Orientador: Lauro Tatsuo Kubota / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-11-01T13:48:03Z (GMT). No. of bitstreams: 1 Kisner_Alexandre_D.pdf: 7108475 bytes, checksum: c8b021ab9cbe477b1fe0b4f1abf6fd48 (MD5) Previous issue date: 2012 / Resumo: Transistores de efeito de campo (FETs) modificados com nanoeletrodos de Au representam uma excelente ferramenta para o estudo eletrofisiológico de células, uma vez que as características da dimensionalidade destes últimos são comparáveis às espécies celulares a serem detectadas e medidas múltiplas podem ser realizadas simultaneamente. Neste trabalho, foram fabricados transistores de efeito de campo com três diferentes tipos de superfície em suas regiões de porta, transistores com somente SiO2, SiO2 e Al2O3 anódica porosa, e ainda SiO2-Al2O3 contendo nanopartículas de Au dentro de seus poros. Os transistores foram caracterizados por microscopia eletrônica de varredura assim como por medidas elétricas convencionais. Estas últimas demonstraram que os processos de anodização e deposição de nanopartículas em sua superfície não comprometem as suas propriedades elétricas. Os transistores com portas nanoporosas foram modificados com torisinase e empregados como biossensores para a detecção de dopamina. Os resultados demonstraram que estes FETs podem detectar dopamina num alcance de concentração normalmente encontrado durante a liberação destas moléculas por células neurais. Transistores com nanopartículas de Au foram empregados na detecção de serotonina utilizando-se uma metodologia de interação eletrostática através de monocamadas auto-organizadas, o que é ainda pouco explorado com FETs. Esta permitiu a detecção de serotonina num alcance linear de 0,1 a 2 µmol L. Experimentos envolvendo a adesão celular e a detecção de prótons liberados por vesículas de células PC12 foram conduzidos, e demonstraram que os processos interfaciais entre as células e os transistores apresentam um dependência das propriedades capacitivas da superfície, e que a presença das nanopartículas pode aumentar a sensibilidade elétrica da porta dos transistores. Estes efeitos interfaciais influenciaram diretamente na razão sinal-ruído da leitura de sinais de exocitose e sugerem que o uso de transistores nanoestruturados representa uma ferramenta promissora para análise deste tipo de célula in vitro / Abstract: Field effect transistors modified with Au nanoparticles represent an excellent tool to electrophysiology analyzes. Because the dimensions of the devices are comparable to the size of the cells, multiple measurements can be performed simultaneously. In this work, field effect transistors were fabricated with three different kinds of surface in their gates, i.e. transistor with only SiO2, SiO2 and porous anodic Al2O3, and SiO2-Al2O3 with Au nanoparticles embedded into the pores of Al2O3. The characterization of the transistors was performed by electron microscopy analysis and conventional electrical characterization. The last one showed that the anodization process and the Au nanoparticles deposition on surface of the transistors did not affect the electrical properties of the devices. The transistors presenting gates with only SiO2-Al2O3 were modified with tyrosinase and employed as biosensors to detect dopamine. The results of these analysis showed that the devices can detect dopamine in a range of concentration usually found when these molecules are released from neuronal cells. Transistors with Au nanoparticles were also applied as biosensors to detect serotonin. In doing so, the surface of the nanoparticles were modified with self-assembled monolayers that were able to interact with serotonin through electrostatic interactions. Although this approach is scarcely exploited with transistors, it showed promising results. For instance, serotonin could be detect in a linear range of concentration from 0,1 to 2 µmol L. Experiments to analyze the cell adhesion on transistors and detect the release of protons from the extruded matrix of vesicles from PC12 cells were performed and demonstrated that the interfacial processes between cells and transistors were dependent on the capacitive properties of the surface. The presence of nanoparticles can enhance the electrical sensitivity of the gates from the devices. These interfacial effects presented a relationship with the signal to noise ratio of the exocytotic signals measured for vesicles release and suggested that the employment of nanostructurated transistors are promising tools to analyze these events from PC12 cells in vitro / Doutorado / Quimica Analitica / Doutor em Ciências

No description available.

