Les carences précoces en donneurs de méthyles (vitamines B9 et B12 notamment) sont à l’origine de malformations congénitales. Elles exercent un effet délétère sur le développement du cerveau et sont associées à une augmentation de l’incidence de pathologies neurologiques et neurodégénératives à l’âge adulte. Un modèle murin de carence en donneurs de méthyles, le modèle MDD, a été développé au laboratoire et a permis d’étudier la réponse à cette carence, et de mettre en évidence des altérations de la structure cérébrale et des défauts de locomotion chez les ratons issus de mères carencées. Ce comportement est contrôlé par le cervelet, dont on sait que le développement est altéré chez les MDD. En revanche, les mécanismes moléculaires mis en jeu dans la réponse à la carence dans le cervelet restent peu compris. Afin d’étudier les gènes et voies de signalisation dérégulés chez les MDD, nous avons réalisé l’étude du transcriptome du cervelet des ratons carencés. Puis, nous nous sommes intéressés aux modifications épigénomiques engendrées par la carence en analysant leur miRnome et les modifications des protéines histones dans leur cervelet. Nous avons mis en évidence des altérations des voies wnt, dans le cervelet des femelles carencées, qui n’ont pas été retrouvées chez les mâles. De même, de nombreux gènes impliqués dans le développement et les fonctions synaptiques sont dérégulés chez les femelles. Nous avons aussi montré des variations de plusieurs marques d’acétylation et de méthylation des histones chez les MDD. Enfin, de manière plus ciblée, nous avons mis en évidence un miARN dont l’expression diminue dans le cervelet des ratons carencés : miR-344-5p. Nos premiers résultats semblent indiquer qu’il est impliqué dans le contrôle de la mort cellulaire. Ces résultats montrent l’implication de dérégulations globales dépendantes du sexe mais aussi des altérations ciblées dans la réponse à la carence. Une amélioration de la compréhension de ces mécanismes moléculaires nous permettra de mieux appréhender le lien qui existe entre carence précoce en donneurs de méthyles, développement cérébral et incidence de pathologies à l’âge adulte / Early methyl-donor deficiencies (e.g. B9 and B12 vitamins) can lead to congenital disabilities. They are behind developmental abnormalities of the brain, and are associated with the development of neurological and neurodegenerative diseases at adulthood as well. In the lab, we developed a methyl donor deficiency rat model called MDD. It has allowed us to show structure alterations of several brain areas and also locomotor coordination impairments in pups born from dams fed a MDD diet. Cerebellum is the brain structure involved in the control of this behavior and we know its development is delayed in MDD. However, the molecular mechanisms underlying methyl donor deficiency still remain misunderstood in this brain structure. In order to study genes and signaling pathways dysregulated in MDD, we performed transcriptomic analysis of deficient pups’ cerebellum. We also led miRnome analyses and histone modifications investigations with the purpose of understanding epigenomic modifications caused by MDD. We showed alterations of wnt signaling pathways in female’s cerebellum which we did not find in males. We also found that several genes involved in cerebellum’s development and synaptic function were dysregulated in females. Regarding epigenomic regulation, acetylation and methylation of histone marks were also modified in females. Finally, we chose miR-344-5p as an interesting candidate to study more specific epigenetic modifications. Its expression is decreased in MDD and it seems to be involved in cellular death control, according to our first results. These results shed light on global dysregulations, in a sex-dependent manner, as a consequence of methyl donor deficiency but also more specific alterations. A better understanding of the molecular mechanisms taking place in response to MDD could help us to link methyl donor deficiency, brain development and neurodegenerative pathologies occurrence at adulthood

