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Caracterização dos efeitos tóxicos do 1,2-dihidroxibenzeno em células do sistema nervoso central: investigação do efeito protetor de derivados de plantasGóes, Lizandra Moreira January 2013 (has links)
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Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / Catecóis são derivados do benzeno, podendo apresentar citotoxicidade, que pode constituir um modelo experimental útil para o desenvolvimento de novos fármacos. No bioma brasileiro inúmeras plantas produzem metabólitos com atividades diversas, como antioxidantes, ou inibidores do crescimento celular. No Brasil, as neoplasias são a segunda causa de óbito, especialmente aquelas derivadas do sistema nervoso, aumentando o interesse por novos antineoplásicos e agentes neuroprotetores. Este trabalho caracteriza efeitos citotóxicos do 1,2-dihidroxibenzeno (CAT) e discretamina (DSC) em células do sistema nervoso in vitro. Determinou-se a EC50 de CAT e DSC usando brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium (MTT), investigou-se sua auto-oxidação por espectrofotometria, avaliou-se mudanças morfológicas e condensação/fragmentação nuclear por microscopia. Avaliou-se a proteção de DSC e 8-metoxipsoraleno (8-MOP) contra a citotoxicidade do CAT. O padrão de morte celular foi analisado por citometria de fluxo. A espoliação de glutation reduzido (GSH) foi analisada usando monoclorobimano. A toxicidade do CAT para células SH-SY5Y e C6 depende da dose e associa-se à formação de quinonas. Houve mudanças morfológicas, condensação/fragmentação da cromatina e morte apoptótica, não relacionada à espoliação de GSH. DSC não foi tóxica para células SH-SY5Y, porém protegeu contra os efeitos do CAT em baixas concentrações. DSC mostrou-se citotóxica para células de glioma (GL-15 e C6) e potencializou o CAT. Pré-tratamento por 30 minutos com DSC protegeu contra a ação do CAT após 72 horas. 8-MOP potencializou os efeitos do CAT, não revertendo seus efeitos na viabilidade celular, morfologia celular, condensação/fragmentação nuclear, e espoliação de GSH. Esses resultados caracterizam um modelo de citotoxicidade que pode ser aplicado no desenvolvimento de novos agentes farmacológicos. Estudos complementares são necessários para elucidar a proteção da DSC. / Catechols are benzene derivatives, which may exhibit cytotoxic activity that can be employed to develop new drugs. Plants are important sources of metabolites with pharmacological activities such as antioxidants, or cell growth inhibitors. In Brazil, cancer is the second leading cause of death, especially those derived from the nervous system, which increase the interest for new antineoplastic and neuroprotective drugs. The cytotoxic effects promoted by 1,2-dihydroxybenzene (CAT) and discretamine (DSC) in nervous system cells were characterized in vitro. The protective effects of DSC and 8-methoxypsoralen (8-MOP) against CAT-induced cytotoxicity were also evaluated. CAT and DSC EC50 was determined by using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT). CAT auto-oxidation was investigated by spectrophotometry. Morphological changes and nuclear condensation/ fragmentation were evaluated by microscopy. The pattern of cell death was obtained by flow cytometry. Reduced glutathione (GSH) depletion was analyzed by using monochlorobimane. CAT induced a dose-dependent toxicity to SH-SY5Y and C6 cells, associated with reactive quinones formation. It also induced morphological changes, nuclear condensation/fragmentation, and apoptotic death not caused by GSH depletion. DSC was not toxic to SH-SY5Y cells, but protected against CAT effects at low concentrations. DSC was be cytotoxic to glioma cells (GL-15 and C6) and potentiated CAT effects. However, pretreatment for 30 minutes with DSC protected them against CAT after 72 hours. 8-MOP also potentiated CAT effects instead to protect cells. These results characterize an experimental model useful for studies searching new pharmacological agents. However, further studies are needed to elucidate the DSC protective effects.
