Au cours de ces années de Doctorat, nous avons étudié les effets ototoxiques de certains médicaments et les moyens de prévenir les surdités neuro-sensorielles quils peuvent induire. Parmi ces molécules, nous nous sommes concentrés sur les plus couramment utilisées en pratique clinique : les antibiotiques de la famille des aminoglycosides et le cisplatine, un agent anti-cancéreux. Lintroduction de notre travail replace la surdité dans son contexte de santé publique. En particulier, nous décrivons pourquoi les médicaments ototoxiques sont utilisés et dans quelles circonstances. Nous présentons la structure de loreille interne et nous tentons dexpliquer sa vulnérabilité aux molécules ototoxiques. Nous abordons ensuite les moyens de prévention et/ou de traitement de ces atteintes neuro-sensorielles pharmaco-induites. Outre les moyens classiques de prévention, que sont les facteurs trophiques et les antioxydants, nous décrivons de nouvelles voies dapproche que sont les voies de signalisation impliquant la protéine kinase C ou la cascade dactivation RhoA/ROCK. La présentation de notre travail original sarticule autour de deux parties. Dans la première partie, nous rapportons les résultats obtenus au cours de notre étude de la toxicité des aminoglycosides et du cisplatine chez la souris et le cobaye in vivo. Nous avons mis en évidence une différence de vulnérabilité significative entre ces deux espèces face à lagression ototoxique. Cette différence existe au niveau fonctionnel, mis en évidence par létude des potentiels évoqués auditifs, et au niveau anatomique, étudié en histologie et en immunohistochimie. Nous en discutons les implications en recherche et en pratique clinique. Dans la seconde partie, nous étudions les moyens de prévenir cette surdité in vivo et in vitro. Nous avons utilisé un modèle de surdité par aminoglycoside chez le cobaye. Nous avons testé et validé une technique de perfusion intra-cochléaire in vivo. Nous avons observé les effets de deux molécules expérimentales : la Bryostatine 1, un activateur de la protéine kinase C, et un inhibiteur de la voir RhoA-ROCK. Leffet protecteur de ces molécules est actuellement limité au ganglion spiral, dont la survie est essentielle à tout traitement dimplantation prothétique et de réadaptation. Nous discutons des perspectives en médecine humaine dans notre conclusion. In this work, we focused our attention on the effects of main ototoxic drugs i.e. aminoglycosides and cisplatin in mammals. We identified new avenues for the prevention of this toxicity. In the introduction, we described how and why ototoxic drugs are used. We then described potential otoprotective strategies in neurosensory deafness. Among them, trophic factors and antioxidant molecules have been widely used. New otoprotective approaches do exist, implying the protein kinase C or RhoA/ROCK signalling. Our original work was presented in two parts. In the first part, we reported the in vivo effects of aminoglycosides and cisplatin in two mammalian species: mice and guinea pigs. Contrarily to guinea pigs, evidence of mice resistance to ototoxicity was found at a functional level, assessed by auditory brainstem responses, and at an anatomical level, studied by immunohistochemistry. We discussed the implication of such differences in research and in clinical practice. In the second part, we studied the effect of two potential otoprotective molecules: Bryostatine 1, an activator of the protein kinase C, and Y-27632, a Rho kinase inhibitor. We showed that these molecules are protecting spiral ganglion neurons both in vitro and in vivo. Survival of spiral ganglion neurons is crucial in the management and rehabilitation of deafness. The potential perspectives of these results in human medicine were discussed.

