Memoria para optar al título de Químico Farmacéutico / En los últimos años se ha descrito y dado énfasis al rol determinante que tiene el fibroblasto cardiaco (FC) en el proceso inflamatorio, dónde cumple un rol clave a fin de mantener la homeostasis del órgano. En los procesos inflamatorios y en cualquier episodio de daño, los neutrófilos son la primera línea de defensa del organismo, los cuales son atraídos al sitio de daño por la quimioquina, interleuquina 8 (IL-8), quien además, provoca su activación. Posteriormente, el reclutamiento de neutrófilos en los sitios de daño es dependiente de las proteínas de adhesión E-selectina, ICAM-1 y VCAM-1. Por lo tanto, controlar la expresión de estas proteínas constituye un nuevo enfoque terapéutico en el tratamiento de los procesos inflamatorios. En ese sentido, el interferón beta (IFN-β), ha demostrado moderar la respuesta inflamatoria; pero hasta la fecha no se ha demostrado su utilidad como agente antiinflamatorio en el corazón y específicamente en el FC. Debido a estos antecedentes, el objetivo general fue estudiar el efecto preventivo del IFN-β sobre la expresión de IL-8, ICAM-1 y VCAM-1 bajo un contexto inflamatorio, inducido por estímulo con LPS. Consecuente con este hallazgo, se observó que el IFN-β disminuye la adhesión de neutrófilos sobre FC inducida por LPS. De esta manera, se demostró que el tratamiento con IFN-β puede modular la cascada inflamatoria, y así, transformarse en una potencial y valiosa herramienta terapéutica capaz de controlar la expresión de estas proteínas de vital importancia partícipes del proceso inflamatorio luego de algún evento de injuria cardiaca como lo puede ser una miocarditis, o bien, un infarto al miocardio / In recent years it is described the determining role that cardiac fibroblast (CF) has in the inflammatory process, where plays a major role to maintain homeostasis. In inflammatory process and any event of damage, neutrophils are the first line of defense, which are attracted to the site of damage through interleukin 8 (IL-8), who initially causes activation. Subsequently, neutrophil recruitment at sites of damage depends on Eselectin, ICAM-1 and VCAM-1 adhesion proteins. Therefore, controlling the expression of these proteins is a new therapeutic approach in the treatment of inflammatory process. In this sense, interferon beta (IFN-β) has shown be a inflammatory moderator; but to date it has not been proved useful as an anti-inflammatory agent in the heart, specifically on CF. On the basis of this background, the overall objective was to study the preventive effect of IFN-β on the expression of IL-8, ICAM-1 and VCAM-1 on CF under an inflammatory stimuli with LPS and evaluate neutrophil adhesion to CF. The results showed that IFN-β prevents the increased expression on IL-8, ICAM-1 and VCAM-1 induced by LPS. Consistent with this finding, we observed that IL-8, ICAM-1 and VCAM-1 decreases the neutrophil adhesion induced also by LPS on CF. Thus, it was shown that IL-8, ICAM-1 and VCAM-1 can modulate the inflammatory cascade, and thus become a potentially valuable therapeutic tool able to manage these important participants proteins of the inflammatory process after an event of cardiac injury as can be a myocarditis, or myocardial infarction

Memoria para optar al Título de Bioquímico / The genus Salmonella belongs to the Enterobacteriaceae family and comprises two species: S. bongori and S. enterica. Both species include more than 2500 serovars. Within S. enterica serovars, the most studied are S. Typhi, S. Typhimurium and S. Enteritidis, the latter being the main causative agent of salmonellosis worldwide. During its infective cycle, Salmonella needs to adapt to variations in different environmental conditions, such as changes in pH, osmolarity and oxygen availability. In addition, these conditions serve as environmental signals that modulate the expression of virulence factors. One of these factors is the lipopolysaccharide (LPS), the main component of the envelope of Gram negative bacteria. The LPS presents three structural domains: the O antigen (AgO), the core region and lipid A, also called endotoxin. The latter anchors the LPS to the outer membrane, and is a known virulence factor responsible for the induction of the immune response. The lipid A domain is capable of undergoing covalent modifications, which modulate its toxicity and helps bacteria to evade antimicrobial agents and the immune system of the host. One of the main environmental cues faced by Salmonella during its infectious cycle is the oxygen availability. During the aerobic-anaerobic transition participates the global regulators ArcA and Fnr. Results from our laboratory show that changes in the polymerization degree of the AgO in response to oxygen availability is modulated by these regulators. This observation led us to examine whether global regulators Fnr and ArcA are involved in the