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Avaliação comparativa do risco mutagênico dos poluentes provenientes dos combustíveis renováveis (álcool e biodiesel) e não renováveis (gasolina e diesel) através do bioensaio Trad-SH / Assessment and comparison of mutagenic risk of pollutants from combustion from renewable fuels (ethanol and biodiesel) and non-renewable (gasoline and diesel) by Trad-SH assayDeuzuita dos Santos Oliveira 22 October 2010 (has links)
As emissões veiculares constituem-se em uma das mais graves ameaças a qualidade de vida da população, principalmente nos grandes centros urbanos. Poluentes de origem veicular podem induzir alterações no material genético de organismos a eles expostos, pois nessas emissões, destacam-se entre outros, os hidrocarbonetos policíclicos aromáticos (HPAs), que são substâncias consideradas carcinogênicas e mutagênicas. A utilização de indicadores sensíveis a ação de agentes genotóxicos serve para avaliar a presença destes compostos no ambiente. Portanto, o objetivo deste trabalho foi avaliar comparativamente o risco mutagênico das emissões provenientes da combustão dos combustíveis fósseis (gasolina e diesel) e renováveis (álcool e biodiesel), utilizando o bioensaio Trad-SH. Os ensaios foram realizados utilizando veículos do ciclo Diesel (Citröen JUMPER 2.8 L, 2006) e do ciclo Otto (VW FOX 1.6 Flex, 2005 sem o conversor catalítico), em um dinamômetro de chassi, submetidos a um ciclo de condução urbano padronizado (FTP-75) modificado, para a coleta dos gases de escapamento. Os gases foram misturados com o ar ambiente e homogeneizados para simular o que ocorre no trânsito urbano. As inflorescências do clone KU-20 de Tradescantia foram expostas a mistura de poluentes, provenientes dos veículos, dentro de uma câmara de fumigação, por duas horas. Para a avaliação do efeito mutagênico foi feita uma comparação entre as inflorescências não expostas aos poluentes (grupo 1) e as inflorescências expostas as emissões do álcool (grupo 2), do biodiesel (grupo 3), da gasolina (grupo 4) e do diesel (grupo 5). Os dados obtidos dos experimentos foram analisados estatisticamente onde se observou que a freqüência média das mutações no grupo 1 (controle), foi significativamente mais baixa do que aquela dos grupos 2, 3, 4, e 5. Os resultados indicam que as emissões dos veículos abastecidos com combustíveis fósseis (gasolina e diesel), são mais mutagênicas do que as emissões provenientes da combustão dos combustíveis renováveis (álcool e biodiesel). / The vehicle emissions constitute a major serious threat to the population\'s quality of life, especially in the large urban areas. Pollutants from vehicle origin may lead to changes concerning the genetic material of some organisms that have been exposed to them, and within these emissions it is stood out among others the polycyclic aromatic hydrocarbons (PAH\'s), which are carcinogenic and mutagenic compounds. The use of sensitive indicators to the action of genotoxic agents is useful to prevent and to evaluate the presence of these compounds in the environment. Therefore, this work is aimed at evaluating comparatively the mutagenic risk of emissions from the burning of fossil fuel (gasoline and diesel) and renewable ones (ethanol and biodiesel) by the use of Trad-SH bioassay. The assays were carried out using vehicles from the diesel cycle (Citröen JUMPER 2.8 L, 2006) and the Otto cycle (VW FOX 1.6 Flex, 2005, without a catalytic converter), in a chassis dynamometer, submitled to a standard urban driving cycle (FTP-75) in order to collect the gases of exhaust. The gases were mixed up to the atmosphere air and homogenized to simulate what happens in urban traffic. The inflorescences of KU-20 clone of Tradescantia were exposed to a mixture of pollutants from vehicles, in a fumigation chamber for two hours. To evaluate the mutagenic effect it was necessary to establish a comparison between the inflorescences which were not exposed to pollutants (group 1) and inflorescences exposed to emissions of alcohol (group 2), biodiesel (group 3), gasoline (group 4) and diesel (group 5). Data obtained from experiments were statistically analyzed in which appears that the average frequency of mutations in group 1 (control) was significantly lower than that of groups 2, 3, 4 and 5. The results indicate that emissions from vehicles fueled by fossil fuels (petrol and diesel) are more mutagenic than emissions from the combustion of renewable fuels (ethanol and biodiesel).