La Proteína Quinasa C (PKC) juega un papel fundamental en la regulación del crecimiento celular. Estas proteínas están implicadas en diferentes vías intracelulares que son consideradas como dianas para el tratamiento contra el cáncer. Atendiendo a las propiedades enzimáticas, las PKC se clasifican en tres grandes subfamilias: clásicas, nuevas y atípicas. En las PKC clásicas, el dominio C2 es un motivo regulador que responde a señales de Ca2+ intracelulares. Este dominio presenta un motivo denominado región rica en lisinas que interacciona con fosfolípidos acídicos. Los resultados de esta tesis demuestran que la afinidad de este dominio por fosfolípidos como el PIP2 es mayor frente a otros de la misma naturaleza. El dominio C2 de las PKC nuevas se une a fosfolípidos cargados negativamente de modo Ca2+-independiente. Nuestro estudio demuestra que la interacción de este dominio con las membranas es principalmente electrostática con una pequeña contribución de interacciones hidrofóbicas. Por otra parte, el estudio de la estructura secundaria del dominio catalítico de la PKC mostró una elevada proporción de hélice . La adición de Mg2+-ATP provocó un mayor efecto protector frente a la desnaturalización térmica. / Protein kinase C (PKC) is a family of related protein kinases that plays an important role in regulating cell growth. These protein kinases are involved in several intracellular pathways that end in transcription and are considered to be potential targets for anticancer therapy. The mammalian isoenzymes have been grouped into three subfamilies according to their enzymatic properties: classical, novel and atypical.The C2 domain is a regulatory sequence motif and is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases. This domain presents a motif named the lysine-rich cluster that interacts with acidic phospholipids. The results demonstrate that PIP2 interacts with the C2 domain of PKCα in a different way to that described for other phospolipids.C2 domain in novel PKC binds to negatively charged phospholipid vesicles in a Ca2+-independent manner. Our study confirms that the main way in which C2-PKC interacts with membranes is electrostatic in nature, with a very small contribution on the part of hydrophobic interactions.The secondary structure of catalytic domain from atypical PKC showed a high contribution of -helix component. In addition, Mg2+-ATP significantly altered the denaturation pattern of this domain because it protected against denaturation.

En esta Tesis Doctoral se ha estudiado el efecto de una serie de ácidos grasos como el araquidónico, docosahexaenoico, eicosapentaenoico y oleico, así como un conjunto de derivados de DAG-lactonas en la activación de diferentes isoenzimas de PKC. El sistema modelo de estudio utilizado han sido diferentes líneas celulares de cáncer de mama (BT-474, MCF-7 y MDA-MB-231). Mediante técnicas de biología molecular y celular, microscopía confocal, ARN de interferencia y microarrays de expresión diferencial de genes se ha estudiado la función de la PKCalpha en la capacidad proliferativa, invasiva y de migración de las células modelo de cáncer de mama, revelando nuevos mecanismos moleculares por los que la PKCalpha se localiza en la membrana plasmática y se activa en dichas células. También se pone de manifiesto el efecto antiproliferativo e inductor de apoptosis de los diversos ácidos grasos estudiados, así como la implicación directa de la PKCalpha en la capacidad proliferativa, migr atoria e invasiva de dichas células / In this Doctoral Thesis, the role of several fatty acids like arachidonic, docosahexaenoic, eicosapentaenoic and oleic, as well as some DAG-lactones derivatives, on the activation of different PKC isoforms has been studied. Some breast cancer cell lines, specifically BT-474, MCF-7 and MDA-MB-231, have been used as a model. The role of PKC in proliferation, invasion and migration has been studied by means of cellular and molecular techniques, confocal microscopy, siRNA and gene expression microarrays. The results obtained reveal new molecular mechanisms of PKCalpha; localization and activation in breast cancer cell lines. It is also interesting the anti-proliferative and pro-apoptosis role of several fatty acids tested, as well as the direct involvement of PKCalpha; in proliferation, migration and invasion of breast cancer cells

The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ± 4 g: X ± S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ± 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ± 7 min) compared to PLAC (115 ± 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo.