A musculatura esquelética tem um papel fundamental para a manutenção da homeostase do organismo. A perda de massa muscular está relacionada a prejuízos na qualidade de vida de indivíduos saudáveis, além de piorar o prognóstico de pacientes com doenças sistêmicas, como o câncer, o diabetes e a insuficiência cardíaca. Em quadros mais graves de insuficiência cardíaca, a perda excessiva de massa muscular associada a um reduzido consumo de oxigênio de pico, são considerados como preditores independentes de mortalidade. O aumento do estresse oxidativo tem sido apontado como um dos principais desencadeadores do aumento da degradação de proteínas na atrofia muscular. Na presente tese, investigamos a contribuição do estresse oxidativo para a ativação das vias de sinalização NF-kB, FOXO e MAPK na atrofia muscular desencadeada pela insuficiência cardíaca. Para compreender melhor os mecanismos envolvidos na ativação dessas vias pelo estresse oxidativo, utilizamos a linhagem de células musculares C2C12. Observamos que o tratamento com peróxido de hidrogênio (1,2mM, 12h) induziu um aumento do estresse oxidativo, o qual foi capaz de aumentar a atividade do proteassoma, desencadeando a atrofia dos miotúbulos. Verificamos também um aumento da expressão proteica de alguns componentes dessas vias de sinalização, como p-p38 e NF-kB; apontando para uma ativação diferenciada dessas vias pelo estresse oxidativo. Para verificar se essas vias de sinalização relacionadas ao estresse oxidativo estavam também relacionadas à atrofia desencadeada pela insuficiência cardíaca, avaliamos um modelo experimental de ratos com insuficiência cardíaca induzida pelo infartado do miocárdio. Observamos uma redução da área de secção transversa do músculo plantar, acompanhada de um aumento da inflamação sistêmica, de p38 e das atividades de NF-kB e do proteassoma. Como o treinamento físico aeróbico tem se apresentado como uma estratégia terapêutica não farmacológica eficaz na redução do estresse oxidativo e no restabelecimento da atividade do sistema ubiquitina proteassoma, submetemos os ratos infartados ao treinamento físico aeróbico em esteira rolante. O treinamento físico aeróbico preveniu a perda de massa muscular, reduzindo a inflamação sistêmica e as atividades de NF-kB e do proteassoma. Em conjunto, os resultados apontam para o estresse oxidativo como um fator preponderante para o aumento da degradação de proteínas relacionada à atrofia muscular, seja por indução de inflamação (TNF-α) ou por sua ação direta. Além disso, observamos que as vias de sinalização são ativadas de forma diferenciada nos dois modelos, sugerindo que a degradação de proteínas nos miotúbulos está relacionada ao controle de qualidade de proteínas e, nos ratos infartados, às alterações do metabolismo, servindo como fonte de energia. Já o treinamento físico aeróbico comprovou sua eficácia no restabelecimento da atividade do proteassoma, reduzindo a inflamação e a atividade de NF-kB, prevenindo assim, a perda de massa muscular / About 40% of human body mass consists of skeletal muscles, which are involved in all aspects of movement including breathing, eating, posture, walking and reflexes. Skeletal muscle is also important as a source of heat generation and as a regulator of intermediary metabolism. Loss of skeletal muscle mass and function (skeletal muscle atrophy) leads to several functional impairments, affecting health and quality of life. It occurs in several chronic diseases such as cancer, diabetes and heart failure. In heart failure, atrophy is considered an independent predictor of poor prognosis. Oxidative stress has a crucial role in atrophy, activating different signaling pathways capable of stimulating the ubiquitin proteasome system to degrade proteins. In this study, we investigated the oxidative stress contribution to NF-kB, FOXO and MAPK signaling pathway activation in heart failure-induced atrophy. To better understand the mechanisms involved with oxidative stress and signaling pathways activation in atrophy, we have used C2C12 skeletal muscle cells. We observed that, even in high hydrogen peroxide concentrations, oxidative stress increased proteasome activity, phosphorylated p38 and NF-kB protein expression, causing myotubes atrophy. In