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Avaliação do potencial de risco mutagênico dos poluentes presentes na exaustão de motor diesel por meio do bioensaio Trad-SH / Evaluation of the mutagenic potential of air pollutants from diesel engines emission using the Tradescantia Stamen Hair assay (TSH)Deuzuita dos Santos Oliveira 04 April 2005 (has links)
A poluição atmosférica já é considerada um caso de saúde pública nos grandes centros urbanos. Freqüentemente as emissões veiculares são as principais causas dessa poluição, principalmente as provenientes dos motores diesel, os quais, além da produção de material particulado (MP), em cuja superfície são adsorvidas substâncias carcinogênicas e mutagênicas, produzem poluentes como os hidrocarbonetos policíclicos aromáticos (HPAs). Contudo não existe uma avaliação direta dos riscos dessas emissões nos seres vivos. Plantas bioindicadoras podem dar idéia desses riscos. Este trabalho teve como objetivo avaliar o potencial de risco mutagênico da exaustão proveniente de um motor diesel, utilizando o bioensaio Trad-SH (clone KU-20) como bioindicador da poluição do ar. Nos experimentos, a exaustão do motor diesel aspirado de 2.0 L de deslocamento volumétrico foi diluído com ar atmosférico de modo a atingir concentrações de uma atmosfera pesadamente poluída (aproximadamente 50, 100 e 150 ppm de CO). Obtidos estes níveis de diluição, as inflorescências foram expostas a esta mistura de poluentes por duas horas (doses agudas). A concentração de CO foi monitorada continuamente por meio de um analisador de gases Horiba Enda utilizando o princípio de absorção seletiva no infravermelho. Para se avaliar o efeito mutagênico foi feita uma comparação entre as inflorescências não expostas aos poluentes (grupo 1) e as inflorescências expostas (grupos 2, 3 e 4) com aproximadamente 50, 100 e 150 ppm de CO respectivamente. Os dados obtidos foram analisados estatisticamente observando-se que, a freqüência média das mutações no grupo 1 (controle), foi significativamente mais baixa do que aquela dos grupos 3 e 4, porém foi similar à do grupo 2. Por sua vez, não houve diferenças significativas nas freqüências de mutações entre os grupos 3 e 4. Os resultados indicam que a exaustão do motor a diesel teve um papel significativo no desenvolvimento de mutações, mas somente quando diluída para concentrações de CO acima de 100 ppm. / Air pollution caused by vehicle emission has been considered as a major public health concern in the urban centers. Emission from diesel engine powered vehicles, in particular, are highly toxic, since carcinogenic and mutagenic compounds can adsorb on the expelled particles leading to the formation of the so-called polycyclic aromatic hydrocarbons (PAH\'s). A detailed investigation on the risks of those compounds on the living beings using the methodology applied in the work has not been carried out so far. This work is aimed at evaluating the mutagenic potential of the emission from a diesel engine using the Tradescantia stamen hair assay (TSH), by monitoring the stamen hair mutation in the clone KU-20. Experimentally, the inflorescence of the KU-20 clones was kept for 2 h under a simulated urban heavily polluted atmosphere, obtained by mixing the emission from a diesel engine and atmospheric air. The CO concentration in the atmosphere was monitored using a Horiba-Enda gas analyzer. The mutagenic effects of the atmosphere were analyzed by comparing a group of non-exposed control inflorescence (group 1) to inflorescences kept under polluted atmospheres containing 50, 100 and 150 ppm of CO, assigned to as groups 2, 3 and 4. The experimental data were analyzed using statistical methods. The frequency of mutations observed in the inflorescences from group 2 was slightly higher than that observed in the group 1. In the groups 3 and 4, however, the frequency of mutations was significantly higher than that exhibited by the control group. The latter suggests that the emission from a diesel engine plays an important role in the development of plants mutation, specially for atmospheres containing more than 100 ppm of CO.