control of probable oxygen-dependent modifications of lipid A in response to oxygen availability. The main goal of this study was to determine the effect of oxygen availability on covalent modifications of lipid A in S. Enteritidis and the participation of transcriptional factors ArcA and Fnr in this process. First, we mounted a method for LPS extraction followed by a hydrolysis step to obtain a highly-purified lipid A that can be used to study the composition of this molecule by MALDI-TOF. Thus, in aerobiosis we observed four major signals corresponding to hexa-acylated and hepta-acylated lipid A molecules and their corresponding hydroxylated species. Under this environmental condition, hydroxylated species were more abundant than non-hydroxylated species. In the case of anaerobic cultures we detected the same four signal, but this relationship is reversed, being more abundant the nonhydroxylated species. To determine the participation of ArcA and Fnr in the modulation of covalent modifications of lipid A, we generated ΔarcA and Δfnr mutant strains and a strain overproducing ArcA (parcA). Our results indicate that both regulators are involved in controlling structural changes of lipid A in response to oxygen availability. Additionally, we observed that these regulators control the incorporation of other covalent modifications into lipid A. LpxO enzyme (encoded by lpxO gene) is responsible of the hydroxylation of lipid A in Salmonella. Therefore, we studied the relative expression (anaerobic/aerobic) of this gene in wild-type S. Enteritidis and their Δfnr and parcA derivatives via qRT-PCR. Our results indicate that the expression of lpxO depends on oxygen availability, an observation that is consistent with changes in the levels of lipid A hydroxylation described herein. Furthermore, ArcA and Fnr participate in the modulation of the expression of this gene. The results of this study reveal a change in the hydroxylation degree of S. Enteritidis lipid A in response to oxygen availability, in which global regulators ArcA and Fnr are involved. These findings contribute to understanding the molecular mechanisms of adaptation to the environment used by Salmonella during infection / Fondecyt

Memoria para optar al título de Químico Farmacéutico / El daño ocasionado por la ocurrencia de un infarto cardiaco es complejo, generando la pérdida de viabilidad de las células cardiacas entre otros efectos deletéreos ocurridos tanto en el periodo isquémico de falta de oxígeno y nutrientes, así como también en la posterior reperfusión sanguínea. Frente a esta condición patológica los fibroblastos cardiacos (FC) son capaces de reaccionar, secretando y renovando la matriz extracelular; lo que los convierte en elementos celulares claves en la cicatrización y remodelamiento del tejido cardiaco dañado post-infarto al miocardio. Esto hace necesario el intentar regular la viabilidad de estas células para una correcta cicatrización y mantención de la función cardiaca. En relación a esto se ha reportado el efecto cardioprotector ejercido por el empleo de LPS en condiciones de daño celular causado por isquemia/reperfusión (I/R). Sin embargo, dicho efecto ha sido descrito principalmente en cardiomiocitos, por lo que su efecto en FC es aún desconocido. Nuestro trabajo estudió la capacidad cito-protectora ejercida por LPS sobre FC de ratas neonatas sometidos a un modelo de I/R in vitro e indagó las vías transduccionales implicadas en este efecto. El tratamiento de los FC con LPS (1 μg/mL) durante la isquemia y reperfusión previno la pérdida de viabilidad inducida por I/R. Sin embargo, en el pre-condicionamiento con LPS durante 24 o 16 h, y en el tratamiento con LPS durante la isquemia o reperfusión, no se observó el efecto cito-protector. En esta misma línea, demostramos que el efecto cito-protector ejercido por LPS es mediado a través del receptor TLR4 vía PI3K/Akt y ERK1/2, ya que el empleo de TAK-242, Ly29002 y PD98059, inhibidores del receptor y de las vías transduccionales respectivamente, bloquearon completamente el efecto cito-protector. La activación de TLR4 por LPS previno, además, el procesamiento de la procaspasa 8 y 3 inducido por I/R. Conjuntamente, demostramos que las vías PI3K/Akt y ERK1/2 participaban en la prevención del procesamiento de la procaspasa 8 ejercido por LPS, pero no tuvieron efecto en la activación de la caspasa 3. Nuestros resultados dan cuenta del efecto cito-protector y antiapoptótico ejercido por LPS a través de TLR4 vía PI3K/Akt y ERK1/2 frente a la muerte de FC inducida por I/R / The damage caused by a myocardial infarct is complex, causing cardiac cell viability loss, among other deleterious effects that occur during the ischemia period, such as lack of oxygen and nutrients; as well as the posterior reperfusion with blood. In this situation, cardiac fibroblasts react by secreting proteins and renewing the extracellular matrix. These properties