Directing differential cellular response by manipulating the physical characteristics of the material is regarded as a key challenge in biomaterial implant design and tissue engineering. In developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of substrate conductivity on cell fate processes is rather limited. The need for such an understanding is based on the fact that specific tissues in the body are electrically active in nature, such as in brain, heart and skeletal muscle. These tissues make use of electrical conductivity as an effective cue for tissue homeostasis, development, regeneration and so on. Moreover, understanding the importance of underlying conductivity in basic biological processes is essential in developing electrically conductive biomaterials with the ability to simulate normal electrophysiology of the body by interfacing with bioelectric fields in cells and tissues. Electrical stimulation and charge conduction can regulate numerous intracellular signalling pathways, can interact with cytoskeleton proteins to modulate the morphology, increase protein synthesis and on the more can favor the ECM protein conformational changes. On these grounds, the present dissertation illustrates that persistent electrical activation influences the multipotency of hMSCs and acts like a promoter towards selective differentiation of hMSCs into neural/cardiomyogenic or osteogenic lineage. Besides, continual exposure to electric field stimulated conducting culture environments lead to growth arrest while enhancing differentiation. In total, this dissertation suggests the dominant role of conductivity in inducing my oblast differentiation and hMSc lineage commitment that involves EF stimulated in vitro culture conditions. Also, a knowledge base with qualitative and quantitative understanding of stem cells and their response to substrate physical properties and external field effect was developed through this comprehensive study. Such an improved understanding of the ability of hMSCs in sensing electrical conductivity may lead to the development of culture additives/conditions that better induce directed stem cell differentiation.

Mature αβ and γδ T cells arise from a common precursor population in the thymus. Much debate has focused on the mechanism of T cell lineage choice made by these multi-potential precursor cells. It is widely believed that the decision of these precursor cells to commit to the γδ or αβ T cell lineages is regulated primarily by a specific instructive signal relayed through the appropriate T cell receptor. Contrary to this model, we present evidence for a TCR-independent lineage commitment process. Comparison of global gene expression profiles from immature αβ and γδ lineage thymocytes identified Sox13, an HMG-box transcription factor, as a γδ T cell-specific gene. Unlike other HMG-box transcription factors such as TCF1, LEF1 and SOX4, that are critical for proper αβ T cell development, Sox13 expression is restricted to early precursor subsets and γδ lineage cells. Importantly, SOX13 appears to influence the developmental fate of T cell precursors prior to T cell receptor expression on the cell surface. Transgenic over-expression of Sox13 in early T cell precursors strongly inhibits αβ lineage development, in part, by inhibiting precursor cell proliferation and concomitantly, leading to increased cell death among αβ lineage subsets. Steady-state γδ T cell numbers, however, appear unaffected. Strikingly, the DP αβ lineage cells that do develop in Sox13 transgenic mice are imprinted with a γδ- or precursor-like molecular profile, suggesting that SOX13 plays an active role in the lineage fate decision process or maintenance. Sox13-deficient mice, on the other hand, have selectively reduced numbers of γδ thymocytes, indicating that SOX13 is essential for proper development of γδ T cells. We present additional data demonstrating that SOX13 is a canonical WNT signaling antagonist modulating TCF1 activity, raising a strong possibility that WNT signals, and their modulators, are at the nexus of γδ versus αβ T cell lineage commitment.

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular. / The aim of the present work was to evaluate the behavior of human dental pulp cells exposed to TGFβ1 and aFGF in culture, at the following concentrations: TGFβ1 1ng/mL, TGFβ1 5ng/mL, TGFβ1 1ng/mL + aFGF 5ng/mL, TGFβ1 5ng/mL + aFGF 5ng/mL e aFGF 5ng/mL. We assessed the cellular morphology, alkaline phosphatase activity, using pNPP as substrate, and expression of osteocalcin, bone sialoprotein, dentin sialophosphoprotein proteins by RT-PCR. After four days, the nucleolus media in the group treated with TGFβ1 1ng/mL was significantly higher than the group treated with aFGF 5ng/mL The alkaline phosphatase activity in the TGFβ1 1ng/mL treated group was significantly higher than the media observed in TGFβ1 5ng/mL + aFGF 5ng/m treated group. Osteocalcin expression was observed in all human dental pulp cell cultures. However, in the aFGF 5ng/mL treated group the osteocalcin expression decreased. The exposure to growth factors did not induced the expression of dentin matrix components such as BSP or DSPP. Our data suggest that the cells exposed to TGFβ1 1ng/mL were stimulated and had a higher cell activity, and that cells exposed to aFGF 5ng/mL were inhibited having a cell activity decrease.

