Patr��n g��nico de fibrosis y apoptosis en nefropat��a obstructiva experimental : : modulaci��n por rosuvastatina

La nefropat��a obstructiva puede ser un desorden renal complejo de tratar debido al severo cuadro inflamatorio, desbalance oxidativo, apoptosis y fibrosis. Estudios previos sostienen que rosuvastatina (Ros) podr��a tener utilidad como una opci��n terap��utica en enfermedades renales que cursar��an con apoptosis y fibrosis. Objetivo: Evaluar los posibles efectos antiapopt��ticos y antifibr��ticos de Ros durante la obstrucci��n ureteral unilateral en ratas neonatas. Materiales y M��todos: Ratas Wistar neonatas de 48 hs. de vida fueron intervenidas quir��rgicamente (grupo experimental) o no (grupo control). Ambos grupos fueron subdivididos en tratadas o no tratadas con Ros (10mg / kg por d��a) v��a oral durante 14 d��as. Posteriormente se procedi�� a nefrectomizar y procesar las cortezas renales para determinar por RT-PCR las expresiones de genes: ��xido nítrico sintasa inducible (iNOS), factor promotor g��nico de chaperonas (hsf1), prote��na de shock t��rmico (hsp70), bax, bcL2, wt1, p53, snail, prote��na morfog��nica del hueso (bmp7), caderina E, factor transformador de crecimiento (tgf-��) y factor de necrosis tumoral (tnf-��). Resultados: La obstrucci��n ureteral unilateral neonatal indujo una marcada fibrosis y apoptosis, mientras que el tratamiento con Ros modul�� el patr��n de genes fibr��ticos y apopt��ticos mediante disminuci��n de la expresi��n de bmp7, caderina E, wt1, p53 y bcl2; adem��s indujo una ca��da en la expresi��n de los genes profibr��ticos y proapopt��ticos (bax, tnf-�� y tgf-��). El an��lisis de los resultados presentados, permiten sugerir que la protecci��n renal de rosuvastatina durante nefropat��a obstructiva de ratas neonatas estar��a asociado a la interacci��n entre hsp70 y la biodisponibilidad del ��xido nítrico con el concomitante descenso en genes pro-apopt��ticos.

Efectos de vecindad de la radiaci��n ionizante y sus implicaciones en radioterapia y radioprotecci��n

En el paradigma cl��sico, los efectos biol��gicos de la radiaci��n ionizante se atribuyen al da��o en el ADN inducido en cada c��lula irradiada. La demostraci��n de efectos de vecindad causados por radiaci��n ionizante (EVIR) ha generado un cambio profundo en la concepci��n actual de la radiobiolog��a. Los EVIR son aquellos efectos causados por la radiaci��n que se producen en c��lulas que no han sido irradiadas. Diversos avances t��cnicos, en particular el empleo de microhaces, han permitido estudiar los EVIR in vitro. Se conocen dos v��as por las cuales las c��lulas irradiadas pueden comunicarse con las no irradiadas, a saber: mediante uniones especializadas (nexos) que comunican los citoplasmas de c��lulas adyacentes, y mediante la secreci��n de factores solubles al medio extracelular. Estos factores incluyen varias citokinas y especies reactivas del ox��geno y nitr��geno. Las v��as de se��alizaci��n en las c��lulas afectadas involucran en particular la activaci��n de prote��na kinasas activadas por mit��genos (MAPK) y del factor de transcripci��n NFciclooxigenasa 2, sintasa de ��xido nítrico 2 y NAD(P)H oxidasa. Los EVIR pueden causar mutaciones puntuales y cambios epigen��ticos. Los efectos sobre las v��as de se��alizaci��n pueden persistir indefinidamente e incluso transmitirse a la descendencia. Parad��jicamente, en ciertas condiciones los EVIR pueden ser adaptativos, es decir que tornan a las c��lulas afectadas m��s resistentes a la radiaci��n. La adaptaci��n exige s��ntesis de prote��nas y mejora la capacidad celular de reparar el ADN y resistir el estr��s oxidativo. Los EVIR tambi��n se han demostrado in vivo. Por tanto, pueden tener implicaciones importantes en radioterapia, tanto para mejorar la eficacia terap��utica como para reducir la incidencia de efectos adversos. Asimismo, su mejor conocimiento puede influenciar las normas internacionales de radioprotecci��n.

