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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Ludwig Erks kritische Liedersammlung und sein "Volkslied"-Begriff die Methoden der Aufzeichnung, Dokumentation, Text- und Melodiekritik und Edition von Volksliedern : ein Beitrag zur Wissenschaftsgeschichte /

Schade, Ernst, January 1971 (has links)
Thesis--Marburg. / Vita. Includes bibliographical references (p. [278]-286).
2

RASA3, a Key Player in Dopamine D2S Receptor-mediated MAPK Signaling

Ma, Xun 10 February 2011 (has links)
The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.
3

RASA3, a Key Player in Dopamine D2S Receptor-mediated MAPK Signaling

Ma, Xun 10 February 2011 (has links)
The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.
4

RASA3, a Key Player in Dopamine D2S Receptor-mediated MAPK Signaling

Ma, Xun 10 February 2011 (has links)
The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.
5

RASA3, a Key Player in Dopamine D2S Receptor-mediated MAPK Signaling

Ma, Xun January 2011 (has links)
The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.
6

Probing the Function of PolyP on Signalling Networks in Mammalian Systems

Nasser, Rawan 12 November 2020 (has links)
Polyphosphates (PolyP) are linear chains of inorganic phosphates joined together by phosphoanhydride bonds. This polyanionic molecule has been found in all organisms and has been implicated in diverse biological processes such as cell growth and blood coagulation. Nonetheless, the mechanism of polyP’s involvement in these processes and its impact at a molecular level is still unknown. In 2015, the addition of polyP chains to lysine residues was found to be a novel post-translational modification. However, a hurdle in studying the impact of polyP in the mammalian system is its low endogenous levels. In this study, I applied an ectopic expression system using E. coli-derived polyphosphate kinase 1 (PPK) in HEK293T cells to induce excess production of polyP. In mammalian cells, increased intracellular polyP levels lead to increased activation of both Erk1/2 and p70s6k. I also expanded our system by utilizing the yeast exopolyphosphatase, Ppx1, to deplete polyP. Overall, this work presents a novel role for polyP in mediating key signalling pathways and will help probe the function of polyP in mammalian cells.
7

Efeito dos laseres de baixa intensidade na proliferação e diferenciação de osteoblastos humanos / Low level laser effects in proliferation and differentiation of human osteoblasts