La connexine 43 (Cx43) est l’unité protéique de base dans la formation des canaux des jonctions gap (JG) responsables des échanges intercellulaires. Toutefois, elle forme aussi des canaux non-jonctionnels à large conductance, nommés hémicanaux (Hc), qui fournissent un accès entre l’intérieure des cellules et le milieu extracellulaire. Bien qu’ils soient beaucoup moins étudiés que les JG, on estime que les Hc restent normalement à l’état fermé, et ce, grâce à la phosphorylation des connexines qui les forment. Suite à un stress ischémique, les Cx43 se déphosphorylent et entraînent ainsi l’ouverture des Hc de Cx43 (HcCx43), un effet qui compromet la survie des cellules. La protéine kinase C (PKC) est l’enzyme de phosphorylation qui possède le plus grand nombre de sites de phosphorylation sur la Cx43 en comparaison avec les autres kinases. Ses fonctions dépendent de la mise en jeu d’un répertoire d’au moins 12 isoformes distinctes. Dans les cardiomyocytes, les isoformes de PKC participent au développement des réponses adaptées ou mésadaptées au stress ischémique. Malgré que la régulation des canaux de Cx43 par la PKC lors d’une ischémie soit bien documentée, il n’existe pas à l’heure actuelle de connaissances sur les effets fonctionnels spécifiques qu’exercent des différentes isoformes de PKC sur les HcCx43, ni sur la valeur thérapeutique de la modulation de ses derniers. Dans ce contexte, nous avons proposé que les HcCx43 sont régulés sélectivement et différentiellement par les différentes isoformes de PKC et que l’inhibition spécifique de ces hémicanaux peut protéger le coeur lors d’un événement ischémique. Le présent travail comporte trois études qui ont été entreprises spécialement dans le but de valider ces hypothèses. Dans la première étude, nous avons profité de l’expertise du laboratoire du Dr Baroudi dans la dissection des isoformes de PKC pour étudier le rôle fonctionnel de chacune d’elles dans la régulation des HcCx43 en utilisant une gamme unique de peptides synthétiques inhibiteurs et activateurs spécifiques des isoformes de PKC, en combinaison avec la technique du patch-clamp. Nous avons démontré, entre autre, que les HcCx43 sont particulièrement inhibés par l’isoforme PKC epsilon, connue pour son effet cardioprotecteur contre les dommages ischémiques lors d’un préconditionnement ischémique. Dans la deuxième étude, nous avons caractérisé l’effet d’un peptide synthétique mimétique structural de la Cx43 sur la fonction des HcCx43. En plus d’avoir élucidé ces effets sur les propriétés fonctionnelles du canal, nous avons démontré d’une manière directe et indéniable que le peptide Gap26 inhibe et spécifiquement les HcCx43 et que son administration in vitro (cardiomyocytes isolés) et ex vivo (coeur intact) confère à ces modèles expérimentaux une résistance importante contre le stress ischémique. Dans la troisième étude, nous avons investigué pour la première fois in vivo le potentiel de deux peptides uniques mimétiques structuraux de la Cx43, Gap26 et Gap27, dans la cardioprotection contre les lésions ischémiques lorsqu’ils sont administrés à basse dose sous forme d’un bolus intraveineux unique. Nous avons démontré que l’injection de ces peptides avant ou après la survenue de l’ischémie réduit significativement la taille de l’infarctus qui en résulte.En conclusion, l’ensemble de ces résultats révèlent le rôle bénéfique de l’inhibition des HcCx43 lors d’une ischémie et dévoilent un potentiel thérapeutique prometteux des mimétiques structuraux de Cx43 dans la prévention et le traitement de l’infarctus du myocarde. / Connexin 43 (Cx43) is the basic unit in the composition of Gap junction channels but also of the non-junctional unapposed hemichannels (Hc). Gap junction channels play key roles in cardiac function by allowing conduction of electrical impulses and exchange of biologically important molecules between cells. The unapposed Hc, however, perform functions different from those achieved by Gap junction channels mainly by providing pathways between the cytosol and the extracellular space allowing movement of ions and other small metabolites. Although they are much less studied than Gap junction channels, Hc are believed to remain normally in a closed state and that phosphorylation is an important factor promoting their closure. Under ischemic stress,the amount of non-phosphorylated Cx43 increases resulting in increasing hemichannels opening, an effect that can lead to irreversible tissue injury and cell death. Protein kinase C (PKC) possesses the largest number of