Neurofibromatosis type two (NF2) is a genetic disorder predisposing those affected to the development of multiple benign tumors in their central and peripheral nervous systems. This is due to the absence of the tumor suppressor protein merlin, which is encoded by the NF2 gene. In nearly all NF2 cases, patients present with bilateral schwannomas of the vestibulocochlear nerve, in addition to other schwannomas throughout the central and peripheral nervous systems, as well as meningiomas and ependymomas. Currently, no therapeutic alternatives to surgical removal and radiation therapy are available for NF2 patients. This study investigated cobimetinib, an inhibitor of the often-deregulated mitogen activated protein kinase (MAPK) pathway in NF2 tumors, and its in vitro mechanism of action in both mouse and human NF2 schwannoma model cell lines. It was demonstrated that the drug decreased 70% and 60% of the viability at 10μM in the mouse and human merlin-deficient cell lines, respectively. It was further demonstrated that this decrease in viability was due to cytostatic and cytotoxic effects of cobimetinib in the case of the mouse NF2 schwannoma model but only due to cytostatic effects of cobimetinib in the human NF2 schwannoma model. These results show promise in targeting the MAPK pathway in NF2 tumors, and the promise of cobimetinib specifically, supporting further cytometric flow and in vivo testing of the inhibitor.

L'épiderme est un tissu stratifié, majoritairement constitué de kératinocytes qui forme la première barrière de l'organisme contre les agressions extérieures. Après blessure cutanée, la migration des kératinocytes est une phase cruciale de la cicatrisation. Le comportement des kératinocytes est placé sous le contrôle des molécules de la matrice extracellulaire ainsi que par les facteurs solubles (facteurs de croissance et cytokines..) sécrétés dans le microenvironnement. Les cellules résidentes ou recrutées sur le site de lésion libèrent également des nucléotides extracellulaires (ATP, UTP) dans l'environnement des kératinocytes. Dans ce travail de thèse, nous avons examiné l'impact des nucléotides extracellulaires et du récepteur purinergique P2Y2 sur la migration des kératinocytes et sur l'activité motogénique de deux facteurs de croissance, l'IGF-I et de l'EGF. Dans un premier temps, nous avons pu montrer que l'activation de P2Y2 et de la protéine hétérotrimérique Gαq inhibe l'activité de l'isoforme p110α de la PI3K sur des cellules stimulées par l'IGF. Cette inhibition de la voie PI3K/Akt aboutit à une perturbation de la mobilisation de la cortactine et de la formation des lamellipodes ainsi qu'une diminution de la vitesse de migration des kératinocytes. Dans un second temps, nous avons mis en évidence que l'activation de P2Y2 inhibe l'activation de la voie ERK1/2 par l'EGF en inhibant la phosphorylation des protéines MEK1/2, ERK1/2 et p90RSK. Nous avons établi que la conséquence de cette inhibition est la stabilisation des hémidesmosomes / The epidermis is a stratified tissue, mainly composed of keratinocytes, that forms the first barrier of the organism. When skin injury occurs, the epidermis structure is altered and many signalling pathways are activated in order to re-establish its homeostasis. Among these signalling pathways, the PI3K and MAPK ERK1/2 pathways play key roles by controlling keratinocyte migration and proliferation. The aim of this thesis was to analyse the regulation of these two signalling pathways by extracellular nucleotides, acting through purinergic receptors P2Y2 and the heterotrimeric Gαq protein and to evaluate the impact of this receptor on keratinocyte migration. Firstly, we showed that P2Y2 receptor activation inhibits PI3K p110α isoform and consequently alters keratinocyte cell shape and migration. Additionally, we showed that purinergic signalling activation inhibits EGF-induced ERK1/2 pathway activation by inhibiting the phosphorylation of MEK1/2, ERK1/2 and p90RSK proteins. As a consequence, P2Y2 stabilizes α6β4 integrin localisation into hemidesmosome-like structures and inhibits keratinocyte migration. The involvement of purinergic signalling pathway in regulation of different signalling events suggests that it may play a central role in regulation of cellular events that occurred during skin wound healing process. Moreover, our present data in association with those of the literature show that extracellular nucleotides can act as a double-edged sword in the regulation of cell migration: either activate or block cell migration in a striking cell-specific manner.

