No / Microglial activation and behavioral abnormalities occur before neuronal loss in experimental murine prion disease; the behavioral changes coincide with a reduction in synaptic plasticity. Because synaptic plasticity depends on an intact perineuronal net (PN), a specialized extracellular matrix that surrounds parvalbumin (PV)-positive GABAergic (gamma-aminobutyric acid [GABA]) inhibitory interneurons, we investigated the temporal relationships between microglial activation and loss of PN and PV-positive neurons in ME7 murine prion disease. Anesthetized C57Bl/6J mice received bilateral intracerebral microinjections of ME7-infected or normal brain homogenate into the dorsal hippocampus. Microglial activation, PrP accumulation, the number of PV-positive interneurons, and Wisteria floribunda agglutinin-positive neurons (i.e. those with an intact PN) were assessed in the ventral CA1 and subiculum at 4, 8, 12, 16, and 20 weeks postinjection. Hippocampal areas and total neuron numbers in the ventral CA1 and subiculum were also determined. Loss of PN coincided with early microglial activation and with a reduction in synaptic plasticity. No significant loss of PV-positive interneurons was observed. Our findings suggest that the substrate of the earliest synaptic and behavioral abnormalities in murine prion disease may be inflammatory microglia-mediated degradation of the PN.

No description available.

L'assemblage des nucléosomes est étroitement couplée à la synthèse des histones ainsi qu’à la réplication et la réparation de l’ADN durant la phase S. Ce processus implique un mécanisme de contrôle qui contribue soigneusement et de manière régulée à l’assemblage de l’ADN en chromatine. L'assemblage des nucléosomes durant la synthèse de l’ADN est crucial et contribue ainsi au maintien de la stabilité génomique. Cette thèse décrit la caractérisation par spectrométrie de masse(SM) des protéines jouant un rôle critique dans l’assemblage et le maintien de la structure chromatinienne. Plus précisément, la phosphorylation de deux facteurs d’assemblage des nucléosome, le facteur CAF-1, une chaperone d’histone qui participe à l'assemblage de la chromatine spécifiquement couplée à la réplication de l'ADN, ainsi que le complexe protéique Hir, jouant de plus un rôle important dans la régulation transcriptionelle des gènes d’histones lors de la progression normale du cycle cellulaire et en réponse aux dommages de l'ADN, a été examiné. La caractérisation des sites de phosphorylation par SM nécéssite la séparation des protéines par éléctrophorèse suivi d’une coloration a l’argent. Dans le chapitre 2, nous demontrons que la coloration à l’argent induit un artéfact de sulfatation. Plus précisément, cet artéfact est causé par un réactif spécifiquement utilisé lors de la coloration. La sulfatation présente de fortes similitudes avec la phosphorylation. Ainsi, l’incrément de masse observé sur les peptides sulfatés et phosphorylés (+80 Da) nécéssite des instruments offrant une haute résolution et haute précision de masse pour différencier ces deux modifications. Dans les chapitres 3 et 4, nous avons d’abord démontré par SM que Cac1, la plus grande sous-unité du facteur CAF-1, est cible de plusieurs sites de phosphorylation. Fait intéréssant, certains de ces sites contiennent des séquences consensus pour les kinases Cdc7-Dbf4 et CDKs. Ainsi, ces résultats fournissent les premières évidences que CAF-1 est potentiellement régulé par ces deux kinases in vivo. La fonction de tous les sites de phosphorylation identifiés a ensuite été évaluée. Nous avons démontré que la phosphorylation de la Ser-503, un site consensus de la DDK, est essentielle à la répréssion transcriptionelle des gènes au niveau des télomères. Cependant, cette phosphorylation ne semble pas être nécéssaire pour d’autres fonctions connues de CAF-1, indiquant que le blocage de la phsophorylation de Cac1 Ser-503 affecte spécifiquement la fonction de CAF-1 aux structures hétérochromatiques des télomères. Ensuite, nous avons identifiés une intéraction physique entre CAF-1 et Cdc7-Dbf4. Des études in vitro ont également demontré que cette kinase phosphoryle spécifiquement Cac1 Ser-503, suggérant un rôle potential pour la kinase Cdc7-Dbf4 dans l’assemblage et la stabilité de la structure hétérochromatique aux télomères. Finalement, les analyses par SM nous ont également permi de montrer que la sous-unité Hpc2 du complexe Hir est phosphorylée sur plusieurs sites consensus des CDKs et de Cdc7-Dbf4. De plus, la quantification par SM d’un site spécifique de phosphorylation de Hpc2, la Ser-330, s’est révélée être fortement induite suite à l’activation du point de contrôle de