make them a key element in the scar and remodeling process of the injured cardiac tissue. Thus, the viability of these cells are required to preserve the correct functioning of the heart. In this regard, the use of LPS as a cytoprotector agent in cardiac Ischemia/reperfusion (I/R) has been reported. However such effect has been described mostly in cardiomyocytes cells, whereas its role in cardiac fibroblasts remains unknown. Our work studied the LPS cytoprotector effect in neonate cardiac fibroblast exposed to an in vitro model of I/R, and transductional pathways involved in this process were explored. Incubation with LPS (1μg/mL) during both ischemia and reperfusion periods prevented the I/R-induced cell loss. However, LPS used only during preconditioning, ischemia or reperfusion did not induced cytoprotection. Furthermore we demonstrated that the LPS-dependent protective effects were completely abolished when TLR4 receptor (TAK-242), and PI3K/Akt and ERK1/2 (Ly29002 and PD98059, respectively) inhibitors were used. These data suggest that LPS-dependent cytoprotector effects are mediated through TLR4 receptor activation and PI3K/Akt and ERK1/2 signalling pathways. Additionally the activation of TLR4 by LPS prevented the cleave of procaspases 3 and 8 induced by I/R. Both PI3K/Akt and ERK1/2 were necessary for the prevention of the caspase 8 activation, but not of caspase 3. Our results conclude that LPS protects cardiac fibroblasts from I/R apoptosis by the activation of TLR4 and both PI3K/Akt and ERK1/2 signalling pathways / FONDECYT

Memoria para optar al título de Químico Farmacéutico / Autorizada por el autor, pero con restricción para ser publicada a texto completo hasta diciembre de 2015, en el Portal de Tesis Electrónicas. / La diferenciación de fibroblastos cardiacos (FC) a miofibroblastos cardiacos (MFC) es gatillada por TGF-β1, la cual señaliza principalmente a través de las proteínas Smad. Los MFC muestran como principal característica la presencia de microfilamentos citoplásmicos de α-SMA, estructuradas como fibras de estrés, lo que les permite la contracción. Por otro lado, LPS es un ligando del receptor TLR4, que señaliza de manera dependiente e independiente de MyD88 teniendo como principal efector el NF-κB, induciendo así la expresión de genes de citoquinas proinflamatorias. Sin embargo, aunque se ha descrito un efecto antagónico entre la señalización inducida por LPS y la señalización canónica de TGF- β, a la fecha no se ha estudiado si en FC y en MFC la expresión de α-SMA inducida por TGF-β1 es antagonizada por LPS. Para responder estas interrogantes se utilizaron FC y MFC de ratas adultas, y se determinó in vitro la capacidad de LPS de inhibir la expresión de α-SMA. Además, utilizando TAK- 242, un inhibidor de las vías intracelulares dependientes de TLR4, se determinó que los efectos gatillados por LPS ocurrían a través de este receptor. La utilización de los inhibidores PD98059, LY29002 y BAY 11-7082 permitió determinar que en FC LPS a través de la vía de señalización PI3K/Akt, disminuye la expresión de α-SMA. Por lo que nuestros resultados demuestran que LPS inhibe la expresión de α-SMA en FC, a través del receptor TLR4 mediante la activación de vía de señalización PI3K/Akt y que LPS es capaz de disminuir la expresión de α-SMA en MFC a través de TLR4 inhibiendo la mantención del fenotipo miofibroblasto / The difference between cardiac fibroblasts (CF) and cardiac myofibroblast (CMF) is triggered by TGF-β1, which mainly signals via Smad proteins. The main characteristic of CMF is cytoplasmic microfilaments of α-SMA; this are structured as stress fibers, which allow the contraction of CMF. Also, LPS is a ligand of the TLR4 receptor that signals via a dependant and independent pathway of MyD88 whose main factor is NF-κB; this induces the expression of inflammatory cytokine genes. However, despite an opposite effect has been described between the signaling induced by LPS and the canonical signaling of TGF-β, the opposite effect that LPS may have on the expression of α-SMA on CF and CMF induced by TGF-β1 has not been studied so far. In order to answer these questions, CF and CMF of adult rats were used; this showed in vitro evidence that LPS is capable of inhibiting the expression of α-SMA. Also, with the use of TAK-242, which is an inhibitor of the dependant intracellular domain of TLR4, we determined that the effects triggered by LPS occurred through said receptor. Using PD98059, LY29002 and BAY11-7082 inhibitors allowed us to determine that, via the PI3K/Akt signaling pathway, LPS decreases the expression of α-SMA in CF. Therefore, our results show that LPS inhibit the expression of α-SMA in CF through the TLR4 receptor via the activation of the PI3K/Akt signaling pathway, and that LPS is capable of decreasing the expression of α-SMA in CMF through TLR4 receptor, which inhibits the maintenance of the myofibroblast phenotype