Plasticidade de células tronco mesenquimais de medula óssea de ratos na presença de meio condicionado do nervo facial e do fator de crescimento fibroblástico 2

A number of evidences show the influence of the growth of injured nerve fibers in Peripheral Nervous System (PNS) as well as potential implant stem cells (SCs) to make it more suitable for nerve regeneration medium. In this perspective, this study aimed to evaluate the plasticity of mesenchymal stem cells from bone marrow of mice in the presence of culture medium conditioned with facial nerve explants (D-10) and fibroblast growth factor-2 (FGF-2). In this perspective, the cells were cultivated only with DMEM (group 1), only with D-10(group 2), only with FGF-2(group 3) or with D-10 and FGF-2(group 4). The growth and morphology were assessed over 72 hours. Quantitative phenotypic analysis was taken from the immunocytochemistry for GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200 on the fourth day of cultivation. Cells cultured with conditioned medium alone or combined with FGF-2 showed distinct morphological features similar apparent at certain times with neurons and glial cells and a significant proliferative activity in groups 2 and 4 throughout the days. Cells cultived only with conditioned medium acquired a glial phenotype. Cells cultured with FGF-2 and conditioned medium expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200. On average, area and perimeter fo the group of cells positive for GFAP and the área of the cells immunostained for OX-42 were higher than those of the group 4. This study enabled the plasticity of mesenchymal cells (MCs) in neuronal and glial nineage and opened prospects for the search with cell therapy and transdifferentiation

Efeito do tratamento com as drogas antipsicóticas haloperidol e clozapina sobre a infecção de toxoplasma gondii em cultura de células retinianas embrionárias

T. gondii is an obligate intracellular protozoan and the main cause of retinochoroiditis in humans. The aim of this study was to evaluate the effect of the antipsychotic drugs haloperidol and clozapine on the course of infection by T. gondii of cultured embryonic retinal cells. Embryo retinas of Gallus gallus domesticus (E12) were used for the preparation of mixed monolayer cultures of retinal cells. Cultures were maintained on plates of 96 and 24 wells by 37°C in DMEM medium supplemented with 5% fetal bovine serum for 2 days. After this period, cultures were simultaneously infected with tachyzoites of T. gondii and treated with the antipsychotics haloperidol and clozapine for 48 hours. Treatment effects were determined by both assessing cell viability with the MTT method and evaluating infection outcomes in slides stained with Giemsa. The treatment with haloperidol and clozapine cells infected with T. gondii resulted in higher viability of these cells, suggesting a possible prevention of neuronal degeneration induced by T. gondii. Additionally, intracellular replication of this protozoan in cells treated with haloperidol and clozapine were significantly reduced, possibly by modulation of the parasite s intracellular calcium concentration

Síntese e caracterização de componentes nanoestruturados de células a combustível de óxidos sólidos

Perovskite-like ceramic materials present the general formula ABO3, where A is a rare earth element or an alkaline metal element, and B is a transition metal. These materials are strong candidates to assume the position of cathode in Solid Oxide Fuel Cells (SOFC), because they present thermal stability at elevated temperatures and interesting chemical and physical properties, such as superconductivity, dieletricity, magnetic resistivity, piezoelectricity, catalytic activity and electrocatalytic and optical properties. In this work the cathodes of Solid Oxide Fuel Cells with the perovskite structure of La1-xSrxMnO3 (x = 0.15, 0.22, 0.30) and the electrolyte composed of zirconia-stabilized-yttria were synthesized by the Pechini method. The obtained resins were thermal treatment at 300 ºC for 2h and the obtained precursors were characterized by thermal analysis by DTA and TG / DTG. The powder precursors were calcined at temperatures from 450 to 1350ºC and were analyzed using XRD, FTIR, laser granulometry, XRF, surface area measurement by BET and SEM methods. The pellets were sintered from the powder to the study of bulk density and thermal expansion

Desenvolvimento de componentes nanométricos para aplicação em células a combustível à base de sólidos estruturados e filmes finos