Oliveira, Flávia Amadeu de 18 November 2013 (has links)
Dentre os vários compostos utilizados na pesquisa e na terapia de doenças osteo-degenerativas, a fototerapia com laseres de baixa potência (LLLT) e os diodos emissores de luz (LEDs) vem sendo investigada com o intuito de avaliar seus efeitos no metabolismo ósseo. Estes, que possuem comprimentos de ondas específicos, atuam na biomodulação das células, funcionando como um agente terapêutico, reequilibrando e normalizando a sua atividade. No entanto, pouco se sabe sobre o efeito dos diferentes espectros na proliferação e diferenciação de osteoblastos humanos, bem como seus efeitos no metabolismo celular como a síntese e a ativação de proteínas sinalizadoras envolvidas nesses processos. Diante disso, o objetivo deste trabalho foi avaliar, comparativamente, a influência da fototerapia com LLLT e LED na proliferação e diferenciação de osteoblastos humanos. Além disso, investigamos o envolvimento da ativação da via de sinalização ERK1,2 nestas respostas, utilizando o seu inibidor específico e/ou avaliando a sua ativação durante a proliferação e após fototerapia. Para esse estudo, osteoblastos humanos (HOAL) foram cultivados em meio de cultura DMEM suplementado com 10% de soro fetal bovino (SFB) e incubados em estufa de CO2. As células foram irradiadas pontualmente com os laseres vermelho (660nm), infravermelho (780nm) e LED (637nm), nas doses de 10, 20 e 50 J/cm2 na potência de 40mW, após adesão celular. Após 24, 48, e 72 horas foram realizados os ensaios de redução do MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5- difeniltetrazólio) e cristal violeta (CV) para avaliar a viabilidade das células e após 72 horas foi realizada a análise da proliferação por citometria de fluxo nos quais os resultados sugerem aumento de células viáveis ou proliferação quando estimuladas pelos diferentes espectros. Após a verificação do efeito positivo dos laseres e LED na viabilidade e/ou proliferação, foi realizada a análise da ativação da proteína intracelular ERK por western blotting usando anticorpo específico após 10 minutos da irradiação pontual. Mostramos que a irradiação das células HOAL com LLLT, na dose de 10 J/cm2, aumentou significativamente a fosforilação da ERK1/2 em relação ao controle. Para os ensaios de diferenciação, as células receberam pontualmente o estimulo a cada 6 dias e após os períodos de 07, 14, 21 e 28 dias foram realizados os ensaios da atividade de fosfatase alcalina (ALP), expressão gênica do colágeno I (COL1A1) e SPARC (osteonectina) por RT-PCR em tempo real e ensaio de mineralização com vermelho de alizarina. De um modo geral, não foram observados diferenças na atividade da ALP entre os grupos tratados em relação ao controle. A expressão gênica do COL1A1 e SPARC foi aumentada, no qual o LED em ambas doses apresentou maior eficácia em aumentar a expressão desses genes. No que se refere à mineralização, foram observadas pequenas diferenças quanto a deposição de cálcio. Dessa forma, a LLLT e LED, nesse regime de aplicação modulou positivamente o metabolismo dos osteoblastos humanos em relação à viabilidade, porém, no processo de mineralização foram observadas poucas diferenças. / Among the various compounds used in research and bone degenerative diseases therapy, phototherapy with low level laser (LLLT) and light emitting diodes (LEDs) has been investigated in order to evaluate its effects on bone metabolism. Those, who have specific wavelengths, act in biomodulation cells functioning as a therapeutic agent, rebalancing and normalizing their activity. However, little is known about the effect of the different spectra in the proliferation and differentiation of human osteoblasts and their effects on cellular metabolism as well as the synthesis and activation of signaling proteins involved in these processes. Therefore, the aim of this study was to compare the influence of LLLT and LED phototherapy in the proliferation and differentiation of human osteoblasts. In addition, we investigated the involvement of activation of ERK1,2 signaling pathway these responses using its specific inhibitor and/or evaluating their activation during the proliferation and after phototherapy. For this study, human osteoblasts (HOAL) were cultured in DMEM culture medium supplemented with 10 % fetal bovine serum (FBS) and incubated in CO2 incubator . Cells were irradiated with punctual red lasers (660nm), infrared (780nm) and LED (637nm) at doses of 10, 20 and 50 J/cm2 in power 40mW, after cell adhesion. After 24, 48, and 72 hours, MTT assay (- (4,5- dimethylthiazol-2- yl) -2,5 - diphenyltetrazolium bromide 3 ) and violet crystal (CV) were performed to assess the viability of cells and after 72 hours, was performed of proliferation analysis by flow cytometry. The results suggest an increase in viable and proliferation of cells when stimulated by different spectra. After checking the positive effect of lasers and LED viability and/or proliferation, analysis of ERK activation of intracellular protein by western blotting using a specific antibody was performed 10 minutes after the spot irradiation. We show that irradiation of HOAL cells with LLLT at a dose of 10 J/cm2, significantly increased the phosphorylation of ERK1/2 compared to control. For differentiation assays, cells promptly received stimulation every 6 days and after periods of 07, 14, 21 and 28 days testing the activity of alkaline phosphatase (ALP), gene expression of type I collagen (COL1A1) and SPARC (osteonectin) were performed by Real Time RT-PCR and mineralization experiment with alizarine red. In general, no differences in the activity of ALP between the treated groups compared to the control were observed. The gene expression was increased, and SPARC COL1A1, where in the LED in both doses showed greater effectiveness in increasing the expression of these genes. With respect to mineralization, small differences in the deposition of calcium were observed. Thus, LLLT and LED, this enforcement regime, positively modulated the metabolism of human osteoblasts during the cell viability, but in the process of mineralization few differences were observed.
8