Abstract 2-oxoglutarate-dependent dioxygenases (2-OGDDs) are an enzyme family that contains many enzymes that modify chromatin in extensive ways. These enzymes include several histone lysine demethylases (KDMs) and TET enzymes that convert methylated cytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) ultimately leading to DNA demethylation. Disturbed DNA and histone methylation are found in many cancers. However, the role of KDMs and TETs behind these oncogenic changes has so far not been fully investigated. This study focused on the role of these chromatin-modifying enzymes in cancers with special emphasis on enzyme kinetic studies. Cancers with inactivating fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) mutations accumulate fumarate, succinate and R-2-hydroxyglutarate, respectively. In this study we showed how these cancer-associated 2-oxoglutarate (2-OG) analogues can inhibit the TET enzymes and many of the KDMs leading to lower 5-hmC levels and increased H3K27 and H3K9 methylation on chromatin, respectively. We also characterized kinetic properties of acute myeloid leukaemia (AML)-associated TET2 mutants and found that their ability to bind 2-OG or iron was impaired leading to diminished catalytic activity. Tumours are often hypoxic due to inadequate vasculature and blood supply. The TET enzymes and KDMs require oxygen for the reactions they catalyse. We determined the oxygen affinity of TETs and many KDMs and found that a H3K27 demethylase KDM6A has a remarkably low affinity for oxygen indicating that it is inactivated in hypoxic tumours and tissues. H3K27 methylation was found to be increased in hypoxic cells and this blocked cell differentiation. Altogether, these studies shed light on the mechanisms behind the altered DNA and histone methylation found in several cancers with hypoxic conditions or FH, SDH and IDH mutations. Altered DNA and histone methylation has previously been associated with progression of cancer, such as epithelial-to-mesenchymal transition (EMT). We now linked catalytic inhibition of 2-OGDDs to disturbed DNA and histone methylation that can account for altered cell differentiation, EMT and increased aggressiveness and invasiveness of cancers. / Tiivistelmä 2-oksoglutaraatista riippuvaiset dioksygenaasit ovat entsyymiperhe, johon kuuluu useita entsyymejä, jotka muokkaavat kromatiinin epigeneettisiä merkkejä monin tavoin. Näitä entsyymejä ovat mm. DNA:n demetylaatioon vaikuttavat TET-entsyymit sekä useat histonidemetylaasit. Vaikka muutoksia DNA:n ja histonien metylaatiotasoissa on havaittu useissa syövissä, ei näiden entsyymien roolia muutosten taustalla ole vielä tutkittu. Tämä tutkimus kohdistui näiden epigenetiikkaan vaikuttavien entsyymien roolin ymmärtämiseen syövissä keskittyen erityisesti kyseisten entsyymien kinetiikkaan. Useissa syövissä on havaittu fumaraattihydrataasin, sukkinaattidehydrogenaasin ja isositraattidehydrogenaasien aktiivisuuteen vaikuttavia mutaatioita, jotka johtavat fumaraatin, sukkinaatin ja R-2-hydroksiglutaraatin kertymiseen syöpäsoluihin. Tässä tutkimuksessa osoitimme, kuinka nämä karsinogeeniset 2-oksoglutaraattianalogit voivat inhiboida TET-entsyymejä ja histonidemetylaaseja, mikä alentaa 5-hydroksimetyylisytosiinitasoja ja lisää histonien metylaatiota. Näytämme myös, kuinka tietyillä akuutissa myelooisessa leukemiassa esiintyvillä TET2-mutanteilla on heikentynyt kyky sitoa 2-oksoglutaraattia tai rautaa, mikä johtaa entsyymien aktiivisuuden laskuun. Kasvainkudoksissa happipitoisuudet ovat usein matalia nopean kasvun ja puutteellisen verisuonituksen vuoksi. TET-entsyymit ja histonidemetylaasit vaativat happea katalysoimissaan reaktioissa. Määritimme TET-entsyymien ja monien histonidemetylaasien riippuvuutta hapesta ja osoitimme, että H3K27-histonidemetylaasi KDM6A on erittäin riippuvainen hapesta, mikä osoittaa, ettei se pysty toimimaan kasvaimissa ja kudoksissa, joissa happipitoisuudet ovat matalia. Huomasimme, että vähähappisissa olosuhteissa solujen H3K27 metylaatio on lisääntynyt, mikä johti erilaistumisen estymiseen soluissa. Tämä tutkimus paljasti uusia mekanismeja useista syövistä löytyneiden muuntuneiden DNA:n ja histonien metylaatiotasojen taustalla. Häiriintynyt DNA:n ja histonien metylaatio on aiemmin yhdistetty syöpien etenemiseen, erityisesti solujen erilaistumisen häiriintymisen kannalta. Tässä tutkimuksessa yhdistimme 2-oksoglutaraatista riippuvaisten entsyymien inhibition häiriintyneeseen DNA:n ja histonien metylaatioon, joka voi johtaa muuntuneeseen solujen erilaistumiseen ja lopulta lisääntyneeseen syöpien aggressiivisuuteen ja invasiivisuuteen.