phosphorylation sites on Cx43 and exerts significant control on Cx43 channels. Its function depends on the involvement of at least 12 distinct isoformes. Various PKC isoforms exert specific cellular and cardiovascular functions, nonetheless the functional role of PKC isoforms in the modulation of the unapposed Cx43 hemichannels has never been assessed, neither has the therapeutic potential of Cx43Hc modulation in the protection of ischemic heart. In this context, three studies have been performed, they form the body of this thesis. In the first study, a unique set of synthetic PKC isoform-selective activator and inhibitor peptides was utilised. In combination with the patch clamp technique, we have demonstrated that Cx43Hc conductance is strongly inhibited by, among many isoforms, epsilon PKC isoforme, known for its cardioprotective effect against ischemic injury. In the second study, we characterized the effect of a synthetic structural mimetic peptide of Cx43. Using patch clamp technique, we have demonstrated that the peptide Gap26 inhibits directly and specifically Cx43Hc, we also showed that Gap26 can confer resistance to cardiomyocytes (in vitro) and intact heart (ex vivo) against ischemia. In the third study, we investigated for the first time in vivo the capability of a unique pair of structural Cx43 mimetic peptides, Gap26 and Gap27, to protect heart from ischemic injury when administered in single low-dose intravenous boluses. We demonstrated that administration of either one or both peptides, before or after the onset of ischemia renders heart more resistant to ischemia and reduces significantly the size of myocardial infarct. Altogether, our results revealed salvatory effect of Cx43Hc inhibition during ischemia and uncovered therapeutique potentials of the synthetic structural mimetic peptides of Cx43 in ischemic heart disease.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The reactivation of a memory results in its destabilization, requiring a process of memory reconsolidation to maintain it. Spermidine is an endogenous aliphatic amine with polycationic structure that modulates N-methyl-D-aspartate (NMDA) receptor activity and improves memory. Recent evidence suggests that systemic administration of spermidine improves the reconsolidation of fear memory. Here we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by intrahippocampal (ih) administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4 mA footshock as unconditioned stimulus. Twenty-four hours after training, animals were re-exposed to the apparatus in the absence of shock (reactivation session). Immediately after the reactivation session, spermidine (2-200 ρmol/site); the PKC inhibitor, 3-[1- (dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl) maleimide hydrochloride (GF 109203X, 0.3 - 30 ρg/site); the antagonist of the polyamine-binding site at the NMDA receptor, arcaine (0.2 - 200 ρmol/site) or the PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.02 - 2 nmol/site) were injected intra-hipocampally (i.h.). Testing was carried out in the same apparatus, twenty-four hours after reactivation. Freezing scores at testing were considered a measure of memory. While the post-reactivation administration of spermidine (20 and 200 ρmol/site) improved, GF 109203X (1, 10 and 30 ρg/site) impaired memory reconsolidation. GF 109203X (0.3 ρg/site) prevented spermidine (200 ρmol/site)-induced improvement of memory reconsolidation. The post-reactivation administration of arcaine (200 pmol/site) impaired and PMA (2 nmol/site) improved memory reconsolidation. PMA (0.2 nmol/site) prevented arcaine (200 ρmol/site)-induced impairment of memory reconsolidation. The protein synthesis inhibitor anisomycin (2 μg/site) prevented spermidine (200 ρmol/site)-induced improvement of memory reconsolidation. These drugs had no effect on memory if they were administered in the absence of reactivation. These results suggest that the enhancement of memory reconsolidation induced by the administration of spermidine involves PKC activation. / A reativação de uma memória resulta na sua desestabilização, que exigem um processo de reconsolidação de memória para mantê-la. A espermidina é uma amina alifática endógena com estrutura policatiônica que modula a atividade do receptor Nmetil- D-aspartato (NMDA) e melhora a memória. Evidências recentes sugerem que a administração sistêmica de espermidina melhora a reconsolidação da memória de medo. No entanto não se sabe se a administração intra hipocampal de espermidina melhora a