an experimental heart failure model of infarcted rats, we evaluated plantaris muscle and verified a reduced cross sectional area, accompanied by increased systemic inflammation, p-38 protein expression and increased both NF-kB and proteasome activities. As aerobic exercise training causes a lot of beneficial effects on skeletal muscle structure and function in chronic diseases, we submitted infarcted rats to 8 weeks of aerobic exercise training on a treadmill. Aerobic exercise training prevented atrophy by reducing inflammation and both NF-kB and proteasome activities. Collectively, our data suggest a differentiated activation by oxidative stress in muscle cells and animal models. In the first case, protein degradation was involved with protein quality control; and, in the other, oxidative stress is a second messenger, stimulating protein degradation to provide substrates to metabolism. Aerobic exercise training re-established proteasome activity by reducing inflammation and NF-kB activity, preventing muscle atrophy

Nous montrons pour la première fois qu’en plus de la MAPK ERK1/2, l’endothéline-1 (ET-1) via les récepteurs ETA et ETB active une autre MAP kinase : la p38 uniquement dans les cellules de léiomyomes utérins de rate (ELT3) mais pas dans les cellules myométriales saines. Dans les cellules ELT3, l’analyse des voies de signalisation montre que malgré les similitudes observées entre les modes d’activation des voies p38 et ERK1/2 par ET-1, celles-ci sont activées de façon indépendante l’une de l’autre. En plus, la forskoline active p38 (mais pas ERK1/2), par contre l’activation de p38 par ET-1 n’implique pas une production d’AMPc. Par ailleurs ERK1/2 et p38 coactivées par ET-1 coopèrent pour augmenter l’expression de COX2 et la production des prostaglandines E2 (PGE2) pour favoriser l’effet antiapoptotique de ET-1. De plus p38 activée par ET-1 contribue à la prolifération des léiomyomes. Nos résultats élucident les mécanismes par lesquels ET-1 contribue à la croissance des léiomyomes. / We demonstrated for the first time, that in addition to the MAPK ERK1/2, Endothelin-1 (ET-1) through ETA and ETB receptors activated another MAP kinase: p38 only in uterine leiomyoma cells (ELT3) but not in normal myometrial cells. In ELT3 cells, analysis of signaling pathways showed that, despite the similarities between the mechanisms involved in the activation of p38 and ERK1/2 pathways by ET-1, these kinases are activated independently one of another. In addition, forskolin (a cAMP inducer), activated p38 (but not ERK1/2), whereas the activation of p38 by ET-1 did not involve production cAMP. Moreover the coactivated ERK1/2 and p38 pathways by ET-1 cooperated to increase expression of COX2 and prostaglandin E2 (PGE2) production. This PGE2 like ET-1 exerted an antiapoptotic effect in ELT3 cells. Furthermore, p38 activated by ET-1 contributes to the proliferation of ELT3 leiomyoma cells. Our data highlight the mechanisms by which ET-1 could promote uterine leiomyoma growth.

Le sepsis est défini par l’apparition de dysfonctions d’organes, multiples et mortelles, causées par une réponse de l’hôte dérégulée suite à une infection. L’hétérogénéité de la maladie représente un défi clinique majeur au regard de la prise en charge thérapeutique, et à ce jour les marqueurs proposés ne suffisent pas à stratifier les patients. Les rétrovirus endogènes humains (HERV) pourraient être des marqueurs pertinents,compte tenu des propriétés immunosuppressives de leurs enveloppes et de leur expression dans des maladies inflammatoires et auto-immunes. Cette thèse a pour objectif de savoir dans quelle mesure les HERV sont exprimés et modulés, dans des conditions d’inflammation et d’immunosuppression. Pour cela,nous avons utilisé une puce à ADN haute densité permettant (i) l’analyse de la transcription de 363 689HERV et 1500 gènes, et (ii) une lecture fonctionnelle de l’activité des LTR. L’expression des HERV a été objectivée (i) dans un modèle ex-vivo de tolérance à l’endotoxine sur des cellules mononuclées du sang périphérique (PBMC) d’individus sains et (ii) sur sang total provenant d’individus sains et de patients en choc