Memoria para optar al título de Químico Farmacéutico / Los fibroblastos cardiacos son los mayores responsables de la secreción y renovación de la matriz extracelular (MEC). Por otro lado, bajo condiciones patológicas como en el post-infarto al miocardio, los fibroblastos cardiacos se diferencian a miofibroblastos, células con un fenotipo mucho más activo en cuanto a la producción de la MEC, además ambos elementos celulares son claves en el desarrollo del remodelamiento cardiaco después de un infarto agudo al miocardio. Una regulación controlada en el crecimiento de la población de fibroblastos y miofibroblastos es importante para una correcta cicatrización y mantención de la función cardiaca. Uno de los mayores reguladores de la población cardiaca es el Sistema Renina-Angiotensina (RAS) y se ha reportado que en una situación post-infarto este sistema se encuentra sobre-activado, específicamente la enzima convertidora de angiotensina y el receptor subtipo AT1 de angiotensina (AT1R). Nuestro modelo experimental consideró sobrexpresar el AT1R en ambos tipos celulares con el uso de adenovirus y evaluar si angiotensina II (Ang II) modula la viabilidad celular. Nuestros resultados demuestran que la estimulación con Ang II conduce a una masiva muerte del fibroblasto cardiaco neonato que sobrexpresa el AT1R (FCN-AdAT1R) de una manera tiempo-dependiente. Ang II gatilla la apoptosis por la vía mitocondrial, promoviendo el aumento de los niveles de Bax, la pérdida del potencial de membrana mitocondrial (mΔψ) y la fragmentación de la caspasa 3. Los efectos producidos por Ang II sobre los FCN-AdAT1R fueron bloqueados por Losartan (antagonista específico AT1R) y por los inhibidores de la vía de la fosfolipasa C -proteínakinasa C (PLC-PKC), U73122 y Gö6976, respectivamente. Por otra parte, los miofibroblastos cardiacos neonatos que sobrexpresan el AT1R (MCN-AdAT1R), fueron menos propensos que los FCN-AdAT1R a los efectos inducidos por Ang II, en cuanto a muerte celular, apoptosis y aumentos en los niveles de Bax. La diferente sensibilidad a la apoptosis producida por Ang II por parte de los MCN-AdAT1R se debió en parte al cambio del fenotipo celular Nuestros resultados demuestran que Ang II ocasiona apoptosis del FCN-AdAT1R por la vía AT1R-PLC-PKC y que esta depende de la vía mitocondrial / The cardiac fibroblasts are main cells involved in secretion and turnover to extracellular matrix protein (ECM). Under pathological conditions characterized by inflammation, as in myocardial infarction, they are differentiated to cardiac myofibroblast, which are cells with a more active phenotype, producing higher ECM protein. Both cellular types are key elements in the development of cardiac remodeling after myocardial infarction. A tight regulation of fibroblast and myofibroblast growth is important to correct wound healing and maintain cardiac function. Renine-angiotensin-system (RAS) is the most important regulator of cardiovascular system, and have been reported that after myocardiac infarction the RAS is up-regulated, specifically angiotensin converting enzyme (ACE) and angiotensin type 1 receptor (AT1R). In our experimental model using adenovirus to overexpress the AT1R in cardiac fibroblast (FCN-AT1R), we evaluated the Ang II effects on cell viability. Ours results show that Ang II triggers FCN-AdAT1R death, in a time-dependent manner. The death cell produced for Ang II in our model was apoptosis by mitochondrial pathway, through an increase Bax expression, loss of mitochondrial membrane potential (mΔψ) and caspase 3 activation. These effects were blocked by Losartan (specific antagonist of AT1R) and by phospholipase C-proteinkinase C (PLC-PKC) inhibitors, U73122 and Gö6976 respectively. For other hand, cardiac myofibroblast that overespressing AT1R (MCN-AdAT1R) were less sensible than FCN-AdAT1R to effect of Ang II on death cell, apoptosis and expression of Bax. The different sensibility to apoptosis Ang II-induced was due to the different phenotype. Ours results show that Ang II triggers apoptosis of FCN-AdAT1R by AT1R-PLC-PKC pathway through mitochondrial disruption

Phosphatidylinositol is a a minor membrane component of eukaryotic cells, however, it plays a crucial role in cell signaling pathways as a precursor for a number of signaling molecules and second messengers. Among the most significant ones are phosphoinositides created by phosphorylation of the hydroxyl groups of phosphatidylinositol at positions 3,4, and 5 of the inositol ring. Despite its significance, the spatial and temporal distribution and dynamics of phosphatidylinositol remains unclear owing mainly to the lack of a specific optical probe (biosensor) to visualize phosphatidylinositol in living cells. Biosensor for inositol phospholipids are based on lipid-binding domains of their effector proteins with high enough affinity and specificity for a given phosphoinositide - but nosuch domain is known for PI. However, an enzyme - phosphatidylinositol-dependent phospholipase C - that specifically recognizes phosphatidylinositol is known. This enzyme catalyzes the hydrolysis of phosphatidylinositol into diacylglycerol and inositol 1-phosphate and unlike eukaryotic homologs does not act upon the phosphorylated forms of phosphatidylinositol. The main aim of this thesis was to solve the structures of several inactive mutant forms of phospholipase C from Bacillus cereus complexed to myo-inositol which...