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Avaliação dos efeitos de acetato em células de neuroblastoma SH-SY5Y e células-tronco humanas de dente decíduo esfoliado cultivadas na presença de glutamatoGraça, Júlio César Gomes 11 August 2017 (has links)
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Previous issue date: 2017-08-11 / O glutamato, aminoácido não-essencial, é um neurotransmissor excitatório do sistema nervoso central (SNC), sendo liberado durante o impulso nervoso. Em situações de patologia cerebral, o acúmulo de glutamato ocorre no espaço extracelular, causando dano neuronal e, eventualmente, apoptose. Muitos trabalhos relataram que a citotoxicidade do glutamato está associada a várias doenças neurológicas. Neste contexto, o acetato, um ácido graxo de cadeia curta, pode beneficiar o SNC de forma energética e estrutural. A acetil-coenzima A, forma metabolicamente ativa de acetato, é utilizada como substrato em vias bioquímicas envolvidas no metabolismo de carboidratos, lipídios e proteínas, além de aumentar a acetilação das histonas, alterando a expressão de genes inflamatórios. A linhagem celular de neuroblastoma humano SH-SY5Y é comumente usada em estudos relacionados a doenças neurodegenerativas, com uma capacidade de expansão em larga escala antes da diferenciação, enquanto que a linhagem de células-tronco de polpa de dente de leite decíduo esfoliado (SHED) é comumente utilizada em modelos de estudo do comportamento celular. O objetivo desta pesquisa foi avaliar os efeitos do acetato mediante a citotoxicidade causada pelo glutamato em células SH-SY5Y e SHED. Células SH-SY5Y e SHED foram cultivadas, respectivamente, em meio DMEM/F12 suplementado com 10% (v/v) de soro fetal bovino (SFB) e 1% (v/v) de penicilina-estreptomicina, e meio alfa-MEM, suplementado com 10% (v/v) de SFB, 1% (v/v), 1% (v/v) de penicilina-estreptomicina, 0,01 mM de aminoácidos não essenciais e 2 mM L-glutamina. A viabilidade celular em diferentes concentrações de acetato (5 a 75 mM) e glutamato (25 a 150mM) foi medida pelo ensaio MTT. A diferenciação foi realizada em SH-SY5Y pela suplementação do meio com 10 μM de ácido retinóico (AR) e redução de SFB para 1% (v/v) durante 4, 7 e 10 dias em cultura, e em SHED pela substituição do meio de cultivo por DMEM Low-Glicose, suplementado com 10% (v/v) de SFB, 1% (v/v) de penicilina-estreptomicina, 10-7 M de dexametasona, 50 μM de 2-fosfato ácido ascórbico e 2 mM de β-glicerolfosfato, a fim de verificar como essas células respondem à mistura de acetato/glutamato. A análise estatística foi realizada teste ANOVA de uma ou duas vias, bem como pelo teste de Kruskal-Wallis, quando apropriado, com p < 0,05 considerado estatisticamente significativo. Após 7 dias de incubação, as concentrações de 5 e 25 mM de acetato apresentaram menor influência sobre a viabilidade de células SH-SY5Y e SHED, enquanto que o IC50% de glutamato ficou em torno de 75mM e 50mM para estas linhagens, respectivamente. Ao submeter as células ao tratamento combinado de acetato e glutamato, observou-se que o acetato não exerceu citoproteção mediante exposição celular ao glutamato. Após análise qualitativa da diferenciação osteogênica em SHEDs, foi observado maior mineralização nas células tratadas com AR e acetato, em comparação com as células controle. Estudos subsequentes, que permitam identificar como tais células respondem ao acetato em nível molecular, considerando a expressão de ciclinas, compactação da cromatina e a presença de marcadores bioquímicos característicos durante a diferenciação de cada linhagem, por exemplo, poderão fornecer um entendimento mais completo de como esse composto atua na dinâmica metabólica e bioenergética celular. / Glutamate, a non-essential amino acid, is an excitatory neurotransmitter of the central nervous system (CNS), being released during the nerve impulse. In situations of cerebral pathology, the accumulation of glutamate occurs in the extracellular space, causing neuronal damage and, eventually, apoptosis. Many studies have reported that glutamate cytotoxicity is associated with various neurological diseases. In this context, acetate, a short chain fatty acid, can benefit the CNS energetically and structurally. Acetyl-coenzyme A, a metabolically active form of acetate, is used as a substrate in biochemical pathways involved in the metabolism of carbohydrates, lipids and proteins, in addition to increasing the acetylation of histones, altering the expression of inflammatory genes. The SH-SY5Y human neuroblastoma cell line is commonly used in studies related to neurodegenerative diseases, with a large scale expansion capacity prior to differentiation, whereas the stem cell line of exfoliated deciduous teeth (SHED) is commonly used in models of cellular behavior. The objective of this research was evaluate the effects of acetate by glutamate-induced cytotoxicity on SH-SY5Y and SHED cells. SH-SY5Y and SHED cells were cultured respectively in DMEM / F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin, and alpha-MEM medium, supplemented with 10% (v/v) FBS, 