septique, stratifiés ou non en fonction du statut immunitaire. (1) De 5,6% à 6,9% des HERV sont exprimés dans le compartiment sanguin et environ 20% des LTR possèdent une fonction promotrice ou polyA, les deux fonctions étant mutuellement exclusives. (2) Le contenu du transcriptome HERV est modulé ex vivo dans le contexte de tolérance à l’endotoxine laissant apparaitre deux grands phénotypes transcriptionnels. L’expression de certains loci HERV est corrélée au statut immunitaire de patient septique.L’évaluation d’une signature moléculaire complexe sur une cohorte de validation, permet la séparation en deux groupes présentant des critères de sévérité distincts, suggérant les HERV/MaLR comme biomarqueurs de stratification. (3) L’analyse de la co-expression des gènes et des HERV a permis d’intégrer ceux-ci au sein de réseaux associées à la réponse de l’hôte et de proposer des hypothèses fonctionnelles. / Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection.The heterogeneity of the disease present a major clinical challenge with regard to the therapeutic coverage,and this day the proposed markers are not enough to stratify patients. The human endogenous retrovirus(HERV) could be relevant markers, considering the immunosuppressives properties of their envelopes andtheir expression in inflammatory and autoimmune disease. The aim of this thesis is to know to what extentthe HERVs are expressed and modulated, in inflammatory and immunocompromised contexts. For this, weused a high density DNA chip allowing (i) the transcription analysis of 363,689 HERV and 1500 genes,and (ii) a functional reading of LTRs activities. The HERVs expression was objectified (i) in endotoxintolerance ex vivo model in peripheral blood mononuclear cells (PBMCs) of healthy volunteers and (ii) inwhole blood of healthy volunteers and septic shock patients, stratified or not according to immunity state.(1) Of 5,6% at 6,9% of HERVs are expressed in the blood compartment and around 20% of LTRs have apromoter or polyA function, both functions being mutually exclusive. (2) The HERV transcriptome ismodulated in ex vivo endotoxin tolerance model letting appear two higher transcriptional phenotypes. Theexpression of some HERVs loci are correlated of the immunity state of the septic shock patients. Theevaluation of molecular signature in validation cohort, allowed to separate in two patients groupspresenting different severity criteria, suggesting HERV/MaLR as biomarkers of stratification. (3) The coexpressedanalysis of genes and HERVs allowed to integrate these within signaling pathways associated atthe host immune response and to provide functional hypothesis.

The Hsp70 family of molecular chaperones is essential for protein folding, re-folding misfolded client proteins, clearance of aberrant client proteins, and can also inhibit programmed cell death. There are two major cytosolic members of this family: the constitutive Hsc70, and the inducible Hsp72. Under stress conditions the Hsp70 family protects the cell from protein related damage by the induction of Hsp72. Hsc70 and Hsp72 are highly homologous with minor differences in substrate binding. In cancers, Hsp72 is commonly induced and this induction is thought to aid in cancer cell survival. In these studies we demonstrate the differential regulation of the prosurvival kinase Akt by Hsc70 and Hsp72. We demonstrate that of the two cytosolic forms, Hsp72 is the primary Akt regulator. Using a phenothiazine class inhibitor of Hsp70-family activity, methylene blue, we demonstrate dose dependent decreases in the levels of Akt; produced breast cancer specific cell death. This cell death could be rescued by the use of an Hsp70 family ATPase stimulating compound, SW02. We also demonstrate a similar phenotype with a rhodacyanine class Hsp70 family inhibitor, YM-1, also capable of reducing Akt and causing cancer specific cytotoxicity. The resulting Akt decreases were sufficient to block a tamoxifen-resistance pathway, allowing previously resistant cells to regain sensitivity to tamoxifen. These results demonstrate the capabilities of Hsp70 family inhibitors as potent compounds for the treatment of breast cancer.