Efeito dos laseres de baixa intensidade na proliferação e diferenciação de osteoblastos humanos / Low level laser effects in proliferation and differentiation of human osteoblasts

Flávia Amadeu de Oliveira 18 November 2013 (has links)
Dentre os vários compostos utilizados na pesquisa e na terapia de doenças osteo-degenerativas, a fototerapia com laseres de baixa potência (LLLT) e os diodos emissores de luz (LEDs) vem sendo investigada com o intuito de avaliar seus efeitos no metabolismo ósseo. Estes, que possuem comprimentos de ondas específicos, atuam na biomodulação das células, funcionando como um agente terapêutico, reequilibrando e normalizando a sua atividade. No entanto, pouco se sabe sobre o efeito dos diferentes espectros na proliferação e diferenciação de osteoblastos humanos, bem como seus efeitos no metabolismo celular como a síntese e a ativação de proteínas sinalizadoras envolvidas nesses processos. Diante disso, o objetivo deste trabalho foi avaliar, comparativamente, a influência da fototerapia com LLLT e LED na proliferação e diferenciação de osteoblastos humanos. Além disso, investigamos o envolvimento da ativação da via de sinalização ERK1,2 nestas respostas, utilizando o seu inibidor específico e/ou avaliando a sua ativação durante a proliferação e após fototerapia. Para esse estudo, osteoblastos humanos (HOAL) foram cultivados em meio de cultura DMEM suplementado com 10% de soro fetal bovino (SFB) e incubados em estufa de CO2. As células foram irradiadas pontualmente com os laseres vermelho (660nm), infravermelho (780nm) e LED (637nm), nas doses de 10, 20 e 50 J/cm2 na potência de 40mW, após adesão celular. Após 24, 48, e 72 horas foram realizados os ensaios de redução do MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5- difeniltetrazólio) e cristal violeta (CV) para avaliar a viabilidade das células e após 72 horas foi realizada a análise da proliferação por citometria de fluxo nos quais os resultados sugerem aumento de células viáveis ou proliferação quando estimuladas pelos diferentes espectros. Após a verificação do efeito positivo dos laseres e LED na viabilidade e/ou proliferação, foi realizada a análise da ativação da proteína intracelular ERK por western blotting usando anticorpo específico após 10 minutos da irradiação pontual. Mostramos que a irradiação das células HOAL com LLLT, na dose de 10 J/cm2, aumentou significativamente a fosforilação da ERK1/2 em relação ao controle. Para os ensaios de diferenciação, as células receberam pontualmente o estimulo a cada 6 dias e após os períodos de 07, 14, 21 e 28 dias foram realizados os ensaios da atividade de fosfatase alcalina (ALP), expressão gênica do colágeno I (COL1A1) e SPARC (osteonectina) por RT-PCR em tempo real e ensaio de mineralização com vermelho de alizarina. De um modo geral, não foram observados diferenças na atividade da ALP entre os grupos tratados em relação ao controle. A expressão gênica do COL1A1 e SPARC foi aumentada, no qual o LED em ambas doses apresentou maior eficácia em aumentar a expressão desses genes. No que se refere à mineralização, foram observadas pequenas diferenças quanto a deposição de cálcio. Dessa