The present work aims the preparation of filmes of strontium-doped lanthanum manganite (perovskita) yttria-stabilized zirconia (LSM-SDC) films deposited on substrate of YSZ by means of spin coating technique having as principal objective their application to solid oxide fuel cells of intermediate temperature. La0,8Sr0,2MnO3 and Ce0,8Sm0,2O1,9 were obtained by modified Pechini method by use of gelatin which act as polymerization agent. The powders obtained were characterized by Xray fluorescence, X ray diffraction, electronic scanning microscopy and the superficial area by BET method. The results obtained by X-ray fluorescence showed that the route adopted for obtention of powders was effective in the obtention of the compositions with close values to the stoichiometrics. Ethyl cellulose was used as pore-forming agent and mixed with the LSM-SDC powders in weight proportions of 1:24, 2:23 and 1:9. The films were sintered at 1150 °C for 4 h and characterized by X-ray diffraction and scanning electron microscopy technique (SEM) and atomic force. The phases quantification of the precursory powders and of the obtained films was carried through Rietveld method. According with the analysis of SEM, as the content of ethyl cellulose was increased, the pore distribution in films become more uniform and the pore size reduced. The methodology used for the obtention of the films was very efficient, considering a material was obtained with characteristics that were proper to the application as electrolyte/cathode system to solid oxide fuel cells

Caracterização dos eletrocatalisadores LaMnO3, LaFeO3, LaFe0,2Mn0,8O3 E La0,5Fe0,5MnO3 preparados por autocombustão assistida por microondas para cátodos de células a combustível do tipo SOFC

Materials consisting of perovskite-type oxides (ABO3) have been developed in this work for applications in fuel cell cathodes of solid oxide type (SOFC). These ceramic materials are widely studied for this type of application because they have excellent electrical properties, conductivity and electrocatalytic. The oxides LaMnO3, LaFeO3, LaFe0.2Mn0.8O3 e La0.5Fe0.5MnO3 were synthesized by the method of microwave assisted combustion and after sintering at 800°C in order to obtain the desired phases. The powders were characterized by thermogravimetry (TG), X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy (SEM) and voltammetric analysis (cyclic voltammetry and polarization curves). The results obtained by XRF technique showed that the microwave synthesis method was effective in obtaining doping oxides with values near stoichiometric. In general, powders were obtained with particle size less than 0.5 μm, having a porous structure and uniform particle size distribution. The particles showed spherical form, irregular and crowded of varying sizes, according to the analysis of SEM. The behavior of the oxides opposite the thermal stability was monitored by thermogravimetric curves (TG), which showed low weight loss values for all samples, especially those of manganese had its structure. By means of Xray diffraction of the samples sintered at 800°C was possible to observe the formation of powders having high levels of crystallinity. Furthermore, undesirable phases such as La2O3 and MnOx were not identified in the diffractograms. These phases block the transport of oxygen ions in the electrode/electrolyte interface, affecting the electrochemical activity of the system. The voltammetric analysis of the electrocatalysts LF-800, LM-800, LF2M8-800 e L5F5M-800 revealed that these materials are excellent electrical conductors, because it increased the passage of electrical current of the working electrode significantly. Best performance for the oxygen reduction reaction was observed with iron-rich structures, considering that the materials obtained have characteristics suitable for use in fuel cell cathodes of solid oxide type

Influência da laserterapia na proliferação de células-tronco do ligamento periodontal humano