Darbo tikslas nustatyti skirstymo baltymo GAB1 ryšį su anti-apoptoziniu PI3K/Akt bei mitogeniniu Ras/MAPK signalo perdavimo keliais ir įvertinti GAB1 įtaką šių kelių sąveikai EGFR signalo perdavimo tinkle. Darbo uždaviniai: įvertinti epitelinių ląstelių endogeninio GAB1 veiksmingumą EGF signalo perdavimo metu; nustatyti sąveikos pobūdį tarp PI3K/Akt ir Ras/MAPK kelių EGF signalo metu; kiekybiškai įvertinti GAB1 svarbą EGF signalo perdavimui per PI3K/Akt ir Ras/MAPK kelių in vivo, rezultatus lyginant su matematinio modelio prognozėmis in silico; nustatyti GAB1 veiksmingumo ir jo reguliacijos grįžtamaisiais ryšiais įtaką PI3K-MAPK sąveikos stiprumui priklausomai nuo EGF dozės ir laiko; ištirti GAB1 svarbą EGFR ir insulino receptoriaus signalo perdavimo tinklų sąveikai; modifikuoti Westerno pernašos metodą palyginamajai kiekybinei ir kokybinei baltymų analizei. Darbo išvados: stimuliavus EGFR, skirstymo baltymas GAB1 tampa veiksmingu; EGF lemia reciprokinę PI3K/Akt ir Ras/MAPK signalo perdavimo kelių sąveiką per GAB1; GAB1 yra pagrindinis teigiamo atgalinio ryšio elementas PI3K kelyje, padedąs pagreitinti, stiprinti ir išlaikyti MEK/ERK kinazių atsaką; PI3K-MAPK sąveikos stiprumas kinta laike ir yra atvirkščiai proporcingas EGF signalo stiprumui; GAB1 reikalingas sinergistiškai stiprinti insulinu ir mažomis EGF dozėmis stimuliuojamų ląstelių Ras/MAPK atsaką; sukurtas „Multi-juostelių“ imunopernašos metodas yra tinkamas palyginamajai kiekybinei ir kokybinei baltymų analizei... [toliau žr. visą tekstą] / The aim of the thesis was to determine a connection of endogenous docking protein GAB1 with anti-apoptotic PI3/Akt and Ras/MAPK signaling pathways and to estimate GAB1 contribution to their crosstalk in EGFR signaling network. The tasks: to evaluate GAB1 efficacy upon EGFR stimulation; to examine the nature of crosstalk between PI3K/Akt and Ras/MAPK pathways; to evaluate the contribution of GAB1 to EGF signaling via PI3K/Akt and Ras/MAPK pathways in vivo comparing the results with prognosis in silico; to estimate the EGF dose- and time-dependent impact of GAB1 efficacy and its feedback regulation on the strength of PI3K-MAPK interaction; to investigate the role of GAB1 for crosstalk of EGFR and insulin receptor signaling networks; to modify Western blotting procedure for comparative quantitative and qualitative protein analysis. The conclusions: the docking protein GAB1 is functional upon EGFR stimulation; PI3K/Akt and Ras/MAPK signaling pathways crosstalk reciprocally via GAB1 in response to EGF; GAB1 is major positive feedback element in PI3K pathway amplifying and sustaining MEK/ERK response to EGF; the strength of PI3K-MAPK interaction depends on time and is inversely proportional to EGF signal strength; GAB1 is required to synergistically potentate the Ras/MAPK response to tandem cell treatment with insulin and low EGF doses; the developed Multistrip immunoblotting method is suitable for comparative quantitative and qualitative protein analysis. In comparison with a... [to full text]

L’hématopoïèse se déroule dans un microenvironnement spécialisé appelé niche où les cellules souches hématopoïétiques (CSH) sont en contact étroit avec les cellules stromales mésenchymateuses. Cette interaction cellulaire associée à d’autres facteurs environnementaux, comme la présence des espèces réactives à l’oxygène, est cruciale pour la régulation des CSH normales, mais aussi leucémiques. Pour étudier ce microenvironnement, il est donc important de développer un modèle in vitro de niche humaine qui mime la physiologie in vivo. Nous avons choisi comme modèle deux lignées mésenchymateuses stromales humaines HS-27a et HS-5, très peu décrites dans la littérature. Le premier objectif a été de déterminer la qualité de cette niche tant du point de vue cellulaire, moléculaire que fonctionnel. Nos résultats montrent clairement que les cellules HS-27a participent à la formation d’une niche « quiescente » alors que les cellules HS-5 représentent une niche « proliférative ». Le deuxième objectif a été de créer une niche contrôlée pour le métabolisme oxydatif en régulant l’expression d’une protéine antioxydante, la glutathion peroxydase 3 ou GPx3. L’originalité de ce travail repose sur l’utilisation d’une méthode non virale de transfert de gène par le transposon piggyBac. Le plasmide porteur du gène d'intérêt a été apporté sous forme d’ADN et une source de transposase, enzyme catalysant la réaction d'intégration sous forme d’ARNm. Notre travail montre que GPx3 est un régulateur clé de l’homéostasie hématopoïétique favorisant le maintien des progéniteurs immatures. Pour la première fois, nous créons par ingénierie in vitro une niche hématopoïétique « calibrée » capable de mimer le microenvironnement normal et leucémique. Ce modèle permet non seulement d’identifier les acteurs clés de la régulation des cellules médullaires, mais aussi de développer des stratégies thérapeutiques ciblées. / Hematopoiesis occurs in a hypoxic microenvironment or niche in which hematopoietic stem cells (HSCs) are in close contact with mesenchymal stromal cells. Cellular interactions as well as microenvironmental factors such as reactive oxygen species are crucial for the maintenance of normal and leukemic HSCs. Developing an in vitro human culture system that closely mimcs marrow physiology is therefore essential to study the niche. Here, we present a model using two human stromal cell lines, HS-27a and HS-5. Previously poorly described in the literature, we have further characterized both of these cell lines. The first objective was to assess the quality of HS-27a and HS-5 niches by investigating their cellular, molecular and functional characteristics. Our results clearly show that HS-27a cells display features of a “quiescent” niche whereas HS-5 cells rather represent a “proliferative” niche. The second objective was to engineer a hematopoietic niche where the oxidative metabolism is optimized for the expression of an antioxidant protein, glutathione peroxidase 3 (GPx3). The originality of this work is the use of a non-viral gene transfer system by using the transposon piggyBac. This strategy was achieved by delivering a DNA plasmid carrying the gene of interest, and an mRNA source of transposase, the enzyme which catalyzes the transgene integration. Functionally, GPx3 was shown to be a key regulator for sustaining hematopoietic homeostasis by maintaining immature progenitor cells. For the first time, an original non-viral gene transfer has been used to create an in vitro hematopoietic niche that recapitulates the complexity of normal and leukemic microenvironment. This niche not only provides a platform to identify regulatory factors controlling medullary cells, but may also help in the development of targeted therapeutic strategies.

Em homens, com o avanço da idade ocorre declínio dos níveis plasmáticos de androgênios, enquanto os níveis de estrogênio permanecem constantes ou aumentados. Isto leva à diminuição da razão androgênio/estrogênio, sugerindo que os estrogênios podem ter um papel no desenvolvimento do câncer de próstata. Os estrogênios estão relacionados à indução da proliferação celular através da sua ligação aos receptores clássicos (ERs) e ao receptor não clássico GPER. Ambos receptores podem ativar as vias de sinalização PI3K e MAPK relacionadas à sobrevivência celular e ter importância no desenvolvimento do câncer de próstata (CaP) e da hiperplasia prostática benigna (HPB). Objetivo: Avaliar a relação dos receptores de estrogênio GPER, ERα, ERβ e suas isoformas com as vias PI3K e MAPK pela análise da expressão gênica e proteica dos receptores de estrogênio ERα, ERβ e GPER, pela análise da expressão de proteínas da via MAPK e PI3K e do estrogênio tecidual nos grupos HPB e CaP. Métodos: Tecidos prostáticos provenientes de CaP e de HPB foram submetidos à extração de RNA, o RNA foi reversamente transcrito para cDNA e avaliado usando q-PCR para a expressão dos receptores de estrogênio GPER, ERα, ERβ e suas isformas. A expressão proteica de GPER, ERα, ERβ, mTOR, PI3K e c-JUN e a ativação da p38α, ERK1/2, JNK, AKT e GSK3β foram analisadas por Western blot. A técnica de imunofluorescência foi utilizada para verificação da colocalização dos receptores GPER e ERα em estroma e epitélio de HPB e CaP. Resultados: Comparações entre os tecidos hiperplásico e de carcinoma indicaram uma maior expressão gênica e proteica de ERα e GPER em amostras de CaP comparadas à HPB. Além disso, a ativação da AKT, GSK3β e JNK e a expressão proteica da PI3K e c-JUN foram significativamente aumentadas nos tecidos de CaP enquanto que a expressão proteica de mTOR e a ativação de p38α e ERK estão diminuídas neste grupo. A expressão gênica do ERβ está aumentada no CaP porém não há diferença na expressão proteica entre os grupos. A análise da expressão gênica das isoformas do ERβ mostrou diminuição de ERβ1 e ERβ6 e aumento das isoformas ERβ2 e ERβ5 no CaP. Não foram