forma, a LLLT e LED, nesse regime de aplicação modulou positivamente o metabolismo dos osteoblastos humanos em relação à viabilidade, porém, no processo de mineralização foram observadas poucas diferenças. / Among the various compounds used in research and bone degenerative diseases therapy, phototherapy with low level laser (LLLT) and light emitting diodes (LEDs) has been investigated in order to evaluate its effects on bone metabolism. Those, who have specific wavelengths, act in biomodulation cells functioning as a therapeutic agent, rebalancing and normalizing their activity. However, little is known about the effect of the different spectra in the proliferation and differentiation of human osteoblasts and their effects on cellular metabolism as well as the synthesis and activation of signaling proteins involved in these processes. Therefore, the aim of this study was to compare the influence of LLLT and LED phototherapy in the proliferation and differentiation of human osteoblasts. In addition, we investigated the involvement of activation of ERK1,2 signaling pathway these responses using its specific inhibitor and/or evaluating their activation during the proliferation and after phototherapy. For this study, human osteoblasts (HOAL) were cultured in DMEM culture medium supplemented with 10 % fetal bovine serum (FBS) and incubated in CO2 incubator . Cells were irradiated with punctual red lasers (660nm), infrared (780nm) and LED (637nm) at doses of 10, 20 and 50 J/cm2 in power 40mW, after cell adhesion. After 24, 48, and 72 hours, MTT assay (- (4,5- dimethylthiazol-2- yl) -2,5 - diphenyltetrazolium bromide 3 ) and violet crystal (CV) were performed to assess the viability of cells and after 72 hours, was performed of proliferation analysis by flow cytometry. The results suggest an increase in viable and proliferation of cells when stimulated by different spectra. After checking the positive effect of lasers and LED viability and/or proliferation, analysis of ERK activation of intracellular protein by western blotting using a specific antibody was performed 10 minutes after the spot irradiation. We show that irradiation of HOAL cells with LLLT at a dose of 10 J/cm2, significantly increased the phosphorylation of ERK1/2 compared to control. For differentiation assays, cells promptly received stimulation every 6 days and after periods of 07, 14, 21 and 28 days testing the activity of alkaline phosphatase (ALP), gene expression of type I collagen (COL1A1) and SPARC (osteonectin) were performed by Real Time RT-PCR and mineralization experiment with alizarine red. In general, no differences in the activity of ALP between the treated groups compared to the control were observed. The gene expression was increased, and SPARC COL1A1, where in the LED in both doses showed greater effectiveness in increasing the expression of these genes. With respect to mineralization, small differences in the deposition of calcium were observed. Thus, LLLT and LED, this enforcement regime, positively modulated the metabolism of human osteoblasts during the cell viability, but in the process of mineralization few differences were observed.
9