réplication (le “checkpoint”) suite au dommage a l’ADN. Nous montrons que la Ser-330 de Hpc2 est phopshorylée par les kinases de point de contrôle de manière Mec1/Tel1- et Rad53-dépendante. Nos données préliminaires suggèrent ainsi que la capacité du complex Hir de réguler la répréssion transcriptionelle des gènes d'histones lors de la progression du cycle cellulaire normal et en réponse au dommage de l'ADN est médiée par la phosphorylation de Hpc2 par ces deux kinases. Enfin, ces deux études mettent en évidence l'importance de la spectrométrie de masse dans la caractérisation des sites de phosphorylation des protéines, nous permettant ainsi de comprendre plus précisement les mécanismes de régulation de l'assemblage de la chromatine et de la synthèse des histones. / Nucleosome assembly entails a controlled mechanism that is tightly coupled to DNA and histone synthesis during DNA replication and repair in S-phase. Importantly, this contributes to the prompt and carefully orchestrated assembly of newly replicated DNA into chromatin, which is essential for the maintenance of genomic integrity. This thesis describes the mass spectrometric characterization of proteins critical in the regulation of nucleosome assembly behind the replication fork and chromatin structure. More specifically, the phosphorylation of Chromatin Assembly Factor 1 (CAF-1), a nucleosome assembly factor that uniquely functions during replication-coupled de novo nucleosome assembly in S-phase and the Hir protein complex, a second nucleosome assembly factor that also contributes to the transcriptional regulation of histone genes during normal cell cycle progression and in response to DNA damage, was examined. We first demonstrated that characterization of protein phosphorylation by mass spectrometry (MS), which often relies on the separation of proteins by gel electrophoresis followed by silver staining for visualization, should be given careful considerations. In chapter 2, we report a potential pitfall in the interpretation of phosphorylation modifications due to the artifactual sulfation of serine, threonine and tyrosine residues caused by a specific reagent used during silver staining. Sulfation and phosphorylation both impart an 80 Da addition of these residues making them distinguishable only with MS systems offering high resolution and high mass accuracy capabilities. Chapter 3 and 4 present the MS characterization of in vivo phosphorylation occurring on CAF-1 and Hir proteins, respectively. We first demonstrated that Cac1, the largest subunit of CAF-1, is phosphorylated on several novel residues containing the consensus sequences recognized by either Cdc7-Dbf4 (DDK) or cyclin-dependent kinases(CDKs). These results have provided the first evidence that CAF-1 is regulated by these two kinases in vivo. The function of all identified Cac1 phosphorylation sites was then assessed. In vivo phenotypic studies showed that the specific phosphorylation of Ser-503, a Cac1 DDK-like site identified in our study, is essential for heterochromatin-mediated telomeric silencing. Cac1-Ser-503 did not appear to be required for other known functions of CAF-1, including DNA damage resistance and mitotic chromosom segregation, indicating that blocking Cac1 phosphorylation on Ser-503 sepcifically cripples CAF-1 function at telomeres. Next, biochemical purifications identified a physical interaction between CAF-1 and Cdc7-Dbf4. Consistent with this physical interaction data, in vitro kinase assay studies showed that Cdc7-Dbf4 specifically phosphorylates Cac1 Ser-503 thereby uncovering a novel role for Cdc7-Dbf4 in heterochromatin assembly and/or stability that is potentially mediated through CAF-1. Finally, MS analysis also showed that the Hpc2 subunit of the Hir protein complex is phosphorylated on several CDK- and DDK-like consensus sites. Furthermore, MS quantification of a specific phosphorylation site,Hpc2 Ser-330, was shown to be highly induced following the activation of the DNA damage checkpoint in response to DNA damage. We show that Hpc2 Ser-330 is phopshorylated by checkpoint kinases in a Mec1/Tel1- and Rad53-dependent manner. Our preliminary data suggest that the ability of the Hir protein complex to regulate the transcriptional repression of histone genes during normal cell cycle progression and in response to DNA damage is mediated through the regulated phosphorylation of Hpc2 by these kinases. Finally, these two studies highlight the importance of mass spectrometry in characterizing protein phosphorylation events, which has yielded novel insights into the regulation of chromatin assembly by CAF-1 and histone synthesis mediated by Hir proteins.