Fertilization Management: Assessing New Techniques and Key Interactions to Increase Crop Yields With Less N2O Emissions

El ��xido nitroso (N2O) es un potente gas de efecto invernadero (GHG) proveniente mayoritariamente de la fertilizaci��n nitrogenada de los suelos agr��colas. Identificar estrategias de manejo de la fertilizaci��n que reduzcan estas emisiones sin suponer un descenso de los rendimientos es vital tanto a nivel econ��mico como medioambiental. Con ese prop��sito, en esta Tesis se han evaluado: (i) estrategias de manejo directo de la fertilizaci��n (inhibidores de la nitrificaci��n/ureasa); y (ii) interacciones de los fertilizantes con (1) el manejo del agua, (2) residuos de cosecha y (3) diferentes especies de plantas. Para conseguirlo se llevaron a cabo meta-an��lisis, incubaciones de laboratorio, ensayos en invernadero y experimentos de campo. Los inhibidores de la nitrificaci��n y de la actividad ureasa se proponen habitualmente como medidas para reducir las p��rdidas de nitr��geno (N), por lo que su aplicaci��n estar��a asociada al uso eficiente del N por parte de los cultivos (NUE). Sin embargo, su efecto sobre los rendimientos es variable. Con el objetivo de evaluar en una primera fase su efectividad para incrementar el NUE y la productividad de los cultivos, se llev�� a cabo un meta-an��lisis. Los inhibidores de la nitrificaci��n dicyandiamide (DCD) y 3,4-dimetilepyrazol phosphate (DMPP) y el inhibidor de la ureasa N-(n-butyl) thiophosphoric triamide (NBPT) fueron seleccionados para el an��lisis ya que generalmente son considerados las mejores opciones disponibles comercialmente. Nuestros resultados mostraron que su uso puede ser recomendado con el fin de incrementar tanto el rendimiento del cultivo como el NUE (incremento medio del 7.5% y 12.9%, respectivamente). Sin embargo, se observ�� que su efectividad depende en gran medida de los factores medioambientales y de manejo de los estudios evaluados. Una mayor respuesta fue encontrada en suelos de textura gruesa, sistemas irrigados y/o en cultivos que reciben altas tasas de fertilizante nitrogenado. En suelos alcalinos (pH ��� 8), el inhibidor de la ureasa NBPT produjo el mayor efecto. Dado que su uso representa un coste adicional para los agricultores, entender las mejores pr��cticas que permitan maximizar su efectividad es necesario para posteriormente realizar comparaciones efectivas con otras pr��cticas que incrementen la productividad de los cultivos y el NUE. En base a los resultados del meta-an��lisis, se seleccion�� el NBPT como un inhibidor con gran potencial. Inicialmente desarrollado para reducir la volatilizaci��n de amoniaco (NH3), en los ��ltimos a��os algunos investigadores han demostrado en estudios de campo un efecto mitigador de este inhibidor sobre las p��rdidas de N2O provenientes de suelos fertilizados bajo condiciones de baja humedad del suelo. Dada la alta variabilidad de los experimentos de campo, donde la humedad del suelo cambia r��pidamente, ha sido imposible entender mecan��sticamente el potencial de los inhibidores de la ureasa (UIs) para reducir emisiones de N2O y su dependencia con respecto al porcentaje de poros llenos de agua del suelo (WFPS). Por lo tanto se realiz�� una incubaci��n en laboratorio con el prop��sito de evaluar cu��l es el principal mecanismo bi��tico tras las emisiones de N2O cuando se aplican UIs bajo diferentes condiciones de humedad del suelo (40, 60 y 80% WFPS), y para analizar hasta qu�� punto el WFPS regula el efecto del inhibidor sobre las emisiones de N2O. Un segundo UI (i.e. PPDA) fue utilizado para comparar el efecto del NBPT con el de otro inhibidor de la ureasa disponible comercialmente; esto nos permiti�� comprobar si el efecto de NBPT es espec��fico de ese inhibidor o no. Las emisiones de N2O al 40% WFPS fueron despreciables, siendo significativamente m��s bajas que las de todos los tratamientos fertilizantes al 60 y 