reconsolidação da memória e nem se a proteína cinase dependente de cálcio (PKC) e síntese proteica estão envolvidas neste efeito. Portanto, no presente estudo verificou-se o envolvimento da PKC e da síntese proteica na melhora da reconsolidação da memória do medo induzida pela administração intrahipocampal (ih) de espermidina em ratos. Ratos Wistar machos foram treinados na tarefa de medo condicionado contextual utilizando-se choque de 0,4 mA como estímulo incondicionado. Vinte e quatro horas após o treino, os animais foram re-expostos ao aparelho na ausência de choque (sessão reativação). Imediatamente após a sessão de reativação foram administradas, por via ih, espermidina (2-200 ρmol/sítio); o inibidor da PKC, 3- [1- (dimetilaminopropil) indol-3-il] -4- (indol-3-il) maleimida (GF 109203X, 0,3-30 ρg/sítio); o antagonista do sítio de ligação das poliaminas no receptor de NMDA, arcaína (0,2-200 ρmol/sítio) ou o ativador de PKC, 12-miristato 13-acetato de forbol (PMA, 0,02-2 nmol/sítio). Os testes foram realizados no mesmo aparelho, 24 horas após a sessão de reativação. O tempo de imobilidade dos animais durante o teste foi considerado como medida de memória. Enquanto a administração de espermidina (20 e 200 ρmol/sítio) melhorou, GF 109203X (1, 10 e 30 ρg/sítio) prejudicou a reconsolidação da memória. O GF 109203X (0,3 ρg/sítio) preveniu a melhora da reconsolidação da memória induzida por espermidina (200 ρmol/sítio). A administração da arcaína (200 pmol/sítio) prejudicou e PMA (2 nmol/sítio) melhorou a reconsolidação da memória. O PMA (0,2 nmol/sítio) preveniu o prejuízo na reconsolidação da memória induzido por arcaína (200 ρmol/sítio). O inibidor de síntese de proteica, anisomicina (2 ug/sítio) preveniu a melhora da reconsolidação da memória induzida por espermidina (200 ρmol/sítio). Estas drogas não tiveram nenhum efeito sobre a memória quando foram administrados na ausência da sessão de reativação. Estes resultados sugerem que a melhora da reconsolidação da memória induzida pela administração ih de espermidina envolve a síntese proteica e a ativação de PKC.

Orientadores: Everardo Magalhães Carneiro, Marcia Queiroz Latorraca / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:35:57Z (GMT). No. of bitstreams: 1 Veloso_RobertoVilela_D.pdf: 1133010 bytes, checksum: ae2bb8d027240ebc20854087fa364a1b (MD5) Previous issue date: 2007 / Resumo: A restrição protéica materna reduz o crescimento e promove alterações permanentes na estrutura e função de órgãos da prole, contribuindo para o desenvolvimento de doenças cardiovasculares, obesidade e diabetes. O consumo de alimentos à base de soja está associado a um menor risco de desenvolver diabetes pelo seu conteúdo de isoflavonas e pela composição de aminoácidos de sua proteína que contribuem para uma melhora na secreção de insulina. Tem sido sugerido que a genisteína, uma isoflavona da soja, modula a secreção de insulina através das vias AMPc/PKA e PLC/PKC. Deste modo, nós avaliamos o valor biológico da dieta à base de farinha de soja e seus efeitos sobre o crescimento de órgãos, o perfil de aminoácidos, insulina e metabólitos séricos em ratos submetidos à restrição protéica na infância e recuperados com essa dieta após o desmame, bem como o efeito da dieta à base de soja sobre a secreção de insulina em resposta a glicose e a ativadores do adenilato ciclase e proteína quinase C, além da expressão da PKAa e PKCa em ilhotas pancreáticas de ratos adultos. Ratos de mães alimentadas com 17% ou 6% de proteína (caseína) durante a gestação e lactação foram mantidos com dieta contendo 17% de proteína à base de caseína (grupos CC e RC) ou dieta com 17% de proteína à base de farinha de soja (grupos CS e RS) e dieta com 6% de proteína (grupo HP). Após 90 dias de idade, proles de mães alimentadas com dieta hipoprotéica exibiram déficit permanente de peso corpóreo e de concentrações séricas de insulina, taurina, glutamina, fenilserina e lisina, porém apresentaram um aumento no peso relativo dos órgãos, exceto do fígado. A dieta à base de farinha de soja reduziu o peso relativo do fígado e aumentou as concentrações séricas de insulina, taurina, ornitina e fenilserina. Embora ratos recuperados com soja (RS) tenham ingerido mais dieta proporcionalmente ao peso corpóreo do que os ratos recuperados com caseína (RC) eles mostraram menor coeficiente de eficácia alimentar, e resultou em peso corpóreo final similar entre esses grupos. As concentrações séricas de albumina e proteínas totais não diferiram entre os grupos RS e RC. A dieta à base de soja