Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-08T18:49:28Z No. of bitstreams: 1 Leandro Cattelan Souza.pdf: 1347752 bytes, checksum: 37af779076bce3e7cf38f50d1bf1dce6 (MD5) / Made available in DSpace on 2016-04-08T18:49:29Z (GMT). No. of bitstreams: 1 Leandro Cattelan Souza.pdf: 1347752 bytes, checksum: 37af779076bce3e7cf38f50d1bf1dce6 (MD5) Previous issue date: 2013-01-17 / A doença de Alzheimer (DA) é uma desordem crônico-degenerativa, caracterizada clinicamente por uma perda gradual e progressiva da memória. Há um substancial corpo de evidências demonstrando o papel central do peptídeo beta-amilóide (Aβ) na fisiopatologia da DA. A administração intracerebral desde peptídeo em roedores tem mostrado ser um modelo bastante útil para o estudo desta doença e intervenções neuroprotetoras. Portanto, o objetivo do presente trabalho foi investigar o efeito de um treinamento de natação (ST) sobre o comportamento cognitivo e marcadores de estresse oxidativo e neuroinflamação em um modelo de doença de Alzheimer induzido pelo peptídeo Aβ1-40 em camundongos Swiss Albinos. Os animais foram distribuídos aleatoriamente em 4 grupos: (1) sedentário/veículo; (2) sedentário/Aβ1-40; (3) exercício/veículo; (4) exercício/Aβ1-40. Os grupos Aβ receberam infusão intracerebroventricular (i.c.v.) do peptídeo Aβ1-40 (3μl/sítio; 400pmol/animal) e os grupos veículo infusão i.c.v. de veículo PBS (phosphate-buffered-saline; 3μl/sítio). Os grupos exercício foram submetidos a um ST com aumento progressivo de intensidade e duração por 8 semanas (5x/semana), e os grupos sedentários mantidos em inatividade física. Após 24 horas da última sessão de exercício, os camundongos receberam Aβ1-40 ou PBS (rota i.c.v.). Sete dias após as infusões i.c.v., os camundongos foram submetidos a testes comportamentais e, finalmente, à eutanásia. O hipocampo (HC) e o córtex pré-frontal (PFC) foram removidos para os ensaios bioquímicos. Nossos resultados demonstraram que o ST foi efetivo em atenuar as seguintes alterações causadas pelo peptídeo Aβ1-40: (1) prejuízos nas memórias de curto e longo prazo no teste de reconhecimento de objetos, sem alterar a atividade locomotora; (2) aumento dos níveis de espécies reativas (RS) e diminuição dos níveis de tióis não-protéicos (NPSH) no HC e PFC; (3) inibição da atividade da superóxido dismutase (SOD) e aumento da atividade da glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa-S-transferase (GST) no HC e PFC; (4) Aumento dos níveis de interleucina-1 beta (IL-1β) e fator de necrose tumor-alfa (TNF-α) e diminuição dos níveis de interleucina-10 (IL-10) no HC e PFC. Em conclusão, nós sugerimos que o exercício regular pode prevenir o declínio cognitivo, o estresse oxidativo e a neuroinflamação induzida pelo peptídeo Aβ1-40 em camundongos, suportando a hipótese de que o exercício pode ser utilizado como uma medida não-farmacológica para reduzir os sintomas da DA. / Alzheimer’s disease (AD) is a chronic degenerative disorder, clinically characterized by gradual and progressive decline in memory. There is a substantial body of evidence demonstrating the central role of beta-amyloid (Aβ) peptide in the pathophysiology of AD. The administration of this peptide into the brain of rodents has been shown to be a useful model for the study of this disease and neuroprotective interventions. Thus, the aim of this study was to investigate the effect of swimming training (ST) on cognitive behavioral and markers of oxidative stress and neuroinflammation in a mouse model of DA induced by Aβ1-40 peptide. The animals were randomly assigned into four groups: (1) sedentary/vehicle; (2) sedentary/Aβ1-40; (3) exercise/vehicle; and (4) exercise/Aβ1-40. Aβ1-40 groups received intracerebroventricular (i.c.v.) injection of Aβ1-40 (3μl/site; 400pmol/mouse) and vehicle groups received i.c.v. injection of PBS (phosphate-buffered-saline; 3μl/site). Exercise groups were submitted to swimming training (ST) with progressive increase in time and duration for 8 weeks (5x/week), and sedentary groups were kept in physical inactivity. After 24h of last bout of ST, mice received Aβ1-40 or vehicle (by i.c.v. route). Seven days after i.c.v. injections, mice were submitted to cognitive behavioural tests and, finally, submitted to euthanasia. The hippocampus (HC) and prefrontal cortex (PFC) were removed for biochemical assays. Our results demonstrated that ST was effective in attenuating the following impairments caused by Aβ1-40 peptide: (1) impairment on short-term and long-term memories in object recognition test, without altering the locomotor activity; (2) increased reactive species (RS) levels and decreased non-protein thiols (NPSH) levels in HC and PFC; (3) inhibition of the superoxide dismutase (SOD) activity and increase in glutathione peroxidase (GPx), glutathione reductase(GR) and glutathione S-transferase (GST) activities in HC and PFC; and (4) increased tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) levels and reduced and interleukin-10 (IL-10) levels in HC and PFC. In conclusion, we suggest that regular exercise may prevent cognitive decline, oxidative stress and neuroinflammation induced by Aβ1-40 peptide in mice, supporting the hypothesis that exercise can be used as a non-pharmacological tool to reduce the symptoms of DA.