MyD88 est une protéine adaptatrice du système immunitaire inné, impliquée dans la défense de l’organisme contre les agents microbiens. Elle est recrutée aux « Toll-like receptors » (TLRs) suite à la reconnaissance par ces derniers de motifs microbiens conservés, les PAMPs (Pathogens-associated molecular patterns). La voie de signalisation ainsi déclenchée va aboutir à la production de cytokines pro-inflammatoires, de chimiokines et d’espèces actives de l’oxygène. De cette façon, les TLRs, via MyD88, constituent la première ligne de défense contre les pathogènes.De nombreuses études ont permis de démontrer que MyD88 est nécessaire pour la réponse inflammatoire, qui promeut la carcinogenèse. Dans le cadre d’une étude sur les TLRs et le cancer, l’équipe a démontré, grâce à une étude in vivo, que MyD88 participe au processus de tumorigenèse médiée par l’oncogène ras et est nécessaire à l’activation de la voie canonique des MAPKs, ainsi qu’à la transformation cellulaire in vitro. Nous avons ensuite déterminé le mécanisme par lequel MyD88 intervient dans la voie de signalisation Ras/MAPKs, en permettant le maintien de l’activation de cette voie. En effet, MyD88 interagit avec une MAPK clé de cette voie : la kinase ERK, et protège cette dernière de sa déphosphorylation par sa phosphatase spécifique MKP-3, MyD88 et MKP-3 se liant à ERK par le même domaine. Nous avons démontré la pertinence de ce mécanisme, grâce à la mise en évidence d’une surexpression de la protéine MyD88 et de son interaction avec la forme phosphorylée d’ERK dans des coupes de tissus tumoraux humains (estomac, poumon, colon).L’ensemble des résultats obtenus au cours de ma thèse ont permis de montrer qu’en plus de son rôle bien défini en tant qu’adaptateur des récepteurs de l’immunité innée dans les processus inflammatoires, MyD88 joue un rôle direct, qui semble être crucial dans la signalisation Ras, le contrôle du cycle cellulaire et la transformation cellulaire / MyD88 is an adaptator protein of the innate immune system, implicated in the défense against microbes. MyD88 is recruited by the Toll-Like Receptors (TLRs) upon there interaction with conserved microbial patterns (PAMPs). Therefore, TLR signaling pathway induces the production of pro-inflammatory cytokines, chemokines and reactive oxygen species. TLRs, via MyD88, form the first line of defense against pathogens. Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for Ras-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, ERK, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical Ras pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated ERK in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in Ras signaling, cell-cycle control, and cell transformation

Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas β3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores β3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade βγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas. / During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of β3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of β3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through βγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies

Bidens pilosa L. is a traditional Chinese herbal medicine of which was considered as a potential COX2 inhibitor and anti-inflammatory agent. The objective of this study is to discriminate the protective effect of aqueous extract of Bidens pilosa L. leaves (BPLAE) against TAA-induced live fibrosis using an animal model. The herb extracts were administrated via intraperitoneal injection once per week (1.25, 2.5 g/kg), and thioacetamide (200 mg/kg) was injected three times per week and the mice were sacrificed at week 4 and week 8, respectively. Immunohistochemistry staining, Hematoxylin-eosin (HE) staining, Sirius red staining were carried out to evaluate the pathological alterations of mouse livers; in addition, Western blotting was performed to measure the differential expression of £\-smooth muscle actin (£\-SMA) between different treatment groups (vehicle, week 4 and week 8). Hepatic hydroxyproline was also detected in order to compare difference in collagen formation of each group. The results showed that Bidens pilosa L. effectively reduced amount of hepatic hydroxyproline and £\-SMA protein in mice with fibrotic liver induced by TAA. Moreover, in histiopathological exam, the BPLAE treated mice demonstrated a lower collagen and £\-SMA expression, which indicated that BPLAE might reduce degree and severity of liver fibrosis in mice. In conclusion, these results suggested that BPLAE potentially against fibrogenesis in TAA- induced mice liver fibrosis. Additionally, we found that BPLAE might involve in the signaling pathway of MAPK (ERK1/ERK2), which reduced the phosporylation level of p44 but not p42. Further studies using cell base assay to confirm the inhibiting role of BPLAE against cell proliferation or migration is warrant.

Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas β3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores β3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade βγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas. / During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of β3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of β3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through βγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies

v-Rel is the acutely oncogenic member of the NF-[kappa]B family of transcription factors and transforms cells through the altered regulation of pathways normally controlled by cellular NF-[kappa]B. Initial studies revealed that expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAP kinases. This induction is critical for the v-Rel transformed phenotype, as suppression of MAPK activity with chemical inhibitors or siRNA severely limited colony formation of v-Rel transformed cell lines of hematopoietic origin. However, signaling must be maintained within a certain range in these cells, as strong additional activation of either pathway through expression of constitutively active MKK mutants also attenuated the transformed phenotype. Studies in primary spleen cells revealed that MAPK signaling is also required for the early stages of v-Rel-mediated transformation. However, constitutive MAPK activity further enhanced the transformation efficiency of v-Rel in primary cells. These studies, as well as analogous experiments in DT40 cells, indicate distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation. The proto-oncoprotein, c-Rel, only weakly activates ERK and JNK signaling compared to v-Rel. Importantly, elevated MAPK activity enhanced transformation by c-Rel, indicating that the ability of v-Rel to induce MAPK signaling is a major contributor to its oncogenic potential. Taken together, this work demonstrates an important role for ERK and JNK activity in transformation by v-Rel. Additional studies examined mechanisms through which MAPK activity is regulated in v-Rel transformed cells. Feedback regulation of the ERK activator, MKK1, at T292 was shown to limit ERK activation in v-Rel transformed cells, preventing the detrimental effects of constitutive activity. This result is the first indication that this regulation may have a role in the maintenance of transformation. Further, several v-Rel induced cytokines were identified that activate ERK and JNK signaling in v-Rel transformed cells, revealing one means by which v-Rel-dependent transcriptional changes lead to MAPK activation. These studies demonstrate the integration of multiple mechanisms in achieving the optimal levels of MAPK activity that are essential for v-Rel-mediated transformation. / text

Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. D486N/+ female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

Activation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling controls the differentiation of mouse embryonic stem (ES) cells. An established modulator of the ERK MAPK pathway is the IRS-1 (Insulin Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder 1). Gab1 is ubiquitously expressed and is activated by a wide range of cell surface receptors, mediating growth factor, cell-cell and cell-substratum interactions. The N-terminal region of Gab1 contains a pleckstrin homology (PH) domain required for membrane binding and a nuclear localisation sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse ES cells preferentially express high levels of a novel form of Gab1 (Gab1β) lacking the N-terminal region. Based on its novel structure and abundance, Gab1β may act in a dominant negative manner by binding and mislocalising downstream effectors. Alternatively, it may have a deregulated function unrestrained by the PH or NLS domains. Data presented here shows that Gab1β is tyrosine phosphorylated in response to the self-renewal factor Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS) stimulation. This then leads to the formation of complexes with Shp2 and the p85 subunit of PI3K. Experiments comparing the responses of wild-type and Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to signalling activity is receptor specific and may imply that the response of ES cells to ERK activation is context specific. By reintroducing fluorescently tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as well as in a perinuclear body. I next investigated the potential role of Gab1β in the differentiation of ES cells into neural precursors. A monolayer differentiation protocol was used to differentiate Gab1β wild-type and knock-out cells into neural precursors. Furthermore, the effect of insulin on the emergence of neural precursors from Gab1β-targeted cells was also explored.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.