80% WFPS. Comparado con la urea sin inhibidor, NBPT+U redujo las emisiones de N2O al 60% WFPS pero no tuvo efecto al 80% WFPS. La aplicaci��n de PPDA increment�� significativamente las emisiones con respecto a la urea al 80% WFPS mientras que no se encontr�� un efecto significativo al 60% WFPS. Al 80% WFPS la desnitrificaci��n fue la principal fuente de las emisiones de N2O en todos los tratamientos mientras que al 60% tanto la nitrificaci��n como la desnitrificaci��n tuvieron un papel relevante. Estos resultados muestran que un correcto manejo del NBPT puede suponer una estrategia efectiva para mitigar las emisiones de N2O. Con el objetivo de trasladar nuestros resultados de los estudios previos a condiciones de campo reales, se desarroll�� un experimento en el que se evalu�� la efectividad del NBPT para reducir p��rdidas de N y aumentar la productividad durante un cultivo de cebada (Hordeum vulgare L.) en secano Mediterr��neo. Se determin�� el rendimiento del cultivo, las concentraciones de N mineral del suelo, el carbono org��nico disuelto (DOC), el potencial de desnitrificaci��n, y los flujos de NH3, N2O y ��xido nítrico (NO). La adici��n del inhibidor redujo las emisiones de NH3 durante los 30 d��as posteriores a la aplicaci��n de urea en un 58% y las emisiones netas de N2O y NO durante los 95 d��as posteriores a la aplicaci��n de urea en un 86 y 88%, respectivamente. El uso de NBPT tambi��n increment�� el rendimiento en grano en un 5% y el consumo de N en un 6%, aunque ninguno de estos incrementos fue estad��sticamente significativo. Bajo las condiciones experimentales dadas, estos resultados demuestran el potencial del inhibidor de la ureasa NBPT para mitigar las emisiones de NH3, N2O y NO provenientes de suelos arables fertilizados con urea, mediante la ralentizaci��n de la hidr��lisis de la urea y posterior liberaci��n de menores concentraciones de NH4 + a la capa superior del suelo. El riego por goteo combinado con la aplicaci��n dividida de fertilizante nitrogenado disuelto en el agua de riego (i.e. fertirriego por goteo) se considera normalmente una pr��ctica eficiente para el uso del agua y de los nutrientes. Algunos de los principales factores (WFPS, NH4 + y NO3 -) que regulan las emisiones de GHGs (i.e. N2O, CO2 y CH4) y NO pueden ser f��cilmente manipulados por medio del fertirriego por goteo sin que se generen disminuciones del rendimiento. Con ese prop��sito se evaluaron opciones de manejo para reducir estas emisiones en un experimento de campo durante un cultivo de mel��n (Cucumis melo L.). Los tratamientos incluyeron distintas frecuencias de riego (semanal/diario) y tipos de fertilizantes nitrogenados (urea/nitrato c��lcico) aplicados por fertirriego. Fertirrigar con urea en lugar de nitrato c��lcico aument�� las emisiones de N2O y NO por un factor de 2.4 y 2.9, respectivamente (P < 0.005). El riego diario redujo las emisiones de NO un 42% (P < 0.005) pero aument�� las emisiones de CO2 un 21% (P < 0.05) comparado con el riego semanal. Analizando el Poder de Calentamiento global en base al rendimiento as�� como los factores de emisi��n del NO, concluimos que el fertirriego semanal con un fertilizante de tipo nítrico es la mejor opci��n para combinar productividad agron��mica con sostenibilidad medioambiental en este tipo de agroecosistemas. Los suelos agr��colas en las ��reas semi��ridas Mediterr��neas se caracterizan por su bajo contenido en materia org��nica y bajos niveles de fertilidad. La aplicaci��n de residuos de cosecha y/o abonos es una alternativa sostenible y eficiente desde el punto de vista econ��mico para superar este problema. Sin embargo, estas pr��cticas podr��an inducir cambios importantes en las emisiones de N2O de estos agroecosistemas, con impactos adicionales en las emisiones de CO2. En este contexto se llev�� a cabo un experimento de campo durante un cultivo de cebada (Hordeum vulgare L.) bajo condiciones Mediterr��neas para