuropathological evaluations: (1) AD group: individuals with clinical symptoms and neuropathological hallmarks of AD (n=13), (2) NN group: individuals without clinical symptoms and neuropathological hallmarks of AD (n=11) and (3) RC group: individuals cognitively normal but with neuropathological hallmarks of AD (n=4). Neuropathological examinations were carried out based on accepted criteria, using immunohistochemistry. Diagnosis of dementia was established through a postmortem interview with an informant. Element concentration in the hippocampus and medium frontal cortex was determined using instrumental neutron activation analysis. Certified reference materials were analyzed to assure the quality of the analytical results. Statistical analysis of the results obtained in the brain tissue was performed using the Minitab v15 and SPSS v11 package. Variables were compared among groups using repeated measurement analyses of the variances. Reference material analysis results demonstrated that neutron activation analysis is a precise and exact method for element concentration analysis of the human brain. The results of the brain analysis demonstrated a decrease of Rb concentration in the brains of AD individuals compared to the NN and RC individuals. Furthermore, there is a decrease of K concentration and an increase of Na concentration in the brains of AD individuals compared to the NN individuals. An increase of Fe and Se was also verified in the AD group when compared to the NN group. Comparison of the hippocampus and frontal area concentration demonstrated a difference in the concentration of these two areas for many elements. The elevated concentration of Rb in the RC group when compared to the DA groups and the reduction in the DA groups when compared to the NN group suggest that Rb has a role in the protection mechanism against AD. However, changes in the Rb concentrations, as well as changes in the Na e K concentrations could be secondary to the pathological process. The elevated concentration of Fe could be a toxic factor in AD and the elevated concentration of Se could be a protection mechanism. Whether element deposition in AD is a primary or secondary event remains to be determined. As a result of the differences found in the concentrations of elements highlight the importance of having ideal sampling to determine element composition of brain tissue