detectadas as expressões das isoformas ERβ3 e ERβ4 em ambos os grupos. Nossos resultados também mostraram que os receptores GPER e ERα estão colocalizados no núcleo de células estromais de HPB e CaP. Além disso, o GPER está expresso com maior intensidade no epitélio de ambos os grupos enquanto o ERα está expresso com maior intensidade no estroma do CaP. A medida do estrogênio tecidual mostrou aumento deste no tecido de CaP em relação ao de HPB. Conclusões: O presente estudo mostrou aumento do estrogênio e de seus receptores ERα e GPER no CaP em relação ao HPB indicando que há modulação estrogênica na próstata. Além disso, proteínas das vias MAPK e PI3K que podem ser moduladas pela ação estrogênica estão expressas de maneiras diferentes em ambos os grupos. O estudo da ação estrogênica parece ser importante para o entendimento da progressão das doenças prostáticas. / In men with advancing age decline in plasma levels of androgens occurs while estrogen levels remain constant or increased. This leads to the decrease of the androgen / estrogen, suggesting that estrogens may play a role in the development of prostate cancer. Estrogens are related to the induction of cell proliferation by binding to classical receptors (ERs) and non-classical receptor GPER. Both receptors can activate signaling pathways PI3K and MAPK related to cell survival and have importance in the development of prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Objective: To evaluate the relationship of estrogen receptors GPER, ERα, ERβ with the PI3K and MAPK pathways was performed analysis of gene and protein expression of ERα, ERβ and GPER, analysis of protein expression to MAPK and PI3K pathways and tissue estrogen in PCa and BPH groups. Methods: Prostatic tissues from PCa and BPH underwent RNA extraction, RNA was reverse transcribed to cDNA and evaluated using q-PCR for the expression of estrogen receptors GPER, ERα, ERβ and their isformas. Protein expression of GPER, ERα, ERβ, mTOR, PI3K and c-JUN and activation of p38α, ERK1/2, JNK, AKT and GSK3β were analyzed by Western blot. The immunofluorescence technique was used to verify the colocalization of GPER and ERα receptors in epithelium and stroma of BPH and PCa. Results: Comparisons between the hyperplastic tissue and carcinoma showed a higher gene and protein expression of ERα and GPER in samples of CaP compared to BPH. Furthermore, activation of AKT and GSK3β and JNK protein expression of PI3K and c-JUN were significantly increased in CaP tissues while mTOR protein expression and ERK activation p38α and are decreased in this group. ERβ gene expression is increased in PCa but there is no difference in protein expression between the groups. The analysis of gene expression of ERβ isoforms showed decreased ERβ1 and ERβ6 and increased isoforms ERβ2 and ERβ5 in CaP. Expressions were not detected isoforms and ERβ3 ERβ4 in both groups. Our results also showed that the GPER and ERα receptors are positively colocalized in nucleus of stromal cells of BPH and PCa. In addition, the GPER is expressed more intensely in the epithelium of both groups while ERα is expressed with greater intensity in the stroma of PCa. The extent of this tissue revealed increased estrogen in PCa tissue compared to BPH. Conclusions: The present study showed an increase of estrogen and its receptors ERα and GPER in CaP compared to BPH indicating that there is involvement modulation of estrogen in the prostate. Moreover, proteins of MAPK and PI3K pathways can be modulated by estrogenic activity are expressed differently in both groups. The study of estrogen action appears to be important for understanding the progression of prostatic diseases.

IRS-2 is the inhibitor of serine proteases from the Ixodes ricinus tick. My task in this thesis was to find out the effect of the IRS-2 on dendritic cells activated by TLR4 ligand or by Borrelia afzelii. This effect was studied on several levels. I focused on the cytokine production, the expression of costimulatory molecules and cell signaling pathways. The results show that the IRS-2 may inhibit the expression of costimulatory molecules CD-80 a CD-86 on the cell surface, but this finding needs to be confirmed again. The production of cytokines was not affected by the IRS-2. The effect of the IRS-2 on the activity of p38, Erk1/2 nor NF-?B in LPS stimulated cells vas not observed. The fosforylation of STAT 3 in cells activated by the B. afzelii was lowered by the IRS-2.