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular. / The aim of the present work was to evaluate the behavior of human dental pulp cells exposed to TGFβ1 and aFGF in culture, at the following concentrations: TGFβ1 1ng/mL, TGFβ1 5ng/mL, TGFβ1 1ng/mL + aFGF 5ng/mL, TGFβ1 5ng/mL + aFGF 5ng/mL e aFGF 5ng/mL. We assessed the cellular morphology, alkaline phosphatase activity, using pNPP as substrate, and expression of osteocalcin, bone sialoprotein, dentin sialophosphoprotein proteins by RT-PCR. After four days, the nucleolus media in the group treated with TGFβ1 1ng/mL was significantly higher than the group treated with aFGF 5ng/mL The alkaline phosphatase activity in the TGFβ1 1ng/mL treated group was significantly higher than the media observed in TGFβ1 5ng/mL + aFGF 5ng/m treated group. Osteocalcin expression was observed in all human dental pulp cell cultures. However, in the aFGF 5ng/mL treated group the osteocalcin expression decreased. The exposure to growth factors did not induced the expression of dentin matrix components such as BSP or DSPP. Our data suggest that the cells exposed to TGFβ1 1ng/mL were stimulated and had a higher cell activity, and that cells exposed to aFGF 5ng/mL were inhibited having a cell activity decrease.

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular. / The aim of the present work was to evaluate the behavior of human dental pulp cells exposed to TGFβ1 and aFGF in culture, at the following concentrations: TGFβ1 1ng/mL, TGFβ1 5ng/mL, TGFβ1 1ng/mL + aFGF 5ng/mL, TGFβ1 5ng/mL + aFGF 5ng/mL e aFGF 5ng/mL. We assessed the cellular morphology, alkaline phosphatase activity, using pNPP as substrate, and expression of osteocalcin, bone sialoprotein, dentin sialophosphoprotein proteins by RT-PCR. After four days, the nucleolus media in the group treated with TGFβ1 1ng/mL was significantly higher than the group treated with aFGF 5ng/mL The alkaline phosphatase activity in the TGFβ1 1ng/mL treated group was significantly higher than the media observed in TGFβ1 5ng/mL + aFGF 5ng/m treated group. Osteocalcin expression was observed in all human dental pulp cell cultures. However, in the aFGF 5ng/mL treated group the osteocalcin expression decreased. The exposure to growth factors did not induced the expression of dentin matrix components such as BSP or DSPP. Our data suggest that the cells exposed to TGFβ1 1ng/mL were stimulated and had a higher cell activity, and that cells exposed to aFGF 5ng/mL were inhibited having a cell activity decrease.