evaluar el efecto de combinar residuos de cosecha de ma��z con distintos inputs de fertilizantes nitrogenados (pur��n de cerdo y/o urea) en estas emisiones. La incorporaci��n de rastrojo de ma��z increment�� las emisiones de N2O durante el periodo experimental un 105%. Sin embargo, las emisiones de NO se redujeron significativamente en las parcelas enmendadas con rastrojo. La sustituci��n parcial de urea por pur��n de cerdo redujo las emisiones netas de N2O un 46 y 39%, con y sin incorporaci��n de residuo de cosecha respectivamente. Las emisiones netas de NO se redujeron un 38 y un 17% para estos mismos tratamientos. El ratio molar DOC:NO3 - demostr�� predecir consistentemente las emisiones de N2O y NO. El efecto principal de la interacci��n entre el fertilizante nitrogenado y el rastrojo de ma��z se dio a los 4-6 meses de su aplicaci��n, generando un aumento del N2O y una disminuci��n del NO. La sustituci��n de urea por pur��n de cerdo puede considerarse una buena estrategia de manejo dado que el uso de este residuo org��nico redujo las emisiones de ��xidos de N. Los pastos de todo el mundo proveen numerosos servicios ecosist��micos pero tambi��n suponen una importante fuente de emisi��n de N2O, especialmente en respuesta a la deposici��n de N proveniente del ganado mientras pasta. Para explorar el papel de las plantas como mediadoras de estas emisiones, se analiz�� si las emisiones de N2O dependen de la riqueza en especies herb��ceas y/o de la composici��n espec��fica de especies, en ausencia y presencia de una deposici��n de orina. Las hip��tesis fueron: 1) las emisiones de N2O tienen una relaci��n negativa con la productividad de las plantas; 2) mezclas de cuatro especies generan menores emisiones que monocultivos (dado que su productividad ser�� mayor); 3) las emisiones son menores en combinaciones de especies con distinta morfolog��a radicular y alta biomasa de ra��z; y 4) la identidad de las especies clave para reducir el N2O depende de si hay orina o no. Se establecieron monocultivos y mezclas de dos y cuatro especies comunes en pastos con rasgos funcionales divergentes: Lolium perenne L. (Lp), Festuca arundinacea Schreb. (Fa), Phleum pratense L. (Php) y Poa trivialis L. (Pt), y se cuantificaron las emisiones de N2O durante 42 d��as. No se encontr�� relaci��n entre la riqueza en especies y las emisiones de N2O. Sin embargo, estas emisiones fueron significativamente menores en ciertas combinaciones de especies. En ausencia de orina, las comunidades de plantas Fa+Php actuaron como un sumidero de N2O, mientras que los monocultivos de estas especies constituyeron una fuente de N2O. Con aplicaci��n de orina la comunidad Lp+Pt redujo (P < 0.001) las emisiones de N2O un 44% comparado con los monocultivos de Lp. Las reducciones de N2O encontradas en ciertas combinaciones de especies pudieron explicarse por una productividad total mayor y por una complementariedad en la morfolog��a radicular. Este estudio muestra que la composici��n de especies herb��ceas es un componente clave que define las emisiones de N2O de los ecosistemas de pasto. La selecci��n de combinaciones de plantas espec��ficas en base a la deposici��n de N esperada puede, por lo tanto, ser clave para la mitigaci��n de las emisiones de N2O. ABSTRACT Nitrous oxide (N2O) is a potent greenhouse gas (GHG) directly linked to applications of nitrogen (N) fertilizers to agricultural soils. Identifying mitigation strategies for these emissions based on fertilizer management without incurring in yield penalties is of economic and environmental concern. With that aim, this Thesis evaluated: (i) the use of nitrification and urease inhibitors; and (ii) interactions of N fertilizers with (1) water management, (2) crop residues and (3) plant species richness/identity. Meta-analysis, laboratory incubations, greenhouse mesocosm and field experiments were carried out in order to understand and develop effective mitigation