Rôle de ERK dans l'activation des osmorécepteurs hypothalamiques

Dine, Julien 27 November 2009 (has links)
La vasopressine (VP), hormone anti-diurétique, joue un rôle fondamental dans l’homéostasie des compartiments liquidiens de l’organisme. Ainsi, la concentration de VP libérée dans la circulation sanguine est proportionnelle à l’osmolalité plasmatique. Or, la libération de la VP est étroitement dépendante de l’activité électrique des neurones magnocellulaires hypothalamiques (MNCs) qui la synthétisent. L’activité électrique des MNCs est donc régulée lors de variations de l’osmolalité plasmatique. Cette régulation implique la modulation concertée de propriétés intrinsèques dépendant de l’expression de canaux ioniques mécanosensibles, mais aussi d’entrées synaptiques excitatrices provenant de structures cérébrales osmosensibles. Les Extracellular signal-regulated kinases (ERK 1 et 2) sont des protéines de la voie des MAPK (Mitogen-Activated Protein Kinases) qui convertissent des stimulus extracellulaires en réponses intracellulaires transcriptionnelles et post-traductionnelles. Elles contribuent en particulier à la plasticité des propriétés membranaires intrinsèques et des synapses excitatrices glutamatergiques dans le système nerveux central. Elles sont exprimées par les MNCs de l’hypothalamus. L’objet de ce travail de thèse a été l’étude du rôle de ERK dans l’activation des osmorécepteurs hypothalamiques. Les expériences d’immunohistochimie montrent qu’en réponse à une stimulation systémique hypertonique, la voie ERK 1/2 est activée rapidement, de façon transitoire et sélectivement dans des structures osmoréceptrices de l’encéphale ou directement impliquées dans l’osmorégulation : noyaux supraoptiques (NSO) et paraventriculaires (NPV), organe subfornical, noyau préoptique médian et OVLT. ERK est aussi activée dans la portion parvicellulaire du NPV, mais pas dans une structure témoin qui ne participe pas à l’osmorégulation, la strie médullaire thalamique. De plus, il existe un codage de l’intensité de la stimulation osmotique par le nombre de neurones exprimant phosphoERK dans l’encéphale. Les Western Blot indiquent qu’il existe également un codage de l’intensité du stimulus hypertonique par le degré d’expression de la protéine phosphoERK dans le NSO. Nous montrons aussi qu’il existe une activation plus importante de ERK dans les neurones à VP que dans les neurones à ocytocine. L’ensemble de ces résultats suggère que phosphoERK pourrait contribuer aux mécanismes de l’osmorégulation. Cette hypothèse a été testée en mesurant les effets de l’inhibition de la phosphorylation de ERK sur l’activation des centres osmorégulateurs. Effectivement, l’administration intracérébroventriculaire d’un inhibiteur sélectif de MEK 1/2, l’U 0126, inhibe la phosphorylation de ERK et l’activation neuronale (mesurée par l’expression de Fos) résultant de l’application d’un stimulus hypertonique. La phosphorylation de ERK joue donc un rôle majeur dans l’activation des centres osmorégulateurs de l’encéphale. En accord avec ces données, les enregistrements électrophysiologiques sur tranches d’hypothalamus montrent que ERK est impliqué dans la réponse cellulaire des neurones magnocellulaires à une stimulation osmotique : l’inhibition de la phosphorylation de ERK réduit la dépolarisation membranaire et diminue l’augmentation de la fréquence de décharge des potentiels d’action ainsi que l’augmentation de l’amplitude des potentiels synaptiques spontanés induits par un stimulus hypertonique. Les données obtenues sur les neurones supraoptiques isolés montrent qu’une partie au moins des effets de l’activation de ERK implique une modulation de la conductance membranaire qu’on sait responsable de l’osmotransduction dans ces neurones. En résumé, l’activation de ERK est nécessaire à la réponse normale des neurones magnocellulaires à une stimulation osmotique. / The release of the anti-diuretic hormone vasopressin into the general circulation varies as a function of plasma osmolality and therefore plays a major role in systemic osmoregulation. It is primarily determined by the firing rate of the hypothalamic magnocellular neurons (MNCs). The regulation of MNC firing rate during changes in systemic osmolality involves the concerted modulation of intrinsic mechanosensitive ion channels in these neurons, as well as excitatory glutamatergic inputs derived from osmosensitive forebrain regions. Extracellular signal-Regulated protein Kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, which include changes in intrinsic neuronal properties and synaptic function. In the present work, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of osmoregulatory networks. First, we found that within 10 minutes of intraperitoneal injections of hypertonic saline (3M, 6M), many phosphoERK-immunopositive neurons were observed within osmosensitive forebrain regions including suparoptic and paraventricular nuclei, subfornical organ, median preoptic nucleus and Organum vasculosum lamina terminalis (OVLT), but not in a control region such as the thalamic stria medullaris. This staining was intensity dependent and was reduced 30 minutes after the stimulus. In the supraoptic nucleus, it predominated in vasopressin neurons compared to oxytocin neurons. Western blotting experiments confirmed that ERK phosphorylation in the supraoptic nucleus was intensity dependent. Inhibition of ERK phosphorylation by a MEK inhibitor reduced both the numbers of phosphoERK-immunopositive neurons and Fos expressing neurons, a measure of neuronal activation after hypertonic stimuli. Inhibition of ERK phosphorylation also decreased the membrane depolarization, the increase in firing frequency and the increase in excitatory and inhibitory synaptic potential amplitude that were osmotically-induced in both oxytocin and vasopressin supraoptic neurons recorded from adult acute hypothalamic slices. It also reduced the increase in mechanically-gated membrane cation conductance evoked by hypertonic stimuli in isolated MNC neurons. Altogether, these findings strongly suggest that ERK phosphorylation is a key step in the transduction and integration of osmotic stimuli into activity changes in osmosensitive hypothalamic neurons.
10