O desmame precoce (DP) causa mudanças na mucosa gástrica, como o aumento da proliferação celular e da expressão do Fator de Crescimento Transformante (TGFa). Neste trabalho, avaliamos o papel desse peptídeo e seu receptor EGFR no controle do crescimento gástrico. Ratos com 15 dias de vida foram divididos em: amamentados (controle) e DP, no qual os filhotes foram separados da mãe e alimentados com pasta de ração. O DP aumentou o número de células marcadas para EGFR, acelerou a diferenciação de células mucosas do colo e elevou a expressão de mucina 6. A inibição do EGFR com AG1478 diminuiu a proliferação celular e o número de células mucosas do colo. Entre as proteínas envolvidas na sinalização de EGFR e ciclo celular, detectamos que o DP elevou os níveis de p-ERK1/2 e p-Src e não alterou p-Akt, p21 e p27. Nós sugerimos que o padrão alimentar influencia a proliferação e diferenciação no epitélio gástrico, e que TGFa/EGFR podem regular esses processos durante o desenvolvimento pós-natal, provavelmente por ativação das vias de sinalização de MAPK e Src. / Early weaning (EW) causes changes in the gastric mucosa, including the increase in cell proliferation and Transforming Growth Factor (TGFa). In the present study, we evaluated the role of this peptide and its receptor EGFR in the control of the gastric growth. 15-d-old rats were divided into two groups: suckling (control) and EW, in which the pups were separated from the dam and fed with powdered chow. EW increased the number of EGFR-positive cells, accelerated the differentiation of mucous neck cells and augmented the expression of mucin 6. EGFR inhibition with AG1478 decreased cell proliferation and the number of mucous neck cells. Among the proteins involved on EGFR signaling pathways and cell cycle, we found that EW increased the levels of p-ERK1/2 and p-Src, but did not change p-Akt, p21 and p27. We suggest that the diet pattern influences proliferation and differentiation in the gastric epithelium, and the TGFa/EGFR can regulate these processes throughout the postnatal development, probably by activating MAPK and Src signaling pathways.

O desenvolvimento do músculo esquelético em vertebrados é um processo bem organizado que envolve diversos eventos, inicia-se na fase embrionária e continua durante toda a vida. Esse processo biológico requer uma adequada sinalização celular e é regulado por inúmeros genes. Em busca do entendimento dos mecanismos moleculares envolvidos na determinação do desenvolvimento e crescimento do tecido muscular foi acompanhada a expressão gênica dos fatores miogênicos (MyoD, Myf5, Miogenina e MRF4); Pax7; Miostatina; e da via de ação Shh (Shh, Ptch1, Smo, Pka, Sufu, Gli2 e Gli3) na musculatura peitoral de duas linhagens de galinhas de composições genéticas distintas. Uma linhagem de corte, selecionada para maior deposição de massa muscular, e outra de postura, caracterizada pela baixa taxa de crescimento e pouca massa muscular. Foram utilizados 90 animais distribuídos nas duas linhagens e cinco estádios de desenvolvimento: 9 e 17 dias da fase embrionária e 1, 21 e 42 dias da fase pós-eclosão. A expressão gênica foi analisada por PCR quantitativa em tempo real. Os genes Myf5, Miogenina, MRF4, Pax7, Shh e Ptch1 foram diferencialmente expressos na ontogenia e entre as linhagens. Já os genes MyoD, Miostatina, Smo, Pka, Sufu, Gli2 e Gli3 foram diferencialmente expressos somente na ontogenia. Foi possível traçar o perfil de expressão dos genes ao longo das fases de desenvolvimento. Os genes MyoD, Myf5 e Pax7 foram mais expressos na fase embrionária, onde há maior proliferação celular. Durante as fases estudadas, os genes da Miogenina e MRF4 apresentaram uma auto-regulação entre eles, indicando a ocorrência do processo de diferenciação celular. O gene da Miostatina demonstrou estar inibindo o crescimento muscular nos dias que antecedem a eclosão. Os genes da via de ação Shh foram mais expressos nas idades embrionárias, onde esta via age como um fator de sobrevivência e proliferação celular e menos expressos nas idades posteriores a eclosão, provavelmente, para que haja o processo de diferenciação dos mioblastos. Verificou-se que esses genes foram diferencialmente expressos sendo coordenados espaço-temporalmente, permitindo assim um ajuste sutil entre proliferação e diferenciação das células musculares, colaborando para as diferenças fenotípicas observadas entre as linhagens. / The development of skeletal muscle in vertebrates is a well-organized process that involves several events, initiating in the embryonic phase and continuing throughout life. This biological process requires a proper cell signaling and is regulated by numerous genes. Aiming to understand the molecular mechanisms involved in determining the development and growth of muscle tissue, we monitored gene expression of myogenic factors (MyoD, Myf5, Myogenin and MRF4); Pax7; Myostatin; and Shh pathway (Shh, Ptch1, Smo, Pka, Sufu, Gli2 and Gli3) in the pectoralis muscle of two strains of poultry from different genetic compositions. A broiler, selected for increased deposition of muscle mass, and a layer, characterized by slow growth and low muscle mass. We used 90 animals divided into two lines and five stages of development: 9 and 17 days of embryo and 1, 21 and 42 days post-hatching. Gene expression was analyzed by quantitative real-time PCR. The genes Myf5, Myogenin, MRF4, Pax7, Shh and Ptch1 are differentially expressed in ontogeny and among strains. The genes MyoD, myostatin, Smo, Pka, Sufu, Gli2 and Gli3 were differentially expressed only in ontogeny. It was possible to design the gene expression profile throughout the different developmental stages. The genes MyoD, Myf5 and Pax7 were more expressed in the embryo, where there is greater cell proliferation. During the four phases, the genes MRF4 and Myogenin showed self-regulation among them, indicating the occurrence of the cell differentiation process. The myostatin gene proved to be inhibiting muscle growth in the days before hatching. The genes of the Shh action were more highly expressed in embryonic ages, where this pathway acts as a survival factor and cellular proliferation, and less expressed in later times after hatching, probably to allow for the differentiation of myoblasts. We found that these genes were differentially expressed being coordinated in space and time, allowing, thus, a subtle tuning between proliferation and differentiation of muscle cells, contributing to the phenotypic differences observed between strains.