1% (v/v), 1% (v/v) penicillin-streptomycin, 0.01 mM non-essential amino acids and 2 mM L-glutamine. Cell viability at different concentrations of acetate (5 to 75 mM) and glutamate (25 to 150 mM) was measured by the MTT assay. Differentiation was performed on SH-SY5Y by supplementing the medium with 10 μM retinoic acid (RA) and reducing FBS to 1% (v/v) for 4, 7 and 10 days in culture, and in SHED by replacing the culture medium with DMEM Low-Glucose, supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, 10-7 M dexamethasone, 50 μM ascorbic acid 2-phosphate and 2 mM β- Glycerol phosphate in order to verify how these cells respond to the acetate/glutamate mixture. Statistical analysis was performed one-way or two-way ANOVA, as well as Kruskal-Wallis test, when appropriate, with p < 0.05 considered statistically significant. After 7 days of incubation, concentrations of 5 and 25 mM acetate had less influence on the viability of SH-SY5Y and SHED cells, whereas the IC50% of glutamate was around 75mM and 50mM for these lines, respectively. By subjecting the cells to the combined treatment of acetate and glutamate, it was observed that acetate did not exert cytoprotection through cellular exposure to glutamate. After qualitative analysis of the osteogenic differentiation in SHEDs, greater mineralization was observed in the cells treated with RA and acetate, in comparison with the control cells. Subsequent studies to identify how these cells respond to acetate at the molecular level, considering the expression of cyclins, chromatin compaction, and the presence of characteristic biochemical markers during differentiation of each lineage, for example, may provide a more complete understanding of how this component acts on the metabolic dynamics and cellular bioenergetics.
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O papel do aminoácido leucina na modulação da atividade do peptídeo beta amiloide em células SH-SY5Y / The role of leucine in the modulation of beta amyloid peptide activity in SH-SY5Y cellsFabio Medici Lorenzeti 04 December 2014 (has links)
Estudos demonstram que a indução do estresse oxidativo pelo peptídeo beta amiloide (A?) exerce um importante papel no desencadeamento da excitotoxicidade neuronal o que pode resultar no desenvolvimento de doenças neurodegenerativas. A formação do peptídeo A? se deve a alterações na proteína precursora de amiloide (APP) que é clivada para a formação do peptídeo A?. Por sua vez, os mecanismos de ação do A? no S.N.C. ocorrem através da sinalização do receptor NMDA (N-metil D-aspartato) receptor este que quando ativado pelo glutamato exerce importante papel fisiológico no S.N.C., visto que apresenta atividade ionotrópica que permite o influxo de Na+ e Ca2+ para as células neuronais, auxiliando nos processos de formação da memória e aprendizagem. Entretanto, apesar do seu papel fisiológico, a ativação excessiva do receptor NMDA é fortemente correlacionada com lesões no S.N.C. decorrente da excessiva permeabilidade do íon Ca2+ para o citosol das células neuronais. Com isso as concentrações de glutamato na fenda sináptica são estritamente controladas para que não haja ativação excessiva dos receptores com atividade glutamatérgica, como o receptor NMDA. Estudos indicam que o transporte de glutamina/glutamato através da barreira hematoencefálica é menor do que de outros aminoácidos, sendo que cerca de 25% a 30% do transporte de aminoácidos dos vasos sanguíneos para o cérebro através da barreira hematoencefálica é ocupado pelo aminoácido leucina, sendo este um grande responsável pela síntese de glutamato/glutamina no S.N.C. Com isso, estudos tem demonstrado que dietas enriquecidas com aminoácidos de cadeia ramificada, dentre eles a leucina, é responsável por alterar o metabolismo do glutamato e aumentar a susceptibilidade à excitotoxicidade de células neurais. A fim de testar esta hipótese utilizamos um modelo de cultura de células de neuroblastoma humano e realizamos o tratamento com diferentes concentrações de aminoácido leucina associado com o tratamento de peptídeo beta-amilóide. Realizamos as analises de citotoxicidade (LDH), viabilidade celular (MTT) e apoptose celular por citometria de fluxo (marcação com PE Anexina V e 7-AAD). Nossos resultados indicam que houve diferenças apenas entre o controle em relação aos demais grupos de tratamento / Studies demonstrate that induction of oxidative stress by beta amyloid peptide (A?) plays an important role in triggering neuronal excitotoxicity which can result in the development of neurodegenerative diseases. The formation of A? peptide are due to changes in the amyloid precursor protein (APP) which is cleaved to form the peptide A?. On the other hand, the mechanisms of action of A? in the C.N.S. occur through signaling of the NMDA (N-methyl-D-aspartate) receptor that when activated by glutamate plays an important physiological role in the C.N.S., as has inotropic activity that allows the influx of Na+ and Ca2+ into the neuronal cells, assisting in procedures of memory formation and learning. However, despite its physiological role, the excessive activation of the NMDA receptor is strongly correlated with C.N.S. lesions due to excess permeability of Ca2+ ions into the cytosol of neuronal cells. Thus the concentrations of glutamate in the synaptic cleft are strictly controlled so that there is excessive activation of receptors with glutamatergic activity, as the NMDA receptor. Studies indicate that the transport of glutamine/glutamate across the blood brain barrier is lower than that of other amino acids, of which about 25% to 30% of the amino acid transport blood vessels to the brain through the blood brain barrier is occupied by leucine this being one largely responsible for the synthesis of glutamate/glutamine in the C.N.S. Thus, studies have shown that diets enriched in branched chain amino acids, including leucine, are responsible for altering the metabolism of glutamate and excitotoxic increase susceptibility to neural cells. To test this hypothesis we used a cell culture model of human neuroblastoma and carry out the treatment with different concentrations of leucine associated with the processing of amyloid-beta peptide. We performed analysis of cytotoxicity (LDH), cell viability (MTT assay) and apoptosis using flow cytometry (Annexin V staining with PE and 7-AAD). Our results indicate that there were differences only between the control compared to the other treatment groups
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Evaluation par ondes guidées de niveaux d'adhésion dissymétriques dans des collages structuraux métal-matériau composite. : Modélisation et approches des cas de Kissing Bonds / Guided wave evaluation of asymmetric adhesion levels in metal-composite structural bondings. : Modeling and approaches of kissing bonds cases.Attar, Latifa 28 June 2019 (has links)
Cette thèse s'intéresse à l'évaluation de la qualité de l'adhésion dans des assemblages structuraux métal/adhésif/composite carbone-époxy, assemblages utilisés dans l'industrie automobile et aéronautique. La qualité du collage est évaluée par méthodes non destructives grâce à des ondes guidées ultrasonores de type Lamb et SH. Un nouveau modèle numérique par éléments finis a été développé au cours de ce travail : il s'agit d'un modèle rhéologique aux interphases qui permet de tracer les courbes de dispersion des modes guidés dans les échantillons étudiés. Il permet aussi de prédire l'évolution de l'amplitude de ces modes et la répartition de l'énergie dans la structure suivant le niveau d'adhésion ou la présence d'un défaut de type kissing bond. Des échantillons à niveau d'adhésion connus et maîtrisés sont réalisés avec l'aide de physico-chimistes, où l'époxy est partiellement ou totalement réticulé, et où l'interface substrat/adhésif a subi différents traitements chimiques. En particulier sont étudiés des échantillons dont la dégradation du collage dans la structure n'est pas symétrique. Les résultats expérimentaux sont confrontés à ceux issus du modèle numérique. Ces résultats montrent qu'il est possible de caractériser deux niveaux d'adhésion proches et aussi de déterminer sur quelle interface (métal/adhésif ou composite/adhésif) le collage a été dégradé. L'utilisation d'un banc de mesure à transducteurs électromagnétiques acoustiques (EMAT) a permis l'étude de l'amplitude des modes SH. Des variations importantes de l'amplitude de certains modes ont été constatées lors du passage de l'onde d'une zone de bonne adhésion à une zone où le collage est dégradé. / This thesis focuses on the evaluation of the quality of adhesion in metal/adhesive/carbon-epox: structural assemblies used in the automotive and aerospace industry. The quality of the bonding i assessed by non-destructive methods using ultrasonic guided waves of the Lamb and SH types. A nev finite element numerical model was developed during this work: it is an interphase rheological mode that allows the dispersion curves of the guided modes to be plotted in the samples studied. It als( makes it possible to predict the evolution of the amplitude of these modes and the distribution o energy in the structure according to the level of adhesion or the presence of a kissing bond type defect. Samples with known and controlled adhesion levels are made with the help of physico chemists, 'where the epoxy is partially or totally crosslinked, and where the substrate/adhesivl interface has undergone different chemical treatments. In particular, samples are studied whos degradation of the bonding in the structure is not symmetrical. The experimental results are compare( with those from the numerical model. These results show that it is possible to characterize two clos levels of adhesion and also to determine on which interface (metal/adhesive or composite/adhesive the bonding has been degraded. The use of a measurement bench with electromagnetic acousti( transducers (EMAT) allowed thè study of the amplitude of the SH modes. Significant variations in th( amplitude of some modes have been observed when the wave passes from an area of good adhesioi to an area where the bonding is degraded.
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Are nAChRs and NMDA receptors involved in low dose ethanol-nicotine toxicity in SH-SY5Y cells?Jonsson, Karl January 2013 (has links)
Consumption of alcohol and tobacco is common all around the world and these drugs are frequently consumed concomitantly. It has been estimated that 70-80 % of alcoholics are smokers and non-alcoholic drinkers are more often smokers than teetotallers. Alcohol and tobacco may affect the risk of developing neurological diseases and might influence this risk differently when combined compared to when only one of these compounds is consumed. Some in vitro-research have shown that non-toxic concentrations of ethanol and nicotine, in combination, can exert toxicity, and might do so in a synergistic way. In this work, investigations were made to see if the neuronal nicotinic acetylcholine receptors (nAChRs) and NMDA receptors are involved in this interactive behaviour between ethanol and nicotine. A human neuroblastoma SH-SY5Y cell line was treated with ethanol and nicotine at different concentrations and cell viability was measured through an MTT-assay. A significant reduction in cell viability was induced by chronic treatment with a low-dose combination of ethanol and nicotine. The cell viability reduction was completely inhibited by pretreatment with the non-specific nAChR antagonist mecamylamine. This suggests that nAChRs are involved in low-dose ethanol-nicotine interactions. The NMDA receptor antagonist memantine did not affect the ethanol-nicotine effect, which implies that NMDA receptors are not involved in low-dose ethanol-nicotine interactions in SH-SY5Y cells. However, it is unclear if the SH-SY5Y cell line expresses fully functional NMDA receptors. The expression of NMDA receptors might vary with cell passage number. Further research has to be done to uncover the contribution of specific nAChR subtypes to the ethanol-nicotine interaction. There also remains to be revealed if human neuroblastoma SH-SY5Y cells express fully functional NMDA receptors and how cell passage number affects the expression of these receptors.
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Vliv chronického působení morfinu na přežití buněk po působení oxidativního stresu u neuroblastomové linie SH-SY5Y buněk / Effect of chronic morphine on cell survival after oxidative stress in the SH-SY5Y neuroblastoma cell lineMoutelíková, Karolína January 2018 (has links)
Morphine is a natural opioid which is used in medicine due to his potent analgesic and sedative effects. In the forefront of scientific interest is a chronic usage of opioids which can lead to a development of drug addiction. Morphine role in oxidative stress was described in last years. It was revealed its protective potencial by many studies. However, some studies described its pro-oxidative effect. The aim of this study was to determinate effect of chronic morphine on cell survival after oxidative stress caused by H202 analog - tBHP in the SH-SY5Y neuroblastoma cell line. The results verified morphine protective effect against oxidative stress. The highest protective effect of morphine was achieved in a concetration of 10 µM. It was desribed that morphine can induce activation of mu-opioid (MOR) and Toll-like 4 (TLR4) receptors signalling pathway on molecular level. The aim of this thesis was to evaluate the role of MOR a TLR4 in protective effect of morphine against oxidative stress by two methods. Firstly, it was used tests of oxidative stress on cell viability. The obtained results demonstrated majority role of TLR4 and minory role of MOR. Afterwards, we assesed changes in the expression of MOR a TLR4 after chronic morphine by SDS-PAGE electrophoresis. Results of these experiments did not...