<p>Alzheimer’s disease is a common and very costly disease in today’s society. The hallmarks of the disease are the formation of two proteinaggregates, amyloid plaques containing Aβ-peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The formation ofneurofibrillary tangles is thought to be promoted by amyloid formation and is why the cellular events surrounding the formation and interactionsof the Aβ-peptide is a prime target for Alzheimer’s research. In this thesis, the gene of the highly aggregation prone form of Aβ-peptide, the Aβ1-42, has been inserted in a Drosophila melanogaster to promote expression in the central nervous system through the use of the Gal4-UAS system.Gene expression analysis was done using a RNA purification kit, translating the RNA into cDNA using RT-PCR and the levels were analyzed usingquantitative real-time PCR. For protein expression analysis the immunological techniques of dot blot and western blot were used combined withan immunoprecipitation step using magnetic beads. A fibrillation experiment was also performed to look into the potential seeding effect onamyloid formation from the Aβ1-42 expressing Drosophila using fluorescence spectroscopy.The aim for this thesis was to look into expression of the Aβ1-42 gene and the impact of ageing on expression levels. Another aim was to try andseparate and detect soluble Aβ-peptide species from tissue homogenates of Drosophila.No amplification could be detected in the quantitative real-time PCR, most likely due to concentration issues of the reaction components. For thisreason gene expression could never be quantified nor could the effect of ageing and gene expression be looked into. Insoluble aggregates but nosoluble Aβ-peptide species could be detected or separated from the tissue of the Drosophila. No seeding effect on the amyloid formation could bestatistically determined by the fibrillation experiment, but interesting quenching effects on the total quantum yield of Aβ fibrils in the presence ofbrain homogenates were noted.</p>

Alzheimer’s disease is a common and very costly disease in today’s society. The hallmarks of the disease are the formation of two proteinaggregates, amyloid plaques containing Aβ-peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The formation ofneurofibrillary tangles is thought to be promoted by amyloid formation and is why the cellular events surrounding the formation and interactionsof the Aβ-peptide is a prime target for Alzheimer’s research. In this thesis, the gene of the highly aggregation prone form of Aβ-peptide, the Aβ1-42, has been inserted in a Drosophila melanogaster to promote expression in the central nervous system through the use of the Gal4-UAS system.Gene expression analysis was done using a RNA purification kit, translating the RNA into cDNA using RT-PCR and the levels were analyzed usingquantitative real-time PCR. For protein expression analysis the immunological techniques of dot blot and western blot were used combined withan immunoprecipitation step using magnetic beads. A fibrillation experiment was also performed to look into the potential seeding effect onamyloid formation from the Aβ1-42 expressing Drosophila using fluorescence spectroscopy.The aim for this thesis was to look into expression of the Aβ1-42 gene and the impact of ageing on expression levels. Another aim was to try andseparate and detect soluble Aβ-peptide species from tissue homogenates of Drosophila.No amplification could be detected in the quantitative real-time PCR, most likely due to concentration issues of the reaction components. For thisreason gene expression could never be quantified nor could the effect of ageing and gene expression be looked into. Insoluble aggregates but nosoluble Aβ-peptide species could be detected or separated from the tissue of the Drosophila. No seeding effect on the amyloid formation could bestatistically determined by the fibrillation experiment, but interesting quenching effects on the total quantum yield of Aβ fibrils in the presence ofbrain homogenates were noted.

No description available.

Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is amyloid β (Aβ) and tau. Previously, people have tried studying Aβ in vivo using green fluorescent protein fusion together with Aβ. However, this is difficult since the aggregation of Aβ will lead to loss of fluorescence. This study aimed to crystallize the fusion protein mNG-A β1-42 and to investigate its properties as a molecular fluorescent Aβ-amyloid specific probe. Dynamic light scattering (DLS) was used to confirm that the majority of the protein was not in the form of soluble aggregates. The DLS experiments were followed by several rounds of crystallization trials. Initial screening and the subsequent narrowing down of potential conditions where mNG-Aβ1-42 could form crystals. Several staining experiments were conducted as well, including staining brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. The DLS experiments showed that the protein used in the crystallization experiments mostly consisted of molecular particles of the same radius. However, there was clear evidence of some larger species present that could have been a potential problem for crystallization. Crystallization experiments suggested that PEG 8000 was the most promising precipitant amongst other conditions identified for crystallization of mNG-Aβ1-42. However, the study was ultimately unsuccessful in developing crystals of sufficient high quality for diffraction studies to commence. The staining experiments demonstrated that mNG-Aβ1-42 could bind both by itself and with another amyloid probe, Congo red, and with antibodies in brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. In conclusion, several characteristics of mNG-Aβ1-42 were revealed in this study.