miRNAs 29b and 181a down-regulate expression of the norepinephrine transporter in PC12 cells. M.X. Deng, G. A. Ordway and M.-Y. Zhu. Dept. of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The noradrenaline transporter (NET) is a noradrenergic marker and regulates neurotransmitter signaling by rapidly clearing released norepinephrine from synapses. Our previous studies demonstrated that rat NET mRNA and protein levels are regulated by chronic stress and by administration of corticosterone. Whether miRNAs are intermediaries in the regulation of NET expression remains to be elucidated. The present study was undertaken to determine possible regulatory effects of miRNAs on NET expression in PC12 cells, a cell model for noradrenergic neurons. Using computational target prediction, we identified several miRNAs potentially related to regulation of NET expression. Mimics of these miRNAs were transfected into PC12 cells. NET protein expression was assayed by Western blotting 48 hours after transfection. miR29b- and miR181a-transfected cells showed significantly reduced NET protein levels. To identify the exact target loci, the 3’-UTR of NET mRNA was amplified by PCR from PC12 genomic DNA and cloned downstream of the red firefly gene of the pmirGlo vector. The NET 3’-UTR-bearing pmirGlo and miR29b or miR181a were co-transfected into PC12 cells and luciferase signals were measured 48 hours after transfection. Consistent with Western blots, co-transfection of these miRNAs with rat NET3’-UTR-containing plasmids resulted in reduced levels of luciferase activity in PC12 cells. We conclude that miR29b and miR181a can function as negative regulators of NET translation in vitro. Further studies to determine whether these miRNAs contribute to the regulation of NET expression induced by antidepressants are under way.

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

Recent studies suggest that carbon monoxide (CO) is another gas molecule that has similar biological actions as nitric oxide (NO). The purpose of this study is to investigate the relationship between NO and CO in the survival of naïve rat pheochromocytoma PC12 cells. Western blot analysis revealed that all three isoforms of nitric oxide synthase (NOS) exhibited low expression and two isoforms of heme oxygenase (HO), especially HO-1, exhibited higher expression in PC12 cells under basal condition. Exposure of PC12 cells for 24 h to the NO scavenger, carboxy-2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxy-1-3-oxide (carboxy-PTIO, 2 £gmol) or HO inhibitor, zinc protoporphyrinIX (ZnPP, 25 nmol) resulted in a progressive reduction in mitochondria dehydrogenase activity reflected cell viability as determined by the WST-1 (4-[3-(4-lodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. On the other hand, incubation with NO donors, amino-3-morpholinyl- 1,2,3-oxadiazolium chloride (SIN-1, 1 £gmol) or S-Nitroso-N-acetyl- penicillamine (SNAP, 1 £gmol), or the CO precursor, hematin (500 nmol), resulted in an elevation in cell viability. The progressive reduction in cell viability induced by carboxy-PTIO (2 £gmol) or ZnPP (25 nmol) was significantly blunted by co-treatment with SIN-1 (1 £gmol). However, incubation with the NO precursor, L-arginine (L-Arg, 2 £gmol), or the selective inhibitors for nNOS, iNOS or eNOS, N£s-propyl-L-arginine (NPLA, 100 pmol), S-methylisothiourea (SMT, 10 nmol) or N5-1-Iminoethyl-L-ornithine dihydrochloride (L-NIO, 4 nmol) did not significantly alter cell viability. Co-treatment with carboxy-PTIO (2 £gmol) and L-Arg (2 £gmol) was also ineffective. These results suggest that NO or CO contributes to the survival of naïve PC12 cells.

­^ ¤å ºK ­n We investigated the role of heat shock proteins (HSPs), particularly HSP60, HSP70 or HSP90 in E. coli lipopolysaccharide (LPS)-induced naïve pheochrommocytoma cell (PC12) death. PC12 cells seeded at a density of 1x105 cells per poly-L-lysine-coated 3.5 cm diameter polystyrene dish were incubated with LPS (1 mg/ml; serotype O55:B5) for 3, 6, 12, or 24 hr. Cell viability was measured by trypan blue test, and expression of HSP60, HSP70, and HSP90 were detected by Western blot analysis. We found that the viability of PC12 cell decreased significantly after treatment with LPS for 12 hr, and viability was only 30% at 24 hr post-treatment. Western blot analysis revealed that LPS-induced PC12 cell death was associated with an increase in HSP70 or HSP60. HSP70 was markedly up-regulation at 12 hr; and both HSP70 and HSP60 increased significantly by over 1000% and 200%, respectively, 24 hr after administration of LPS. There was no significant change in HSP90 level 3, 6, 12, or 24 hr after LPS treatment. To further investigate the role of HSP70, 60, or HSP90 in LPS-induced PC12 cell death, we treated PC12 cells with hsps antisense oligonucleotide (AODN) for 24 hr. The effects of LPS on cell viability and HSP60, HSP70, or HSP90 expression were again tested. We found that suppression of HSP70 or HSP60 expression accelerated the process of LPS-induced cell death. A reduction in HSP90 level, however, had little effect. The study revealed that HSP70 and HSP60 played an anti-death role during LPS-induced PC12 cell death, and HSP90 did not appear to be involved.

Universiẗat, Diss., 2003--München.