Low level laser irradiation (LLLI) has been used in Dentistry to promote wound healing and tissue regeneration. The literature shows a positive effect of LLLI on cell proliferation, but little is known about their effectiveness in promoting stem cells proliferation. The aim of this study was to evaluate the effect of LLLI on the proliferative rate of human periodontal ligament stem cells. Extracts of periodontal ligament were isolated from two third molars removed by surgical and/or orthodontic indication. After enzymatic digestion, the cells were grown in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. On the third subculture, the cells were irradiated with a InGaAlP-diode laser, using two different energy densities (0,5J/cm 2 - 16 seconds and 1,0J/cm² - 33 seconds), with wavelength of 660nm and output power of 30mW. A new irradiation, using the same parameters, was performed 48h after the first. A control group (non irradiated) was kept under the same experimental culture conditions. The Trypan blue exclusion test and the mitochondrial activity of the cells measured by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] essay were performed to assess the cell proliferation in the intervals of 0, 24, 48 e 72 h after irradiation. The data of cell counts were submitted to nonparametrical statistical tests (Kruskal-Wallis and Mann-Whitney), considering a confidence interval of 95%. DAPI (4 -6-Diamidino-2-phenylindole) staining of the cells was performed at 72h interval to evaluate possible nuclear morphological changes induced by LLLI. The results of this study show that the energy density of 1,0 J/cm² promoted greater cell proliferation compared to the other groups (control and 0,5 J/cm²) at intervals of 48 and 72h. The mitochondrial activity measured by MTT essay showed similar results to the Trypan blue cell counting test. The group irradiated with 1,0J/cm² exhibited a significantly higher MTT activity in the intervals of 48 and 72h, when compared to the group irradiated with 0,5J/cm². No nuclear morphological change was observed in the cells from the three groups studied. It is concluded that LLLI has stimulatory effects on the proliferation of human periodontal ligament stem cells. Therefore, the use of laser irradiation in this cell type may be important to promote future advances in periodontal regeneration

Atividade biológica de células-tronco da polpa de dentes decíduos humanos submetidas à criopreservação

Dental pulp stem cells have been widely investigated because of their ability to differentiate into both dental and non-dental cells, with potential use in therapies involving tissue engineering. The technique of cell cryopreservation represents a viable alternative for the conservation of these cells, since it stops reversibly, in a controlled manner, all of cell biological functions in an ultra low temperature. The present study aimed to evaluate, using in vitro experiments, the influence of a cryopreservation protocol on the biologic acti vity of stem cells from human exfoliated deciduous teeth (SHED). Cells obtained from the pulp of three deciduous teeth on end-stage exfoliation or with indicated extraction were expanded in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. At second subculture (P2), a group of cells were submitted to cryopreservation for 30 days in 10% DMSO diluted in fetal bovine serum, at -80º C, while the remind cells continued under normal conditions of cell culture. Cell proliferation was evaluated in both groups (not cryopreserved or cryopreserved) by Trypan blue stain essay at intervals of 24, 48 and 72h after plating. Cell cycle analysis of SHEDs submitted or not to the cryopreservation protocol was performed in the same intervals. Events related to cell death were studied by Annexyn V and PI expression under flow cytometry at the intervals of 24 and 72h. The presence of nuclear morphological changes was evaluated by DAPI staining at 72h interval. It was observed that both groups exhibited an upward cell proliferation curve, without considerable changes in cell viability throughout the experiment. The distribution of cell in the cell cycle phasis was consistent with cell proliferation in both groups. There were no nuclear morphological damages in the end range of the experiment. therefore, it is concluded that the proposed cryopreservation protocol is efficient for storing the studied cell type, allowing its use in future experimental studies

Ativação das células hepáticas estreladas e fibrose hepática em crianças portadoras de hepatite autoimune tipo 1: estudo imuno-histoquímico de biópsias hepáticas pareadas antes do tratamento e após a remissão clínica

The activation of hepatic stellate cells (HSC) is considered the most important event in hepatic fibrogenesis. The precise mechanism of this process is unknown in autoimmune hepatitis (AIH), and more evidence is needed on the evolution of fibrosis. The aim of this study was to assess these aspects in children with type 1 AIH. We analyzed 16 liver biopsy samples from eight patients, paired before treatment and after clinical remission, performed an immunohistochemical study with anti-actin smooth muscle antibody and graded fibrosisand inflammation on a scale of 0:4 (Batts and Ludwig scoring system). We observedthere was no significant reduction in fibrosis scores after 24± 18 months (2.5 ± 0.93 vs. 2.0± 0.53, P = 0.2012). There was an important decrease in inflammation: portal (2.6 ±0.74 vs. 1.3± 0.89, P = 0.0277), periportal/periseptal (3.0 ±0.76 vs. 1.4 ± 1.06, P = 0.0277), and lobular (2.8 ± 1.04 vs. 0.9± 0.99, P =0.0179). Anti-actin smooth muscle antibodies were expressed in the HSC of the initial biopsies (3491.93 ±2051.48 lm2), showing a significant reduction after remission (377.91 ±439.47 lm2) (P = 0.0117). HSC activation was demonstrated in the AIH of children. The reduction of this activation after clinical remission, which may precede a decrease in fibrosis, opens important perspectives in the follow-up of AIH.