A fototerapia com laser em baixa intensidade (FTLBI) é capaz de aumentar o metabolismo celular, o que poderia influenciar na diferenciação ósseo/odontogênica das células-tronco da polpa dentária humada (hDPSCs). O PDGF e o BMP-2 são fatores de crescimento envolvidos na dentinogênese e na reparação tecidual. O PDGF tem papel importante durante o desenvolvimento embrionário, na proliferação e migração celular e na angiogênese, enquanto o BMP-2 está fortemente associado à diferenciação celular em tecidos mineralizados, como o osso e a dentina. Sendo assim, o objetivo do estudo foi analisar os efeitos da FTLBI e dos fatores de crescimento (PDGF-BB ou BMP-2), isolados ou em associação, na diferenciação ósseo/odontogênica das hDPSCs. Para o estudo hDPSCs foram cultivadas em meio regular (G1) e irradiadas (G2), meio mineralizante (G3) e irradiadas (G4), meio mineralizante contendo PDGF-BB (G5) e irradiadas (G6), meio mineralizante contendo BMP-2 (G7) e irradiadas (G8). Para os grupos irradiados, a FTLBI foi realizada no modo pontual e em contato, com um laser de diodo semi-condutor, com área de feixe de 0,028cm2 e comprimento de onda 660nm (InGaAlP-vermelho), utilizando-se os seguintes parâmetros: potência de 20mW, densidade de energia de 5J/cm2, tempo de irradiação de 7 segundos por ponto e 0,14J de energia por ponto. A expressão dos genes relacionados à diferenciação ósseo/odontogênica (DSPP, DMP-1 e OCN) através do PCR quantitativo em tempo real (qRT-PCR), a atividade da fosfatase alcalina e os depósitos de cálcio foram analisados em 3, 7 e 14 dias. Os dados obtidos foram comparados pelo teste ANOVA complementado pelo teste de Tukey (p<0,05). As culturas tratadas com meio mineralizante contendo BMP-2 e irradiadas (G8) foram as que mostraram os maiores índices de diferenciação ósseo/odontogênica nos testes realizados. As expressões de DSPP, OCN e DMP-1, ao menos em 14 dias, foram significantemente maiores no G8 que nos demais grupos experimentais, exceto os grupos G3 e G7. Estes grupos apresentaram expressões de DSPP e OCN semelhantes às do G8 em 14 dias. A maior atividade de ALP foi observada no G8 em 3 dias e a menor no mesmo grupo aos 14 dias. A maior quantidade de depósitos de cálcio também foi encontrada no G8 em 14 dias. A associação de FTLBI e BMP-2 se mostrou capaz de induzir a diferenciação ósseo/odontogênica em células-tronco de polpa dentária humana de forma mais marcante que as demais terapias isoladas ou associadas estudadas. Portanto, o uso de uma terapia associando FTLBI e BMP-2 poderia ser de relevância para o restabelecimento da fisiologia pulpar quando aplicada em casos de exposição deste tecido, uma vez que poderia favorecer a diferenciação das células indiferenciadas da polpa dentária. / Laser phototherapy (LPT) is able to increase cellular metabolism, which in turn could influence the odontogenic differentiation of dental pulp stem cells (hDPSCs). PDGF and BMP-2 are growth factors involved in dentinogenesis and tissue repair. PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis, whereas BMP-2 is strongly associated with cell differentiation in mineralized tissues such as bone and dentin. The aim of this study was to analyze the effects of LPT and the growth factors PDGF-BB and BMP-2 combined or not on the odontogenic differentiation of hDPSCs. These cells were grown in regular medium (G1) and irradiated (G2), mineralizing medium (G3) and irradiated (G4), mineralizing medium containing PDGF-BB (G5) and irradiated (G6), mineralizing medium containing BMP-2 (G7) and irradiated (G8). For irradiated groups, LPT was performed in punctual and contact mode with a semiconductor diode laser, with a beam spot area of 0.028 cm2 and wavelength of 660nm (InGaAlP-visible red), using the following parameters: power of 20mW, energy density of 5J/cm2 and irradiation time of 7 seconds per point (0,14 J per point). Differentiation was assessed by the following analysis: expression of genes related to odontogenic differentiation (DSPP, DMP-1 and OCN) using quantitative real time PCR (qRT-PCR); alkaline phosphatase activity and calcium deposition using alizarin red staining in 3, 7 and 14 days. Data were compared by ANOVA and Tukey´s test (p<0.05). The cultures treated with mineralizing