melhorou a resposta de células beta de ratos controles em concentrações fisiológicas de glicose, enquanto em ilhotas de ratos recuperados isso ocorreu na presença de concentrações suprafisiológicas de glicose. A presença de PMA induziu uma resposta secretória com potência similar em ilhotas dos grupos RS e CS e a expressão de PKC foi similar em todos os grupos, exceto no grupo HP, que expressou menores concentrações dessa proteína. A adição de forskolin ao meio de incubação aumentou a secreção de insulina em ratos recuperados e naqueles mantidos com caseína e a expressão de PKAa foi maior no grupo RS em relação ao grupo CS. Esses resultados sugerem que dieta à base de farinha de soja é capaz de promover a recuperação nutricional em animais submetidos à restrição protéica em fases críticas de desenvolvimento, melhorando o perfil de aminoácidos séricos que estimulam a secreção de insulina. Além disso, a melhora na secreção de insulina parece não ser devido a ativação das vias AMPc/PKA e inositol fosfato/PKC / Abstract: Maternal protein restriction leads to reduction in the growth of organs, permanent changes in their structure and functions contributing to development of cardiovascular disease, obesity and diabetes. Consumption of soy-based foods is associated to lower risk of diabetes by its isoflavone content and amino acid composition of its protein that contribute to improve of insulin secretion. It has been suggested that genistein, soy isoflavone, modulates the insulin secretion through of cAMP/PKA and PLC/PKC pathways. Thus, we evaluated the biological value of soybean flour diet and its effects on organ growth, serum amino acids, insulin and metabolites profile in rats submitted to protein restriction in early life and recovered with those diet after weaning, as well as, the effect of soybean diet on insulin secretion in response to glucose and activators of adenylate cyclase and PKC, besides the expression of PKA and PKC in pancreatic islets from adult rats. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet and with 6% casein (LP groups) diet. At 90d of age offspring of protein-restricted-mothers exhibited permanent deficit of body weight, serum insulin, taurine, glutamine, phenylserine and lysine concentrations, but increased relative organs¿ weight, except liver. Soybean flour diet reduced the relative liver weight and increased serum insulin, taurine, ornithine and phenylserine concentrations. Although SR rats had eaten proportionally to body weight more diet than CR rats they showed lower feed conversion efficiency which resulted in the final body weight similar between these groups. The SR and CR also exhibited similar serum albumin and protein concentrations. Soybean diet improved the response of ß-cells from control rats to a physiological concentration of glucose, whereas in islets from recovered rats this occurred in presence of a supra-physiological glucose concentration. PMA induced similar potent secretory response in islets from SR and SC groups and PKC expression was similar in all groups, except LP that expressed lower levels this protein. Forskolin increased the insulin secretion in recovered rats and in those maintained with casein diet and PKA expression was higher in SR than in SC rats. These results suggest that soybean flour diet is able to promote the nutritional recovery in animals submitted to protein restriction in critical phase of development improving serum amino acid levels that have the stimulatory effect on insulin release. Moreover the improve of insulin secretion seemed does not to be due the activation of the cAMP/PKA and inositol phosphate/PKC pathways / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Células tronco embrionárias (CTE) são capazes de proliferar indefinidamente mantendo a sua pluripotência, isto é, a capacidade de se diferenciar em diversos tipos celulares perante estímulos adequados. Esse potencial tem sido intensamente estudado, de modo a permitir a utilização dessas células em terapias de reposição celular. Trabalhos anteriores demonstraram que as proteínas kinases C (PKC) são importantes moduladores moleculares de cascatas de sinalização que levam ao processo de proliferação e auto-renovação das CTE. Porém o papel exato das diferentes isoenzimas das PKCs ainda não foi elucidado. Isso ocorre porque a família das PKCs é composta por pelo menos dez isoenzimas e apenas, recentemente, desenvolveram-se moduladores específicos para as diferentes isoenzimas, o que permitirá estudar o papel específico dessas