ALVES, A. A. Efeito do ácido tânico sobre a toxicidade induzida pela 6-OHDA em células PC12, um modelo in vitro de doença de Parkinson. 2016. 81 f. Dissertação (Mestrado em Farmacologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade federal do Ceará, Fortaleza, 2016. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-08-23T12:23:17Z No. of bitstreams: 1 2016_dis_aaalves.pdf: 1559996 bytes, checksum: 3da3b4c9a2ebe9af6ce74998408001e3 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-08-23T12:23:25Z (GMT) No. of bitstreams: 1 2016_dis_aaalves.pdf: 1559996 bytes, checksum: 3da3b4c9a2ebe9af6ce74998408001e3 (MD5) / Made available in DSpace on 2016-08-23T12:23:25Z (GMT). No. of bitstreams: 1 2016_dis_aaalves.pdf: 1559996 bytes, checksum: 3da3b4c9a2ebe9af6ce74998408001e3 (MD5) Previous issue date: 2016-07-21 / Parkinson's disease (PD) is the second most common neurodegenerative disease in the elderly. It is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta, leading to a severe reduction in striatal dopamine content. The model using PC12 cells, a cell line of rat pheochromocytoma exposed to toxins that recapitulate different mechanisms of cell death in PD has been used as a screening test for neuroprotective agents. Among the principal mechanisms leading to cell death in neurodegenerative diseases including periodontal disease, inflammation, apoptosis and oxidative stress play an important role. The tannic acid (TA) is a polyphenol antioxidant effect of the chelator type and free radical scavenger. The aim of this study was to evaluate the possible effect of citoproteror AT cytotoxicity induced by 6-OHDA on PC12 cells. The cell morphology was evaluated using the fast Panotic color. Cell viability was assessed by the MTT test, cell death by propidium iodide by flow cytometry and ethidium bromide and acridine orange using optical microscopy. The determination of oxidative stress was done by measuring nitrite and nitrate and malondialdehyde. Apoptosis was assessed by evaluating the expression of caspases 3 and 7 and GSK-3β. Treatment with AT increased cell viability tests such as MTT, ethidium bromide / laraja acridine and membrane integrity by propidium iodide, retained cellular morphology, in addition to reducing the levels of nitrite and malondialdehyde, proving its antioxidant activity . Exposure of cells to the AT also decreased significantly the activation of effector caspases 3 and 7, resulting in a decrease in apoptosis. The results suggest that AT is a cytoprotective substance that can prevent the degeneration of dopaminergic neurons. However more studies must be done to prove the effects of the AT, so that it can be used therapeutically in PD. / A doença de Parkinson (DP) é a segunda forma mais comum de doença neurodegenerativa nos idosos. É caracterizada pela perda progressiva de neurônios dopaminérgicos na substância negra pars compacta, levando a uma severa redução do conteúdo de dopamina no estriado. O modelo utilizando células PC12, uma linhagem celular de feocromocitoma de rato, exposta a diferentes toxinas que recapitulam mecanismos de morte celular na DP, vem sendo utilizado como screening para testar agentes neuroprotetores. Dentre os principais mecanismos que levam à morte celular nas doenças neurodegenerativas, incluindo a DP, a inflamação, a apoptose e o estresse oxidativo assumem um papel importante. O Ácido Tânico (AT) é um polifenol com efeito antioxidante do tipo quelante e sequestrador de radicais livres. O objetivo do presente estudo foi avaliar o possível efeito citoproteror do AT sobre a citotoxicidade induzida por 6-OHDA em células PC12. A morfologia celular foi avaliada utilizando a coloração panótico rápido. A viabilidade celular foi avaliada pelo teste do MTT, morte celular por Iodeto de propídio por citometria de fluxo e brometo de etídio e laranja de acridina utilizando microscopia optica. A determinação do estresse oxidativo foi feito pela dosagem de nitrito e nitrato e malonaldeído. A apoptose foi avaliada através da avaliação da expressão das caspases 3 e 7 e GSK-3β. O tratamento com AT aumentou a viabilidade celular em testes como o do MTT, brometo de etídio/laraja de acridina e integridade de membrana por iodeto de propídio, manteve a morfologia celular, além de reduzir os níveis de nitrito e malonaldeído, comprovando sua atividade antioxidante. A exposição das células ao AT também diminuiu de forma significativa a ativação das caspases efetoras 3 e 7, resultando em um decréscimo da apoptose. Os resultados obtidos sugerem que o AT é uma substância citoprotetora que pode prevenir a degeneração de neurônios dopaminérgicos. Contudo mais estudos devem ser feitos para comprovar os efeitos do AT, afim de que ele possa ser usada terapeuticamente na DP.

No description available.