Efeitos da laserterapia na atividade biológica de células de carcinoma epidermóide de língua humano

The low level laser therapy (LLLT) has shown to be effective in promoting the proliferation of different cells in vitro, including keratinocytes, osteoblasts, endothelial cells and stem cells. It has been speculated that the biostimulatory effect of LLLT could cause undesirable enhancement of tumor growth in neoplastic diseases, since the malignant cells are more susceptible to proliferative stimuli. Within this context, this study evaluated the effect of LLLT on epidermoid carcinoma of the tongue cell line (SCC25) proliferation and invasion. Cultured cells were irradiated with an InGaAIP diode laser, 660nm, 30mW using two energy densities (0.5J/cm2 and 1.0J/cm2). Proliferative activity was assessed through trypan blue staining method and through cell cycle analysis using flow cytometry. The invasive potential was measured through cell invasion assay using matrigel. Cyclin D1, E-cadherin, -catenin and MMP-9 expressions were analyzed by immunofluorescence and flow cytometry and related to the investigated biological activities. Proliferation curve demonstrated that SCC25 irradiated with 1.0J/cm2 had the highest proliferative rate when compared to the control group and the group irradiated with 0.5J/cm2 (p<0.05). LLLT affected cell cycle distribution and energy density of 1.0 J/cm2 promoted a higher percentage of cells in S/G2/M phases, with statistically significant differences at 24h interval (p<0.05). LLLT, mainly with 1.0J/cm2, revealed significantly higher potential for invasion and influenced the expression of cyclin D1, E-cadherin, -catenin and MMP-9, promoting the malignant phenotype. In conclusion, our results indicate that LLLT has an important stimulatory effect on proliferation and invasion of SCC25 cells, likely due to altered expression of proteins associated with these processes

Efeitos da sinalização por Sonic Hedgehog sobre a proliferação de células-tronco neurais e gliogênese no córtex cerebral em desenvolvimento

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Análise comparativa da imunoexpressão de GLUT-1, GLUT-3 e M-CSF em lesão periférica e central de células gigantes

The peripheral giant cell lesion ( PG CL ) and the central giant cell lesion ( CGC L) are lesions histologically similar affecting the head and neck region . The study aimed to analyze the immunohistochemical expression of markers GLUT - 1 , GLUT - 3 and M - CSF in a series of cases of PGCL and CGCL , in trying to understand the different biological behavior of these pathologies . The sample consisted of 20 tissue specimens of PGCL 20 central lesion of not aggressive giant cell ( CLNAGC) and 20 central lesi on of aggressive giant cell ( CLAGC), coming from the Pathology Unit of Oral Pathology of the Department of Dentistry of UFRN . W as performed the s emi - quantitative and qualitative analysis of immunohistochemical expression of the markers in giant cells and m ononuclear cells . In relation to the GLUT - 1, it was found a statistically significant difference (p < 0.05) in the number of mononuclear cells immunomarked between the PGCL and the CLNAGC and between the PGCL and CLAGC . Regarding the intensity of staining w as also observed a statistically significant difference both at the mononuclear cells as in giant cells between PL and CLNAGC and between PGCL and CLAGC , at the giant cells there was also a statistically significant difference between the CLNAGC and CLAGC . In relation to GLUT - 3 , was found a statistically significant difference between PGCL and CLAGC and between CLAGC and CLNAGC in amount of mononuclear cells immunomarked . Regarding the intensity of labeling for such protein was found a statistically signifi cant difference at the giant cells between PL and CLAGC . To the M - CSF was observed only a statistically significant difference in the intensity of labeling at the mononuclear cells between PGCL and CLNAGC and between PGCL and CLAGC . Based on these results, we can conclude the participation of GLUT - 1, GLUT - 3 and M - CSF in the pathogenesis of the lesions studied. The bigger immunostaining of these proteins in mononuclear cells show that these cells perform a higher metabolic activity and osteoclastogenic, espe cially in CLAGC . It was found that the mononuclear cells were more related to the pathogenesis of the studied lesions than properly the giant s cell s.