medium containing BMP-2 and irradiated (G8) showed the highest rate of odontogenic differentiation. The expressions of DSPP, DMP-1 and OCN genes, at least in 14 days, were significantly higher in G8 compared to all other groups, except for the groups G3 and G7. These groups showed similar expressions of DSPP and OCN than G8 in 14 days. G8 showed the highest ALP activity in 3 days and the lowest in 14 days compared to all other groups. The largest amount of calcium deposits was observed in G8 in 14 days. The most striking feature on induction of odontogenic differentiation of hDPSCs was observed when LPT was applied in association with BMP-2. Therefore, the use of a combined LPT and BMP-2 therapy could be of relevance for the re-establishment of pulp physiology when applied in cases of dental pulp exposure by promoting the differentiation of hDPSCs.

Could shareholder primacy, with its assumed short-termist practices, have had its day when it comes to managerial activity centered on creating corporate value? Many business and opinion leaders appear to take this position, not least Jack Welch who famously declared 'shareholder primacy is the dumbest idea in the world!' Indeed, in a post-Crash economy has a wider stakeholder focus with a longer-term outlook superseded any business notions of shareholder primacy and wealth maximization? This research examines these possibilities through a consideration of the narrative companies produce, such as annual reports. From this corpus material, an assessment is made of whether UK managers' perceptions about corporate value generation changed over the period covering the worldwide financial crisis, with respect to their relative favouring of shareholders and stakeholders. The corpus of narrative material used is visualized as a conceptual space in which a conversation reflecting perceptual bias to the generation of corporate value occurs. To explore such corpuses, in order to compare narratives at points either side of the 2008 Crash, a new methodology was devised called narrative staining. Hence, a detection and visual mapping over the period was made possible of managers' changing perceptions concerning primacy (shareholder or stakeholder orientation) with its mediation by termism (a short or long-term bias). Termism is also originally conceived as part of a larger temporal category, which includes a sense of urgency to act (urgent versus non-urgent) that is similarly examined. The investigation reveals that over time perceptual change about value creation happened, though in unanticipated ways. Companies pre-Crash were often short-term stakeholder oriented then moved post-Crash to a long-term shareholder orientation. A focus for this study was the corporate domain, consisting of a selection of FT250 companies. However, managerial perceptions about corporate value creation are influenced not simply by the conversation of the corporate domain but rather by a multi-actor conversation taking place throughout the business environment. To comprehend this effect, the research mines further corpuses that comprise the UK's regulatory domain (hard and soft law), the press (Financial Times and other newspapers), and relevant peripheral stakeholder organizations (including the Confederation of British Industry, the Institute of Directors, and the Trades Union Congress). These organizations demonstrated more complex, unforeseen, perceptual effects as the financial crisis proceeded with many aligning according to their political or business agenda, which also impacted any sense of urgency to act they had. There appears to be no previous attempt at an extensive and multivariate analysis of this nature. And the findings challenge prevalent characterizations of shareholder and stakeholder behaviour. Moreover, the research shows that utilizing a wide set of stakeholder corpuses acts a viable proxy for broader financial perspectives amongst UK organizations. The technique of narrative staining therefore provides insights, hitherto inaccessible, for assessing and consolidating large-scale perceptual bias regarding value creation across the economy. The technique also has significant potential for other applications.

No description available.

No description available.