quinases. No presente trabalho verificamos que a ativação da PKC&#948; induziu a proliferação de CTE indiferenciadas sem induzir a diferenciação das mesmas. Para tentar elucidar as vias de sinalização mediadas pela PKC&#948 que levam à proliferação das CTE indiferenciadas realizamos estudos de fosfoproteômica o que possibilitou a identificação de potenciais alvos diretos e indiretos da PKC&#948. Dentre os alvos identificados foram encontradas diversas proteínas relacionadas com proliferação, transcrição, tradução e resposta ao stress (chaperonas), contribuindo para a hipótese de que a ativação da PKC&#948; leva à proliferação das CTE indiferenciadas. Em diversos sistemas, a ativação da PKC&#948; leva à ativação da MAPK, em particular das ERK1/ 2, sendo essa via capaz de induzir a proliferação de diversas linhagens celulares. Identificamos diversas proteínas alvos da PKC&#948, que interagem também com componentes da via das MAPKs. Desta forma, verificamos a influência da ativação da PKC&#948 na via das MAPKs. De fato, a ativação da PKC&#948 na linhagem de CTE murinas indiferenciadas, E14TG2a, ativou a MEK, ERK1/ 2 e o fator de transcrição ELK-1. Como estudos anteriores demonstraram que a inibição da ERK1/ 2 mantém CTE indiferenciadas e que a ativação desta via poderia levar à diferenciação de CTE, investigamos a cinética de ativação da ERK pela PKC&#948. Demonstramos que a ativação da ERK pela PKC&#948 se da de modo transiente e que apesar da PKC&#948 não translocar para o núcleo, sua ativação induz a fosforilação e translocação nuclear da ERK, que atuará na fosforilação do fator de transcrição ELK-1. Desta forma, concluímos que a PKC&#948 induz a proliferação das CTE murinas indiferenciadas ativando transitoriamente a via das ERK1/ 2, que translocam para o núcleo fosforilando fatores de transcrição como a ELK1 e levando possivelmente ao aumento de proliferação dessas células. A ativação transiente das ERK1/ 2 pela PKC&#948 é importante para a auto-renovação das CTE. / Embryonic stem cells (ESC) are able of proliferating indefinitely maintaining their pluripotency, which is the capability to differentiate in different cell types upon appropriate stimuli. Pluripotency has been intensely investigated in order to allow the use of these cells in cellular replacement therapies. Previous work has demonstrated that the serine/ threonine kinases, such as, Protein kinases C (PKC) are important modulators of signaling cascades that lead to the process of proliferation and self-renewal of ESC. However, the exact role of the different PKC isoenzymes still remains to be elucidated. Due to the fact that the PKC family is composed of at least ten different isoenzymes and only recently isoenzyme specific modulators have been developed, which now allows the elucidation of these kinases roles. In the present work we verified that activation of PKC&#948 induced undifferentiated ESC have their proliferation rate increased. Trying to elucidate the signaling pathways mediated by PKC&#948 that lead to the proliferation increase we performed phosphoproteomic studies to identify potential PKC&#948 targets. Between the targets identified we found several proteins related with proliferation, protein transcription, translation and stress response (chaperones). These targets contributed to the hypothesis that PKC&#948 activation leads to undifferentiated ESC proliferation. In different cell lines, PKC&#948 activation leads to MAPK activation, through ERK1/ 2 activation, which are frequently involved with cellular proliferation. We also identified several targets of PKC&#948 that Interact with several components of MAPK`s signaling cascade. PKC&#948 activation in murine undifferentiated ESC line, E14TG2a, led to MEK, ERK1/ 2 and the transcription factor Elk-1 activation. Some articles demonstrate that the inhibition of ERK1/2 are responsible to maintains ESC undifferentiated and that it`s activation could lead to ESC differentiation. Analysing the kinetics of ERK activation in the ESC by PKC&#948, we show that ERK activation was transient and despite the fact that PKC&#948 does not translocated to the nucleus upon activation, but induces ERK activation and it`s nuclear translocation, where ERK could phosphorylate the transcription factor Elk-1. In conclusion PKC&#948 induces undifferentiated murine ESC proliferation increase by a transient ERK activation and it`s nuclear translocation.

<p>The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines.<p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished