Le vieillissement croissant de la population mondiale augmente les risques d’incidence de maladies dégénératives comme la maladie d’Alzheimer (AD). L’AD représente la forme de démence la plus commune. C’est plus de 40 millions de personnes qui sont affectés à l’échelle mondiale, créant ainsi un lourd fardeau économique annuel de plusieurs centaines de milliards de dollars. L’AD est une maladie dégénérative du cerveau qui se traduit par une perte graduelle de la mémoire et des fonctions cognitives. Cette dernière se caractérise par trois symptômes pathophysiologiques intimement reliés qui sont : (1) un système cholinergique défectueux, (2) une accumulation excessive de plaques séniles toxiques composées de peptides β–amyloïdes et (3) une hyperphosphorylation de la protéine Tau qui résulte en des enchevêtrements neurofibrillaires. Il n’existe à ce jour aucune cure contre l’AD et les traitements actuellement retrouvés sur le marché n’ont qu’un effet transitoire. En revanche, il y a de plus en plus d’indices qui suggèrent que l’utilisation de certains facteurs de croissance comme l’IGF-2, le bFGF et les neurotrophines pourraient agir à titre de traitement. Parmi ces derniers, la protéine morphogénétique osseuse 9 (BMP-9) a montré un fort potentiel thérapeutique. Cette dernière est toutefois coûteuse à produire en plus d’être volumineuse, ce qui limite fortement son acheminement au cerveau en raison de la barrière hématoencéphalique. Dans cette optique, nous avons développé deux peptides de 23 acides aminés dérivés de l’épitope knuckle de la BMP-9, pBMP-9 et SpBMP-9, 300 fois moins chers à produire. Les travaux de ce projet de doctorat portent sur le développement d’un système de libération du SpBMP-9, le plus efficace des deux peptides, comme stratégie de traitement contre l’AD. Tout d’abord, il a été montré que le SpBMP-9 permettait d’induire la différenciation neuronale de cellules humaines SH-SY5Y de façon plus efficace que la protéine native par la présence d’une croissance plus importante des neurites et l’expression de marqueurs de différenciation neuronaux. De plus, le SpBMP-9 pouvait agir sur au moins deux symptômes de l’AD en orientant la différenciation vers le phénotype cholinergique et en inactivant la GSK3β, une Tau kinase. Ensuite, un système de libération sous forme de nanoparticules à base de chitosane et d’alginate a été développé, puis caractérisé. Ce système de libération a permis d’encapsuler efficacement le SpBMP-9. Le suivi et la modélisation des cinétiques de libération à l’aide d’un modèle mathématique prenant en considération la distribution de taille des particules a permis d’identifier que la libération était principalement diffusive, mais qu’il existait de fortes interactions entre le peptide et l’alginate. Les tests de viabilité ont montré que les nanoparticules n’étaient pas toxiques sur des cellules SH-SY5Y. Enfin, le SpBMP-9 libéré à partir des nanoparticules était toujours bioactif et permettait une différentiation neuronale. Finalement, une étude exploratoire a permis de mettre en évidence que le SpBMP-9 pouvait agir en synergie avec le bFGF et le NGF. La combinaison du SpBMP-9 avec le bFGF ou le NGF a montré la présence d’une différenciation neuronale accrue mise en évidence par la présence d’une croissance de neurites supérieure, par l’expression de plusieurs marqueurs de différenciation ainsi que par un niveau de calcium intracellulaire plus important. Pour conclure, ces travaux de recherche suggèrent que le SpBMP-9 possède un fort potentiel thérapeutique dans le contexte de l’AD. Une étude plus approfondie de ses mécanismes d’action est requise ainsi que la validation de son effet in vivo sur modèle animal.

Bipolar disorder (BD) is a chronic mood disorder affecting ~1-2% of the global population, characterized by cycling moods of mania and depression. The exact pathogenesis of BD is unknown; however, it has been established that endoplasmic reticulum (ER) stress plays an important role. It is known that BD patients have abnormal activity and expression of ER stress proteins in several brain regions. There exists a need for a definitive diagnostic test for the early detection of BD as it is often misdiagnosed for other conditions including unipolar depression and schizophrenia. Understanding the underlying mechanisms and therapeutic targets being used by BD treatments will be helpful in establishing a diagnostic test. The current gold standard for BD treatment includes Lithium (LiCl) prescription, along with other mood stabilizers such as Valproic acid (VPA) and antipsychotics such as Olanzapine. Current therapies only relieve symptoms and are unable to stop disease progression. Neurotrophic factors (NTFs) are naturally occurring proteins that are responsible for the maintenance, differentiation, and survival of neurons. Cerebral dopamine NTF (CDNF) and Mesencephalic astrocyte-derived NTF (MANF) belong to a novel class of NTFs specific to dopaminergic neurons. This study investigated the role of CDNF and MANF as therapeutic targets for bipolar disorder in SH-SY5Y cells and Sprague Dawley rats, as well as determining the endogenous mRNA levels of CDNF and MANF in BD patients. We demonstrated that common BD mood stabilizers – LiCl and VPA – significantly increased the mRNA expression of MANF and CDNF in vitro. Additionally, we also established that these mood stabilizers alter the NTFs expression in different rat brain regions including pre-frontal cortex (PFC) and cortex. These findings suggest that BD drug treatments potentially act via NTFs in order to relieve symptoms. Thus it highlights the importance of further investigating the interaction between neurotrophic factors and bipolar disorder. / Thesis / Master of Science (MSc)

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

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-07T19:19:35Z No. of bitstreams: 1 Lizandra Moreira Góes... Caracterização dos efeitos tóxicos....pdf: 1602592 bytes, checksum: 16b9153b36fb34585abb21b40bd4bdef (MD5) / Made available in DSpace on 2014-02-07T19:19:35Z (GMT). No. of bitstreams: 1 Lizandra Moreira Góes... Caracterização dos efeitos tóxicos....pdf: 1602592 bytes, checksum: 16b9153b36fb34585abb21b40bd4bdef (MD5) 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.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-09-28T12:03:28Z No. of bitstreams: 1 juliocesargomesgraca.pdf: 1848019 bytes, checksum: f98e75e5c5ca605913fabb43b02b8b5b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-09-28T14:14:08Z (GMT) No. of bitstreams: 1 juliocesargomesgraca.pdf: 1848019 bytes, checksum: f98e75e5c5ca605913fabb43b02b8b5b (MD5) / Made available in DSpace on 2017-09-28T14:14:08Z (GMT). No. of bitstreams: 1 juliocesargomesgraca.pdf: 1848019 bytes, checksum: f98e75e5c5ca605913fabb43b02b8b5b (MD5) 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.

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

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.

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...

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.

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>