L'assemblage des nucléosomes est étroitement couplée à la synthèse des histones ainsi qu’à la réplication et la réparation de l’ADN durant la phase S. Ce processus implique un mécanisme de contrôle qui contribue soigneusement et de manière régulée à l’assemblage de l’ADN en chromatine. L'assemblage des nucléosomes durant la synthèse de l’ADN est crucial et contribue ainsi au maintien de la stabilité génomique. Cette thèse décrit la caractérisation par spectrométrie de masse(SM) des protéines jouant un rôle critique dans l’assemblage et le maintien de la structure chromatinienne. Plus précisément, la phosphorylation de deux facteurs d’assemblage des nucléosome, le facteur CAF-1, une chaperone d’histone qui participe à l'assemblage de la chromatine spécifiquement couplée à la réplication de l'ADN, ainsi que le complexe protéique Hir, jouant de plus un rôle important dans la régulation transcriptionelle des gènes d’histones lors de la progression normale du cycle cellulaire et en réponse aux dommages de l'ADN, a été examiné. La caractérisation des sites de phosphorylation par SM nécéssite la séparation des protéines par éléctrophorèse suivi d’une coloration a l’argent. Dans le chapitre 2, nous demontrons que la coloration à l’argent induit un artéfact de sulfatation. Plus précisément, cet artéfact est causé par un réactif spécifiquement utilisé lors de la coloration. La sulfatation présente de fortes similitudes avec la phosphorylation. Ainsi, l’incrément de masse observé sur les peptides sulfatés et phosphorylés (+80 Da) nécéssite des instruments offrant une haute résolution et haute précision de masse pour différencier ces deux modifications. Dans les chapitres 3 et 4, nous avons d’abord démontré par SM que Cac1, la plus grande sous-unité du facteur CAF-1, est cible de plusieurs sites de phosphorylation. Fait intéréssant, certains de ces sites contiennent des séquences consensus pour les kinases Cdc7-Dbf4 et CDKs. Ainsi, ces résultats fournissent les premières évidences que CAF-1 est potentiellement régulé par ces deux kinases in vivo. La fonction de tous les sites de phosphorylation identifiés a ensuite été évaluée. Nous avons démontré que la phosphorylation de la Ser-503, un site consensus de la DDK, est essentielle à la répréssion transcriptionelle des gènes au niveau des télomères. Cependant, cette phosphorylation ne semble pas être nécéssaire pour d’autres fonctions connues de CAF-1, indiquant que le blocage de la phsophorylation de Cac1 Ser-503 affecte spécifiquement la fonction de CAF-1 aux structures hétérochromatiques des télomères. Ensuite, nous avons identifiés une intéraction physique entre CAF-1 et Cdc7-Dbf4. Des études in vitro ont également demontré que cette kinase phosphoryle spécifiquement Cac1 Ser-503, suggérant un rôle potential pour la kinase Cdc7-Dbf4 dans l’assemblage et la stabilité de la structure hétérochromatique aux télomères. Finalement, les analyses par SM nous ont également permi de montrer que la sous-unité Hpc2 du complexe Hir est phosphorylée sur plusieurs sites consensus des CDKs et de Cdc7-Dbf4. De plus, la quantification par SM d’un site spécifique de phosphorylation de Hpc2, la Ser-330, s’est révélée être fortement induite suite à l’activation du point de contrôle de réplication (le “checkpoint”) suite au dommage a l’ADN. Nous montrons que la Ser-330 de Hpc2 est phopshorylée par les kinases de point de contrôle de manière Mec1/Tel1- et Rad53-dépendante. Nos données préliminaires suggèrent ainsi que la capacité du complex Hir de réguler la répréssion transcriptionelle des gènes d'histones lors de la progression du cycle cellulaire normal et en réponse au dommage de l'ADN est médiée par la phosphorylation de Hpc2 par ces deux kinases. Enfin, ces deux études mettent en évidence l'importance de la spectrométrie de masse dans la caractérisation des sites de phosphorylation des protéines, nous permettant ainsi de comprendre plus précisement les mécanismes de régulation de