Avaliação dos efeitos da retirada do etanol em curto e longo prazo sobre respostas comportamentais relacionadas à ansiedade e sobre células imunorreativas para a serotonina no núcleo dorsal da Rafe em ratas

Ethanol withdrawn individuals present a wealth of signs and symptoms, some of them related with anxiety. To better understand brain areas involved in anxiety caused by ethanol abstinence, preclinical studies have been employing models of ethanol consumption followed by withdrawal in rodents submitted to behavioral tests of anxiety, such as the elevated plus-maze. The aim of this study was to investigate if short- or long-term ethanol withdrawal could alter both anxiety-related behaviors in the elevated plus-maze (EPM) and open field tests and the number of serotonin immunorreactive cels in the dorsal raphe nucleus, a midbrain area associated with anxiety. Female Wistar rats (90 days old) were submitted to increasing concentrations of ethanol (2% for 3 days, 4% for 3 days and 6% for 15 days) as the only source of liquid diet and the control group received water ad libitum. Both groups received food ad libitum. In the behavioral experiments, on 21st day of consumption, ethanol was substituted by water (withdrawal) and 72 h or 21 days after withdrawal animals were submitted to the EPM, where it was evaluated the percentage of time and entries in the open arms and the entries in the enclosed arms during 5 minutes. Twenty and four hours after testing in the EPM, animals were submitted to the open field test for 15 minutes, where the distance traveled by the animals was observed along this period. During the first 5 minutes, the distance traveled, entries and time spent in the center of the test were analyzed. In the immunohistochemistry study, animals were submitted to 21 days of consumption of ethanol followed or not by 72 hours and 21 days of withdrawal previously perfusion, brain tissue preparation and quantification of serotonin dyed cells in the dorsal and caudal portions in the dorsal raphe nucleus. Behavioral data showed that both short- and long-term ethanol withdrawals reduced the open arms exploration in the EPM. In the open field test there were no locomotor activity changes during the total 15 minutes; however, longterm ethanol withdrawal reduced the exploration in the center of the open field during the first 5 minutes. In the immunohistochemistry step, there were no differences, when short- and long-term withdrawn groups were compared with control group; nevertheless, the chronic consumption of ethanol decreased the number of serotonergic immunorreactive cells in the dorsal part of dorsal raphe nucleus. Taken together, results here obtained suggest that both short- and long-term ethanol withdrawals promoted an anxiogenic-like effect that was not related with changes in the serotonin immunorreactivity in the dorsal and caudal parts of the dorsal raphe nucleus.

Reprogramação de células-tronco mesenquimais em neurônios utilizando genes pró-neurais

A possibilidade de repor células perdidas em doenças neurodegenerativas através de transplantes com células-troncos das mais diversas fontes vem sendo amplamente estudada. As células-tronco adultas (CTA) podem ser facilmente isoladas e sua utilização na pesquisa não envolve questões éticas e religiosas. Além disso, estas células são menos propícias à transformação tumoral do que células-tronco embrionárias, outra importante fonte de células para terapias celulares. No entanto, as CTA são, em estados fisiológicos, restritas a geração de células dos seus tecidos de origem, o que poderia limitar a sua utilização. Porém, nos últimos anos, uma série de técnicas vem sendo descritas com o objetivo de reverter tais limitações. Neste trabalho, nós investigamos a capacidade das células-tronco mesenquimais adultas, isoladas de camundongos ou do cordão umbilical humano, serem induzidas a adquirir um fenótipo neuronal de forma direta, sem passar por um estágio de célula progenitora ou pluripotente, através da reprogramação genética com genes pró-neurais. Nossos resultados indicam que tanto células-tronco mesenquimais adultas murinas quanto humanas podem ser reprogramadas em neurônios após a expressão combinada de Sox2 e Ascl1 ou Sox2 e Neurog2. As células reprogramadas exibem morfologias compatíveis com o fenótipo neuronal, expressam proteínas típicas de neurônios maduros, apresentam a capacidade de gerar potenciais de ação repetitivos e formam conexões sinápticas com outros neurônios presentes no cultivo. Portanto, nosso trabalho apresenta a primeira evidência de reprogramação direta de células-tronco mesenquimais humanas em neurônios funcionais.