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Caracterização redox-ativa do ácido úsnico e seu efeito citotóxico em células SH-SY5YRabelo, Thallita Kelly 08 February 2013 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Usnic acid (UA) is the most common and abundant lichenic secondary metabolite with potential therapeutic application. Anti-inflammatory and antitumour properties have already been reported and UA-enriched extracts are widely used to treat several diseases in the folk medicine. On the other hand, a growing body of evidence has suggested that UA present pro-oxidant properties, which might induce cellular damage mediated by reactive species. Based on this data, first we performed in silico evaluation of UA interactions with genes/proteins and important compounds for cellular redox balance. Then, we assessed UA redox properties against different reactive species (RS) generated in vitro, and evaluated its action on SH-SY5Y neuronal-like cells subjected to hydrogen peroxide (H2O2) treatment, since no in vitro neurotoxicological data has been reported so far. Total reactive antioxidant potential index (TRAP) showed a significant antioxidant capacity of UA at 20 μg/mL; UA was also effective against hydroxyl radicals and reduced nitric oxid formation. However, in vitro lipoperoxidation was enhanced by UA, and cell viability was decreased along 24 hours of treatment, according to MTT assay (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and morphological analysis. Moreover, UA did not display protective effects against H2O2-induced cell death in any case. The DCFH- DA (2,7-dichlorfluorescein-diacetate) based assay indicated that UA enhanced basal reactive species production at 20 μg/mL for 1 hour and from 2 ng/mL to 20 μg/mL for 4 and 24 hours. In addition, UA appears to potentiate H2O2-induced reactive species production. Our results suggest that UA displays variable redox-active properties, acting either as antioxidant or pro-oxidant agent according to different system conditions and/or cellular environment. These pro-oxidant properties in SH-SY5Y might be responsible by potential neurotoxic effects of UA / O ácido úsnico (AU) é um dos mais comuns e abundantes metabólitos secundários liquênicos com potencial aplicação terapêutica. As propriedades anti-inflamatória e antitumoral já foram relatadas e extratos de liquens enriquecidos com ácido úsnico, são amplamente utilizados para tratar diversas doenças na medicina popular. Entretanto, um crescente número de estudos tem sugerido que o AU apresenta propriedades pró-oxidantes em sistemas biológicos, as quais podem induzir dano celular mediado por espécies reativas. Baseado nesses dados, primeiro foi realizado a avaliação in silico das interações do AU com genes / proteínas e compostos importantes para o equilíbrio celular redox. Além disso, analisamos as propriedades redox-ativa do AU contra diferentes espécies reativas (SR) geradas in vitro, e seu efeito em células neuronais SH-SY5Y na presença de peróxido de hidrogênio (H2O2), já que nenhum dado neurotoxicológico in vitro tem sido relatado até agora. O índice de potencial antioxidante reativo total (TRAP) mostrou uma capacidade antioxidante significativa do AU a 20 μg/mL; AU também foi eficaz contra os radicais hidroxila e reduziu a formação do óxido nítrico. No entanto, a lipoperoxidação in vitro foi induzida pelo AU, e a viabilidade celular foi diminuída ao longo de 24 horas de tratamento, de acordo com ensaio de MTT (brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio) e análise morfológica. Além disso, o AU não protegeu a célula contra a morte celular induzida pelo H2O2. O ensaio DCFH-DA (2 7 diacetato de diclorofluoresceína) mostrou que o tratamento de AU (20 μg/mL) por 1 hora e (2 ng/mL a 20 μg/mL) durante 4 e 24 horas, aumentou a produção basal de espécies reativas e quando as células foram co-tratadas com o H2O2, o AU parece potencializar o H2O2 induzindo a produção de espécies reativas. Nossos resultados sugerem que o AU exibe variáveis propriedades redox-ativas, atuando como um agente antioxidante e pró-oxidante, de acordo com as diferentes condições do sistema e / ou ambiente celular. Estas propriedades pró-oxidantes em células SH-SY5Y podem ser responsáveis por possíveis efeitos neurotóxicos do AU.
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Effects of ellagic acid in human neuroblastoma cellsFjæraa Alfredsson, Christina January 2013 (has links)
A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems. Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma, being an embryonal tumor, show loss of function of genes controlling differentiation and apoptosis. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments. A common model for human neuroblastoma is the SH-SY5Y cell line resembling immature neuroblasts that can be differentiated in vitro with several agents including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the vitamin A-derivative all-trans retinoic acid. Here, the effect of ellagic acid on proliferation, cell detachment and apoptosis in non-differentiated and in vitro-differentiated SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Apoptosis was partly mediated by the intrinsic pathway. 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid both induced morphological differentiation, while only the latter induced G0/G1-arrest. Single-cell analysis revealed that 12-O-tetradecanoylphorbol-13-acetate-treated cells continued cycling during neuritogenesis while these two read-outs were mutually exclusive in all-trans retinoic acid-treated cells. 12-O-tetradecanoylphorbol-13-acetate- and especially all-trans retinoic acid-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis. / <p>Artikel 4 ("Altered sensitivity...") ingick som manuskript i avhandlingen, nu publicerad.</p>
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