l'assemblage de la chromatine et de la synthèse des histones. / Nucleosome assembly entails a controlled mechanism that is tightly coupled to DNA and histone synthesis during DNA replication and repair in S-phase. Importantly, this contributes to the prompt and carefully orchestrated assembly of newly replicated DNA into chromatin, which is essential for the maintenance of genomic integrity. This thesis describes the mass spectrometric characterization of proteins critical in the regulation of nucleosome assembly behind the replication fork and chromatin structure. More specifically, the phosphorylation of Chromatin Assembly Factor 1 (CAF-1), a nucleosome assembly factor that uniquely functions during replication-coupled de novo nucleosome assembly in S-phase and the Hir protein complex, a second nucleosome assembly factor that also contributes to the transcriptional regulation of histone genes during normal cell cycle progression and in response to DNA damage, was examined. We first demonstrated that characterization of protein phosphorylation by mass spectrometry (MS), which often relies on the separation of proteins by gel electrophoresis followed by silver staining for visualization, should be given careful considerations. In chapter 2, we report a potential pitfall in the interpretation of phosphorylation modifications due to the artifactual sulfation of serine, threonine and tyrosine residues caused by a specific reagent used during silver staining. Sulfation and phosphorylation both impart an 80 Da addition of these residues making them distinguishable only with MS systems offering high resolution and high mass accuracy capabilities. Chapter 3 and 4 present the MS characterization of in vivo phosphorylation occurring on CAF-1 and Hir proteins, respectively. We first demonstrated that Cac1, the largest subunit of CAF-1, is phosphorylated on several novel residues containing the consensus sequences recognized by either Cdc7-Dbf4 (DDK) or cyclin-dependent kinases(CDKs). These results have provided the first evidence that CAF-1 is regulated by these two kinases in vivo. The function of all identified Cac1 phosphorylation sites was then assessed. In vivo phenotypic studies showed that the specific phosphorylation of Ser-503, a Cac1 DDK-like site identified in our study, is essential for heterochromatin-mediated telomeric silencing. Cac1-Ser-503 did not appear to be required for other known functions of CAF-1, including DNA damage resistance and mitotic chromosom segregation, indicating that blocking Cac1 phosphorylation on Ser-503 sepcifically cripples CAF-1 function at telomeres. Next, biochemical purifications identified a physical interaction between CAF-1 and Cdc7-Dbf4. Consistent with this physical interaction data, in vitro kinase assay studies showed that Cdc7-Dbf4 specifically phosphorylates Cac1 Ser-503 thereby uncovering a novel role for Cdc7-Dbf4 in heterochromatin assembly and/or stability that is potentially mediated through CAF-1. Finally, MS analysis also showed that the Hpc2 subunit of the Hir protein complex is phosphorylated on several CDK- and DDK-like consensus sites. Furthermore, MS quantification of a specific phosphorylation site,Hpc2 Ser-330, was shown to be highly induced following the activation of the DNA damage checkpoint in response to DNA damage. We show that Hpc2 Ser-330 is phopshorylated by checkpoint kinases in a Mec1/Tel1- and Rad53-dependent manner. Our preliminary data suggest that the ability of the Hir protein complex to regulate the transcriptional repression of histone genes during normal cell cycle progression and in response to DNA damage is mediated through the regulated phosphorylation of Hpc2 by these kinases. Finally, these two studies highlight the importance of mass spectrometry in characterizing protein phosphorylation events, which has yielded novel insights into the regulation of chromatin assembly by CAF-1 and histone synthesis mediated by Hir proteins.