O sistema imune dos mamíferos forma uma complexa rede de populações celulares especializadas e vias de sinalização extremamente reguladas. Linfócitos T naïve podem diferenciar-se após encontro com o antígeno em pelo menos duas sub-populações distintas, Th1 ou Th2, sendo que o papel do circuito p38MAPK/MSK1 durante este período inicial de ativação não é completamente entendido. Linfócitos T CD4+ naïve humanos foram estimulados in vitro em condições não-polarizantes (Tnp), Th1 ou Th2, na presença de inibidor específico da p38MAPK. As células ativadas e mantidas em condições diferenciadoras Th1 ou Th2 na presença do inibidor SB203580, apresentaram menor produção de IFN-<font face=\"symbol\">g e maior produção de IL-4. Através do bloqueio do RNAm da MSK1 por siRNA, observamos o mesmo efeito resultante da inibição da p38MAPK, fato que foi confirmado em experimentos com linfócitos T de camundongos MSK1-deficientes. A alteração da produção das citocinas características de cada população parece ser decorrente da alteração da expressão da IL12R<font face=\"symbol\">b2 e IL4R-<font face=\"symbol\">a dos receptores de citocinas da IL-12 e IL-4, respectivamente. Desta forma, os nossos dados sugerem que o circuito p38MAPK/MSK1 participa do processo de ativação dos linfócitos T mantidos em condições diferenciadoras Th1/2. / The mammalian immune system form a complex network of highly regulated signaling pathways and populations of specialized cells. After meeting with the antigen naïve T cells differentiate into at least two distinct sub-populations, Th1 or Th2, and the role of the circuit p38MAPK/MSK1 during this initial period of activation is not completely understood. Human CD4+ T lymphocytes were stimulated in vitro under non-polarized, Th1 or Th2 conditions, in the presence of a specific p38MAPK inhibitor. The cells activated and differentiated under Th1 or Th2 condition in the presence of inhibitor SB203580, had decreased production of IFN-<font face=\"symbol\">g and increased IL-4. By silencing MSK1 through siRNA, we observed the same effect due to inhibition of p38MAPK, an observation that was confirmed in experiments with T lymphocytes from mice deficient of MSK1. The change in the production of cytokines appears to be a result of altered expression of IL12R-<font face=\"symbol\">b2 and IL4R<font face=\"symbol\">a receptors of the cytokines IL-12 and IL-4, respectively. Taken together, our data suggest that the circuit p38MAPK/MSK1 plays a key role in the activation of human T cells maintained under Th1/2 differentiation conditions.