Bakgrund: Alzheimers sjukdom (AD) utgör vanligaste formen av demenssjukdomar i Sverige. Alzheimers sjukdom klassas som en neurodegenerativ sjukdom och karaktäriseras av proteininlagringar samt av hjärnatrofi. Ungefär 150 000 personer i Sverige lever idag med demenssjukdomar, varav 90 000 lider av AD och varje år insjuknar ytterligare 20 000 i demens. Vitamin B12 och folat anses utgöra en viktig del med avseende på hjärnans kognitiva funktioner. Låga nivåer av vitamin B12 och/eller folatbrist medför en höjning av Homocystein (Hcy) i blodet. Syfte: Syftet med examensarbetet var att undersöka det eventuella sambandet mellan nivåerna av vitamin B12, folat respektive Hcy och risken för att drabbas av Alzheimers sjukdom. Utifrån syftet ställdes 2 frågeställningar upp. Frågeställning 1: Finns det en risk att utveckla AD vid förändrade nivåer av folat, vitamin B12 och Hcy. Frågeställning 2: Finns det ett samband mellan förändrade nivåer av folat, vitamin B12 och Hcy vid diagnosticerad AD. Metod: Detta arbete är en litteraturstudie baserat på 7 vetenskapliga artiklar. Resultat: Samtliga fem studier utifrån frågeställning 2 visade att AD-patienter hade höga nivåer av Hcy än kontrollpatienterna. Två av dessa studier visade även att AD-patienterna hade lägre nivåer av både vitamin B12 och folat än kontrollindividerna. Två ytterligare studier av de fem samtliga visade också lägre nivåer av folat än kontrollgruppen.  Totalt 4 av 5 studier visade lägre nivåer av folat. Utifrån frågeställning 1 visade studie 1 att det var enbart de låga nivåerna av folat från baslinjen som förespråkade till insjuknande i AD. Studie 2 indikerade att individer med låga nivåer av vitamin B12 och folat har 2 gånger högre risk för att insjukna i AD. Slutsats: Det är fortfarande oklart idag huruvida nivåerna av vitamin B12, folat och Hcy påverkar riskerna för att utveckla AD. Studierna visar att det finns ett samband mellan markörerna kopplat till AD. Det krävs mer forskning för att fastställa det eventuella sambandet mellan vitamin B12, folat och Hcy. Fler patienter måste undersökas och flera studier bör utföras för att kunna fastställa associationerna av dessa ämnen med AD. Studierna visar att det finns ett samband mellan markörerna kopplat till AD. Fler studier som tar hänsyn till inklusionskriterierna med avseende på nutritionsstatus bör göras.  Detta eftersom lågt BMI (Body Mass Index) kan vara av betydelse för uppkomsten samt riskerna för att insjukna i AD. / Background: Alzheimer's disease (AD) is the most common form of dementia in Sweden. Alzheimer's disease is classified as a neurodegenerative disease and is characterized by protein deposits in certain parts of the brain accompanied with brain atrophy. 150 000 people in Sweden today suffer from dementia, of whom 90 000 suffer from AD and every year another 20 000 dementia cases reside. The highest risk factor of developing AD is old age. After the age of 65, the risk is considerably higher for developing AD. Vitamin B12 and folate is considered to be an important part with respect to the brain's cognitive functions. Folate acts as a donor of the methyl groups in a reaction catalysed by methionine synthase, which produce methyl cobalamin which needs to be metabolised to methionine from Homocystein (Hcy). Low levels of vitamin B12 and/or folate deficiency leads to an increase of Hcy in the blood. Objective: The aim of this study was to investigate the possible link between low levels of vitamin B12, folate and Hcy and the risk of developing Alzheimer's disease. Two question based on the objective of the study were set. Question 1: Is there any risk of developing AD with altered levels of folate, vitamin B12 and Hcy? Question 2: Is there a relationship between altered levels of folate, vitamin B12 and Hcy in diagnosed AD? Methods: This work is a literature study based on 7 scientific articles. Results: All five studies from question 2 showed that AD patients had high levels of Hcy compared to control patients. Two of these studies also revealed that AD patients had lower levels of both vitamin B12 and folate than control individuals. Furthermore 2 out of the 5 studies also displayed lower levels of folate compared to the control group, resulting in a total of 4 out of 5 studies showing lower levels of folate. Based on question 1, study 1 presented that only low levels of folate at baseline correlated with developing AD. Study 2 indicated that individuals with low levels of vitamin B12 and folate had twice times higher risk of developing AD. Conclusion: It is still unclear whether the levels of vitamin B12, folate and Hcy affect AD. The studies investigated show that there is a correlation between the investigated markers linked to AD. However, more research is needed to be able to determine the possible link between vitamin B12, folate and Hcy. More patients must be investigated and several studies should be performed to determine the associations of these subjects with AD.  More studies that take in consideration of nutritional status must be carried out, due to the fact that low BMI (Body Mass Index) may be of importance for the risk of developing AD.

There is currently no definitive biomarker for Alzheimer's Disease (AD), confirmation of diagnosis is only possible post-mortem. Magnetic resonance spectroscopy (MRS) has potential in aiding diagnosis, an MRS scan can be performed during an MRI scan, only adding around 10 minutes to scan time. Use of data from the two scans may allow more accurate diagnosis of AD. This thesis investigates a transgenic mouse model of AD, the TASTPM mouse, using in vitro and in vivo MRS as well as matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI MS Imaging). The first aim of the study was to search for a biomarker of AD that may allow better diagnosis or further our understanding of the pathology of the disease. The second aim was to evaluate the TASTPM mouse as a model of AD for use in preclinical testing of amyloid lowering agents. The third aim was to investigate a thalamic pathology in the TASTPM mice using MALDI MS Imaging. Metabolically, we found differences between the brains of TASTPM mice and their wild type base strain in both in vitro and in vivo scans. These differences may be exploited in the preclinical testing of novel amyloid lowering therapies. We also found similarities with human AD and other mouse models, lower N-acetylaspartate, lower glutamate and higher myo-inositol are all observed in human AD, as well as the TASTPM mice in vivo. We also found further evidence of impaired neuronal energy metabolism in TASTPM mice, such as lower succinate. Cerebral hypometabolism is a symptom of human AD. The TASTPM mouse seems to be a fairly good approximation of the human disease, sharing several traits. In our investigation of the thalamic pathology, we discovered a peptide which was strongly localised to the regions of the pathology and isolated it, but were unable to identify it, the work in this area will continue.