MECHANISMS OF NEUROPROTECTION IN SCN2.2 CELLS

Karmarkar, Sumedha 01 May 2012 (has links)
As the major excitatory neurotransmitter, glutamate (Glu) is physiologically important in brain function. Excessive Glu release, however, is a critical underlying pathological mechanism in neurodegenerative disease, especially stroke. Strategies to protect neurons from cell death under these conditions are scarce; in part because of incomplete understanding of inherent neuroprotective mechanisms. The suprachiasmatic nucleus (SCN) is a region of the brain that exhibits endogenous resistance to Glu excitotoxicity. A previous study demonstrated that SCN2.2 cells (an immortalized SCN cell line) were resistant to Glu excitotoxicity as compared to GT1-7 neurons (from the neighboring hypothalamus). This thesis explored the cellular mechanisms underlying this endogenous neuroprotection in SCN2.2 cells. Extracellular regulated kinase (ERK) is expressed in the SCN, activated by Glu, and is anti-apoptotic in other systems. Therefore, this thesis was designed to test the following central hypothesis: SCN2.2 cells are dependent on ERK signaling for survival in the presence of an excitotoxic insult. Glu increased ERK activity in SCN2.2 cells and importantly, resistance to Glu excitotoxicity in SCN2.2 cells was compromised by pre-treatment with an ERK inhibitor (PD98059; PD). ERK inhibition + Glu mediated SCN2.2 cell death in an N-methyl-D-aspartate receptor (NMDAR)-dependent manner; specifically via the NMDAR 2B (NR2B) subunit. Glu treatment increased expression of NR2B, phosphorylated NR2B and NR1 proteins and decreased NR2A and NR2D mRNA in the GT1-7 cells. Glu-treated SCN2.2 cells showed decreased NR2B, phosphorylated NR2B, increased NR2C proteins and increased NR2A and NR2D mRNA levels. These data are consistent with varied NMDAR responses to Glu in GT1-7 vs. SCN2.2 cells, which might underlie the different physiological responses to Glu in the two cell types. Further experiments investigated the role of several signaling kinases, e.g. protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CaMK-II) and c-Jun N-terminal kinase-II (JNK-II) in regulation of ERK activation and on SCN2.2 cell fate. PKA and PKC inhibition together, CaMK-II inhibition and JNK-II inhibition resulted in SCN2.2 cell death in the presence of Glu. PKA + PKC inhibition and CaMK-II inhibition resulted in a corresponding decrease in Glu-induced ERK phosphorylation. Combined inhibition of ERK, CaMK-II and JNK-II resulted in exacerbation of cell death as compared to when the inhibitors were used individually. These results suggest that ERK activity is regulated by a number of different kinases. Glu treatment resulted in a persistent increase in ERK phosphorylation (activation) for up to 48 h in the SCN2.2 cells whereas the pro-apoptotic p38 was phosphorylated (activated) in the GT1-7 cells exposed to Glu. JNK-II was transiently phosphorylated (activated) in the SCN2.2 cells. This suggests an activation of a short-term stress response which can result in activation of a long-term neuroprotective response in these cells. Pro-apoptotic Bid mRNA and cleaved Bid protein levels were increased in the Glu-treated GT1-7 cells. The effect of Glu treatment on the expression of several downstream effector molecules of ERK activation was also explored. Neuritin mRNA was increased with Glu treatment in the SCN2.2, but not in the GT1-7 cells. However, there was no change in the neuritin protein levels in either cell type with Glu treatment. Bcl2 levels remained unchanged in the Glu-treated GT1-7 cells. Although there was no change in the Bcl2 mRNA levels in the SCN2.2 cells, Bcl2 protein was significantly increased with Glu treatment, thus suggesting a post-translational mechanism of neuroprotection involving Bcl2. Taken together, these results are consistent with activation of an apoptotic mechanism in the GT1-7 cells exposed to Glu as opposed to a pro-survival effect in similarly treated SCN2.2 cells. Future studies should be able to take advantage of these mechanisms in developing therapeutic strategies in the treatment of neurodegenerative disorders.

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