A fototerapia com laser em baixa intensidade (FTLBI) é capaz de aumentar o metabolismo celular, o que poderia influenciar na diferenciação ósseo/odontogênica das células-tronco da polpa dentária humada (hDPSCs). O PDGF e o BMP-2 são fatores de crescimento envolvidos na dentinogênese e na reparação tecidual. O PDGF tem papel importante durante o desenvolvimento embrionário, na proliferação e migração celular e na angiogênese, enquanto o BMP-2 está fortemente associado à diferenciação celular em tecidos mineralizados, como o osso e a dentina. Sendo assim, o objetivo do estudo foi analisar os efeitos da FTLBI e dos fatores de crescimento (PDGF-BB ou BMP-2), isolados ou em associação, na diferenciação ósseo/odontogênica das hDPSCs. Para o estudo hDPSCs foram cultivadas em meio regular (G1) e irradiadas (G2), meio mineralizante (G3) e irradiadas (G4), meio mineralizante contendo PDGF-BB (G5) e irradiadas (G6), meio mineralizante contendo BMP-2 (G7) e irradiadas (G8). Para os grupos irradiados, a FTLBI foi realizada no modo pontual e em contato, com um laser de diodo semi-condutor, com área de feixe de 0,028cm2 e comprimento de onda 660nm (InGaAlP-vermelho), utilizando-se os seguintes parâmetros: potência de 20mW, densidade de energia de 5J/cm2, tempo de irradiação de 7 segundos por ponto e 0,14J de energia por ponto. A expressão dos genes relacionados à diferenciação ósseo/odontogênica (DSPP, DMP-1 e OCN) através do PCR quantitativo em tempo real (qRT-PCR), a atividade da fosfatase alcalina e os depósitos de cálcio foram analisados em 3, 7 e 14 dias. Os dados obtidos foram comparados pelo teste ANOVA complementado pelo teste de Tukey (p<0,05). As culturas tratadas com meio mineralizante contendo BMP-2 e irradiadas (G8) foram as que mostraram os maiores índices de diferenciação ósseo/odontogênica nos testes realizados. As expressões de DSPP, OCN e DMP-1, ao menos em 14 dias, foram significantemente maiores no G8 que nos demais grupos experimentais, exceto os grupos G3 e G7. Estes grupos apresentaram expressões de DSPP e OCN semelhantes às do G8 em 14 dias. A maior atividade de ALP foi observada no G8 em 3 dias e a menor no mesmo grupo aos 14 dias. A maior quantidade de depósitos de cálcio também foi encontrada no G8 em 14 dias. A associação de FTLBI e BMP-2 se mostrou capaz de induzir a diferenciação ósseo/odontogênica em células-tronco de polpa dentária humana de forma mais marcante que as demais terapias isoladas ou associadas estudadas. Portanto, o uso de uma terapia associando FTLBI e BMP-2 poderia ser de relevância para o restabelecimento da fisiologia pulpar quando aplicada em casos de exposição deste tecido, uma vez que poderia favorecer a diferenciação das células indiferenciadas da polpa dentária. / Laser phototherapy (LPT) is able to increase cellular metabolism, which in turn could influence the odontogenic differentiation of dental pulp stem cells (hDPSCs). PDGF and BMP-2 are growth factors involved in dentinogenesis and tissue repair. PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis, whereas BMP-2 is strongly associated with cell differentiation in mineralized tissues such as bone and dentin. The aim of this study was to analyze the effects of LPT and the growth factors PDGF-BB and BMP-2 combined or not on the odontogenic differentiation of hDPSCs. These cells were grown in regular medium (G1) and irradiated (G2), mineralizing medium (G3) and irradiated (G4), mineralizing medium containing PDGF-BB (G5) and irradiated (G6), mineralizing medium containing BMP-2 (G7) and irradiated (G8). For irradiated groups, LPT was performed in punctual and contact mode with a semiconductor diode laser, with a beam spot area of 0.028 cm2 and wavelength of 660nm (InGaAlP-visible red), using the following parameters: power of 20mW, energy density of 5J/cm2 and irradiation time of 7 seconds per point (0,14 J per point). Differentiation was assessed by the following analysis: expression of genes related to odontogenic differentiation (DSPP, DMP-1 and OCN) using quantitative real time PCR (qRT-PCR); alkaline phosphatase activity and calcium deposition using alizarin red staining in 3, 7 and 14 days. Data were compared by ANOVA and Tukey´s test (p<0.05). The cultures treated with mineralizing medium containing BMP-2 and irradiated (G8) showed the highest rate of odontogenic differentiation. The expressions of DSPP, DMP-1 and OCN genes, at least in 14 days, were significantly higher in G8 compared to all other groups, except for the groups G3 and G7. These groups showed similar expressions of DSPP and OCN than G8 in 14 days. G8 showed the highest ALP activity in 3 days and the lowest in 14 days compared to all other groups. The largest amount of calcium deposits was observed in G8 in 14 days. The most striking feature on induction of odontogenic differentiation of hDPSCs was observed when LPT was applied in association with BMP-2. Therefore, the use of a combined LPT and BMP-2 therapy could be of relevance for the re-establishment of pulp physiology when applied in cases of dental pulp exposure by promoting the differentiation of hDPSCs.