No description available.

Aging and kinase kinetics of Y77E11.A and Y17G7B.10 in C. Elegans Jacob Goodlaxson, Department of Biomedical Sciences, College of Medicine, East Tennessee State University, Johnson City, TN. The free radical theory of aging suggests that free radical induced oxidative damage may play a role in the pathogenesis of age-related neurological diseases. The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) protects against redox stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin. A measurement of the kinase kinetics of nicotinamide adenine dinucleotide demonstrated a decline in the rate of conversion from NAD into NADP. A homeostatic relationship of NADPH and NADP+ in mitochondria and cytosol may prevent the progression of aging due to the amelioration of the reactive oxygen species (ROS), and other charged particles. The NAPD+ cation is the chief acceptor of negatively charged particles which ionize and create free radical ions. Two previously uncharacterized proteins, Y77E11.A (NADK1) and Y17G7B.10 (NADK2), were studied for their possible kinetic role in producing and maintaining NADPH levels. The roles of NADK1 and NADK2 were determined by taking whole worm lysates of Caenorhabditis elegans deficient of these proteins, followed by supplementation with NAD, then monitoring of NADP levels. These two proteins were statistically important in the conversion of NAD to NADP+. These NADK’s displayed statistically significant reduction in NADP production which could lead to more redox stress. This research indicates that pro-longevity therapies should aim to maintain elevated levels of NADPH and NADP+ to hinder the aging process.

Alzheimer's disease is a widespread disease that has devastating effects on the human brain and mind. Ultimately, it leads to death and there are currently no treatment methods available that can stop the disease progression. The mechanisms involved behind the disease are not fully understood although it is known that amyloid fibrils play an important role in the disease development. These fibrils are able to form plaques that can trigger neuronal death, by interacting with receptors on the cell surface and the synaptic cleft or by entering the cell and disturb important functions such as metabolic pathways. To study the plaque formation of amyloid proteins, both in vitro and in vivo methods are used to investigate the characteristics of the protein. Luminescent conjugated oligothiophene probes are able to bind in to amyloid beta fibrils and emit light when excited by an external light source. This way fibrillation properties of the protein can be studied. Developing probes that can serve as biomarkers for detection of amyloid fibrils could change the way Alzheimer's is treated. Being able to detect the disease in its early disease course, and start treatments early, is suggested to stop the progression of neural breakdown. In this project a software is developed to analyze fluorescent microscopy images, taken on tissue stained with these probes. The software is able to filter out background noise and capture parts of the picture that are of interest when studying the amyloid plaques. This software generates results similar to if the images were to be analyzed using any software where the regions to analyze are selected manually, suggesting that the software developed produce reliable results unbiased by background noise.

No description available.

Linear mixed model (LMM) has the advantage of modeling the corelated data. Alzheimer’s disease (AD) is a chronic neurogenerative disease that affects the brain of the subject. No study was found to study the longitudinal effect of apolipoprotein E epsilon 4 (APOE4) genotype on the logical memory test total score in AD. A longitudinal data of 844 with AD, 2167 with cognitive normal (CN), and 4472 with mild cognitive impairment (MCI) participants who underwent logical memory examination test in the Alzheimer's Disease Neuroimaging Initiative (ADNI) were investigated. Episodic memory of the study participants was monitored based on a short story told to the participants and then participants asked to recall what was told. The multivariate LMM was used to determine the longitudinal changes in the logical memory test total score adjusting for age and sex. The Akaike information criterion (AIC) statistic and the Bayesian information criterion (BIC) statistic were used to select the best covariance structure. The repeated measures longitudinal analysis was performed using PROC MIXED in SAS 9.4. Both AIC and BIC statistics favor the unstructured correlated structure (UN). Using a UN model in the LMM, the APOE gene was is significantly associated with logical memory test total score (pUN covariance structure is the best. This study provided the first evidence of the effect of APOE4 genotype on the logical memory related to AD.

No description available.