O tratamento minimamente invasivo tem sido cada vez mais empregado no tratamento das lesões de cárie dental, especialmente em crianças jovens. Com isso, a remoção do tecido cariado pelo método químico-mecânico permite uma maior conservação das estruturas dentais saudáveis. O Papacárie Duo® é um material de fácil aplicação e possui propriedades bactericidas e anti-inflamatórias. Este material é aplicado sobre a dentina cariada, a fim de promover um amolecimento desta, facilitando a sua remoção. Os efeitos celulares de Papacárie Duo® são pouco conhecidos, dessa forma esta pesquisa teve como objetivo avaliar o efeito do Papacárie Duo® em células indiferenciadas da polpa dental (Capítulo 1) e a capacidade do Papacárie Duo® induzir a ativação de macrófagos e a síntese de mediadores inflamatórios (Capítulo 2). O Papacarie Duo® foi preparado nas concentrações de 0,5 e 5% por meio de diluição seriada, a partir do gel obtido comercialmente. Células OD-21 e macrófagos J774.1 foram mantidas em cultura com os diferentes tratamentos por um período de estimulação de 24 horas para realização do teste de citotoxicidade (Ensaio LDH) e por 36 horas para avaliação da proliferação celular (Ensaio Colorimétrico MTT). A seguir foi realizada avaliação da expressão gênica relativa dos genes Ibsp, Runx2 e Spp1 em células OD-21; e dos genes Il10, Mmp9, Ptgs2 e Tnf em células J774.1, pelo método de transcrição reversa e reação em cadeia de polimerase em tempo real (qRT-PCR), utilizando o sistema TaqMan® após estimulação o período de 24 horas. O Papacárie Duo® a 5% foi citotóxico às células da polpa dental e inibiu a proliferação celular, assim como a expressão de Runx2 e Ibsp. Porém em ambas as concentrações estimulou a expressão de Spp1, a qual foi maior na concentração de 5%. Em macrófagos, o Papacárie Duo® foi citotóxico na concentração de 5%, mas não influenciou a proliferação celular em nenhuma das concentrações (0,5 e 5%). O LPS inibiu a proliferação celular na presença ou não de Papacárie Duo®, sem apresentar citotoxicidade. O Papacárie Duo® induziu a expressão de Ptgs2 e Il10, sem alterar Tnf e Mmp9. Portanto, o Papacárie Duo® foi citotóxico, dependendo da concentração, e apresentou efeito inibitório na diferenciação de células da polpa (OD-21), sem entretanto influenciar a proliferação celular. Papacárie Duo® não impediu a proliferação de macrófagos, porém foi citotóxico na concentração de 5% mas não à 0,5%. Adicionalmente, Papacárie Duo® modulou a ativação de macrófagos pela indução da expressão de Ptgs2 e Il10, sem alterar a expressão de Tnf e Mmp9. / The minimally invasive treatment has been increasingly used in the treatment of dental caries, particularly in young children. Thus, the removal of carious tissue by chemical-mechanical method allows greater conservation of healthy tooth structure. The Papacárie Duo® is an easily applied material, and has bactericidal and anti-inflammatory properties. This material is applied over the carious dentin in order to promote a softening thereof, facilitating its removal. The cellular effects of Papacárie Duo® are little known, therefore this research was to evaluate the effect of Papacárie Duo® in undifferentiated cells from dental pulp (Chapter 1) and the capacity of Papacárie Duo® to induce macrophage activation and synthesis inflammatory mediators (Chapter 2). The Papacárie Duo® was prepared at concentrations of 0.5 and 5% by serial dilution from the gel obtained commercially. OD-21 cells and J774.1 macrophages were maintained in culture with the different treatments for a period of 24 hours to perform the cytotoxicity assay (LDH assay) and for 36 hours for evaluation of cell proliferation (MTT colorimetric assay). The following, was carried out the assessment of the relative gene expression of Ibsp, Runx2 and Spp1 genes in OD-21 cells, and Il10, Mmp9, Tnf and Ptgs2 in J774.1 cells by reverse transcription method and reaction in real time polymerase chain reaction (qRT-PCR) using the TaqMan system after the stimulation period of 24 hours. The 5% Papacárie Duo® was cytotoxic to cells of dental pulp and inhibited cell proliferation and the expression of Runx2 and Ibsp. However, in both concentrations stimulated the expression of Spp1, which was higher at a concentration of 5%. In macrophages, the Papacárie Duo® was cytotoxic at concentrations of 5%, but did not affect the cell proliferation in any of the concentrations (0.5 and 5%). LPS inhibited cell proliferation in the presence or absence of Papacárie Duo® without giving cytotoxicity. The Papacárie Duo® induced the expression of Ptgs2 and Il10, without changing Tnf and Mmp9. Therefore, the Papacárie Duo® was cytotoxic, depending on the concentration, and showed inhibitory effect on the differentiation of pulp cells (OD-21), but without influencing cell proliferation. Papacárie Duo® not prevent macrophage proliferation, but was cytotoxic at a concentration of 5% but not more than 0.5%. Additionally, Papacárie Duo® modulated the activation of macrophages by inducing Il10 and Ptgs2 expression without altering the expression of ,Tnf, and Mmp9.