Die vorliegende Arbeit liefert einen Beitrag zum Verständnis der elektrochemischen Prozesse an der Elektrodengrenzfläche und im Festkörper graphitischer Anoden für Lithium-Ionen-Batterien. Der Zusammenhang zwischen den intrinsischen Eigenschaften des Aktivmaterials und den resultierenden Eigenschaften von Kompositelektroden stand dabei im Fokus der Untersuchungen. Die Temperaturabhängigkeit von Materialeigenschaften (Diffusionskoeffizient, Austauschstromdichte) und Elektrodeneigenschaften (Verhalten unter Strombelastung) wurde in einem Bereich von 40 °C bis -10 °C erfasst. Dazu werden elektrochemische Charakterisierungsmethoden aus der Literatur vorgestellt und hinsichtlich ihrer Gültigkeit für die Anwendung an realen Elektroden evaluiert. Die elektrochemisch aktive Oberfläche wurde bestimmt und stellte sich als ausschlaggebender Parameter für die Bewertung der Elektrodenprozesse heraus. Auf Basis korrigierter Elektrodenoberflächen konnten Austauschstromdichten für die konkurrierenden Prozesse Lithium-Interkalation und -Abscheidung ermittelt werden. Zusammen mit Kennwerten zur Keimbildungsüberspannung für Lithium-Abscheidung flossen die ermittelten Kennwerte in eine theoretische Berechnung des Zellstroms ein. Es konnte gezeigt werden, dass die Lithium-Abscheidung kinetisch deutlich gegenüber der Lithium-Interkalation bevorzugt ist, nicht nur bei niedriger Temperatur. Die Übertragbarkeit wissenschaftlicher Grundlagenexperimente auf kommerzielle Systeme war bei allen Versuchen Gegenstand der Untersuchungen. In einem separaten Beispiel einer Oberflächenmodifikation mit Zinn wurde diese Problematik besonders verdeutlicht. Zusätzlich wurde die parasitäre Abscheidung von Lithium auf graphitischen Anoden hinsichtlich der Nachweisbarkeit und Quantifizierung evaluiert. Hierfür wurde eine neue Untersuchungsmethode im Bereich der Lithium-Ionen-Batterie zur besseren Detektion von Lithium-Abscheidung und Grenzflächen-Morphologie mittels Elektronenmikroskopie entwickelt. Die Osmiumtetroxid (OsO4) Färbung ermöglichte eine deutliche Verbesserung des Materialkontrasts und erlaubte somit eine gezielte Untersuchung von graphitischen Anoden nach erfolgter Lithium-Abscheidung. Darüber hinaus konnte die selektive Reaktion des OsO4 für eine genauere Betrachtung der Solid Electrolyte Interphase genutzt werden. Eine Stabilisierung der Proben an Luft und im Elektronenstrahl konnte erreicht werden. / This work sheds light on the electrochemical processes occurring at commercially processed graphitic anodes. It raises the question whether values published in literature for mostly ideal electrode systems can be readily taken for simulation and design of real electrodes in high-energy cells. A multiple step approach is given, evaluating different methods to determine electrode and material properties independently. The electrochemically active surface area was shown to be a crucial parameter for the calculation of electrode kinetics. Using exchange current densities corrected for the electrode surface area, the overall charging current in a cell could be calculated. The resulting part of lithium deposition in the charging process is strikingly high, not only at low temperatures. To further investigate lithium deposition in terms of morphology and quantity, a method was developed for graphitic anodes. Osmium tetroxide (OsO4) staining serves well as a tool to strongly increase material contrast in electron microscopy. Thus lithium dendrites could be made visible in an unprecedented manner. Furthermore, the selective chemical reaction of osmium tetroxide allows for a better investigation of the multi-layer solid electrolyte interphase as was shown in transmission electron microscopy. Using the staining method, a stabilization of the sample under air and in the electron beam could be achieved.