Made available in DSpace on 2014-12-17T15:28:52Z (GMT). No. of bitstreams: 1 ThemisTSS_DISSERT.pdf: 1249174 bytes, checksum: 23a39272c35586e8f475b1aa239af353 (MD5) Previous issue date: 2012-12-21 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The cortical development requires a precise process of proliferation, migration, survival and differentiation of newly formed neurons to finally achieve the development of a functional network. Different kinases, such as PKA, CaMKII, MAPK and PI3K, phosphorylate the transcription factors CREB, and thus activate it, inducing CREB-dependent gene expression. In order to identify the involvement of such signaling pathways mediated by CREB over neuronal differentiation and survival, in vitro experiments of cell culture were conducted using pharmacological kinase inhibitors and genetic techniques to express different forms of CREB (A-CREB and CREB-FY) in cortical neurons. Inhibition of PKA and CaMKII decreased the length of neuronal processes (neurites); whereas inhibition of MAPK did not affect the length, but increased the number of neurites. Blockade of PI3K do not appear to alter neuronal morphology, nor the soma size changed with the kinase blockades. CREB activation (CREB-FY) along with MAPK and PI3K blockades presented a negative side effect over neuritic growth and the expression of A-CREB leaded to a significant decrease in neuronal survival after 60h in vitro and mimicked some of the effects on neuronal morphology observed with PKA and CaMKII blockade. In summary the signaling through CREB influences the morphology of cortical neurons, particularly when phosphorylated by PKA, and CREB signaling is also important for survival of immature neurons prior to the establishment of fully functional synaptic contacts. Our data contribute to understanding the role of CREB signaling, activated by different routes, on survival and neuronal differentiation and may be valuable in the development of regenerative strategies in different neurological diseases / O desenvolvimento cortical requer um minucioso processo de prolifera??o, migra??o, sobreviv?ncia e diferencia??o celular para que se possa alcan?ar a elabora??o de uma rede neuronal funcional. Diferentes kinases, tais quais a PKA, CaMKII, MAPK e PI3K, fosforilam o fator de transcri??o CREB, ativando-o, e induzindo em ultima inst?ncia a express?o de genes CREB-dependentes. A fim de identificar o envolvimento de tais vias de sinaliza??o mediadas por CREB sobre a diferencia??o e sobreviv?ncia neuronal, experimentos in vitro de cultura celular foram conduzidos fazendo-se uso de f?rmacos bloqueadores das kinases e de t?cnicas gen?ticas para expressar diferentes formas do CREB (A-CREB e CREB-FY) em neur?nios corticais. A inibi??o da PKA e da CAMKII diminuiu o comprimento dos neuritos; enquanto a inibi??o da MAPK n?o afetou o comprimento, mas aumentou o numero de neuritos. O bloqueio da PI3K n?o pareceu alterar a morfologia neuronal, nem o tamanho do soma foi afetado pelo bloqueio dessas kinases. A ativa??o de CREB (CREB-FY) na presen?a de bloqueadores da MAPK e PI3K teve um efeito negativo sobre o crescimento neur?tico e a express?o do A-CREB provocou uma redu??o significativa da sobreviv?ncia neuronal a partir de 60h in vitro e revelou similaridades quanto ? morfologia neuronal observadas com o bloqueio da PKA e CaMKII. Em suma, a sinaliza??o mediada por CREB influi na morfologia de neur?nios corticais, principalmente quando fosforilado pela PKA, e o bloqueio da sinaliza??o via CREB interfere na sobreviv?ncia neuronal mesmo antes do aparecimento de atividade sin?ptica. Nossos resultados contribuem para o entendimento da sinaliza??o por CREB, ativado por diferentes vias, sobre a sobreviv?ncia e diferencia??o neuronal, podendo ser de grande valia na elabora??o de estrat?gias regenerativas em diferentes doen?as neurol?gicas