Avaliação de polimorfismos funcionais nos genes de reparo XRCC1, APEX1, XPD e XPF em carcinomas de células escamosas orais

Base excision repair (BER) and nucleotide excision repair (NER) pathways play critical role in maintaining genome integrity. Polymorphisms in BER and NER genes which modulate the DNA repair capacity may affect the susceptibility and prognosis of oral cancer. This study was conducted with genomic DNA from 92 patients with oral squamous cell carcinomas (OSCC) and 130 controls. The cases were followed up to explore the associations between BER and NER genes polymorphisms and the risk and prognosis of OSCC. Four single-nucleotide polymorphisms (SNPs) in XRCC1 (rs25487), APEX1 (rs1130409), XPD (rs13181) and XPF (rs1799797) genes were tested by polymerase chain reaction – quantitative real time method. The GraphPad Prism version 6.0.1 statistical software was applied for statistical analysis of association. Odds ratio (OR), hazard ratio (HR), and their 95 % confidence intervals (CIs) were calculated by logistic regression. Kaplan-Meier curve and Cox proportional hazard model were used for prognostic analysis. The presence of polymorphic variants in XRCC1, APEX1, XPD and XPF genes were not associated with an increased risk of OSCC. Gene-environment interactions with smoking were not significant for any polymorphism. The presence of polymorphic variants of the XPD gene in association with alcohol consumption conferred an increased risk of 1.86 (95% CI: 0.86 – 4.01, p=0.03) for OSCC. Only APEX1 was associated with decreased specific survival (HR 3.94, 95% CI: 1.31 – 11.88, p=0.01). These results suggest an interaction between polymorphic variants of the XPF gene and alcohol consumption. Additionally APEX1 may represent a prognostic marker for OSCC.

Caracterização de células de lugar no hipocampo e de suas relações com oscilações do potencial de campo local

The main inputs to the hippocampus arise from the entorhinal cortex (EC) and form a loop involving the dentate gyrus, CA3 and CA1 hippocampal subfields and then back to EC. Since the discovery that the hippocampus is involved in memory formation in the 50's, this region and its circuitry have been extensively studied. Beyond memory, the hippocampus has also been found to play an important role in spatial navigation. In rats and mice, place cells show a close relation between firing rate and the animal position in a restricted area of the environment, the so-called place field. The firing of place cells peaks at the center of the place field and decreases when the animal moves away from it, suggesting the existence of a rate code for space. Nevertheless, many have described the emergence of hippocampal network oscillations of multiple frequencies depending on behavioral state, which are believed to be important for temporal coding. In particular, theta oscillations (5-12 Hz) exhibit a spatio-temporal relation with place cells known as phase precession, in which place cells consistently change the theta phase of spiking as the animal traverses the place field. Moreover, current theories state that CA1, the main output stream of the hippocampus, would interplay inputs from EC and CA3 through network oscillations of different frequencies, namely high gamma (60-100 Hz; HG) and low gamma (30-50 Hz; LG), respectively, which tend to be nested in different phases of the theta cycle. In the present dissertation we use a freely available online dataset to make extensive computational analyses aimed at reproducing classical and recent results about the activity of place cells in the hippocampus of freely moving rats. In particular, we revisit the debate of whether phase precession is due to changes in firing frequency or space alone, and conclude that the phenomenon cannot be explained by either factor independently but by their joint influence. We also perform novel analyses investigating further characteristics of place cells in relation to network oscillations. We show that the strength of theta modulation of spikes only marginally affects the spatial information content of place cells, while the mean spiking theta phase has no influence on spatial information. Further analyses reveal that place cells are also modulated by theta when they fire outside the place field. Moreover, we find that the firing of place cells within the theta cycle is modulated by HG and LG amplitude in both CA1 and EC, matching cross-frequency coupling results found at the local field potential level. Additionally, the phase-amplitude coupling in CA1 associated with spikes inside the place field is characterized by amplitude modulation in the 40-80 Hz range. We conclude that place cell firing is embedded in large network states reflected in local field potential oscillations and suggest that their activity might be seen as a dynamic state rather than a fixed property of the cell.