strategies. Nitrification and urease inhibitors are proposed as means to reduce N losses, thereby increasing crop nitrogen use efficiency (NUE). However, their effect on crop yield is variable. A meta-analysis was initially conducted to evaluate their effectiveness at increasing NUE and crop productivity. Commonly used nitrification inhibitors (dicyandiamide (DCD) and 3,4-dimethylepyrazole phosphate (DMPP)) and the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) were selected for analysis as they are generally considered the best available options. Our results show that their use can be recommended in order to increase both crop yields and NUE (grand mean increase of 7.5% and 12.9%, respectively). However, their effectiveness was dependent on the environmental and management factors of the studies evaluated. Larger responses were found in coarse-textured soils, irrigated systems and/or crops receiving high nitrogen fertilizer rates. In alkaline soils (pH ��� 8), the urease inhibitor NBPT produced the largest effect size. Given that their use represents an additional cost for farmers, understanding the best management practices to maximize their effectiveness is paramount to allow effective comparison with other practices that increase crop productivity and NUE. Based on the meta-analysis results, NBPT was identified as a mitigation option with large potential. Urease inhibitors (UIs) have shown to promote high N use efficiency by reducing ammonia (NH3) volatilization. In the last few years, however, some field researches have shown an effective mitigation of UIs over N2O losses from fertilized soils under conditions of low soil moisture. Given the inherent high variability of field experiments where soil moisture content changes rapidly, it has been impossible to mechanistically understand the potential of UIs to reduce N2O emissions and its dependency on the soil water-filled pore space (WFPS). An incubation experiment was carried out aiming to assess what is the main biotic mechanism behind N2O emission when UIs are applied under different soil moisture conditions (40, 60 and 80% WFPS), and to analyze to what extent the soil WFPS regulates the effect of the inhibitor over N2O emissions. A second UI (i.e. PPDA) was also used aiming to compare the effect of NBPT with that of another commercially available urease inhibitor; this allowed us to see if the effect of NBPT was inhibitor-specific or not. The N2O emissions at 40% WFPS were almost negligible, being significantly lower from all fertilized treatments than that produced at 60 and 80% WFPS. Compared to urea alone, NBPT+U reduced the N2O emissions at 60% WFPS but had no effect at 80% WFPS. The application of PPDA significantly increased the emissions with respect to U at 80% WFPS whereas no significant effect was found at 60% WFPS. At 80% WFPS denitrification was the main source of N2O emissions for all treatments. Both nitrification and denitrification had a determinant role on these emissions at 60% WFPS. These results suggest that adequate management of the UI NBPT can provide, under certain soil conditions, an opportunity for N2O mitigation. We translated our previous results to realistic field conditions by means of a field experiment with a barley crop (Hordeum vulgare L.) under rainfed Mediterranean conditions in which we evaluated the effectiveness of NBPT to reduce N losses and increase crop yields. Crop yield, soil mineral N concentrations, dissolved organic carbon (DOC), denitrification potential, NH3, N2O and nitric oxide (NO) fluxes were measured during the growing season. The inclusion of the inhibitor reduced NH3 emissions in the 30 d following urea application by 58% and net N2O and NO emissions in the 95 d following urea application by 86 and 88%, respectively. NBPT addition also increased grain yield by 5% and N uptake by 6%, although neither increase was statistically significant. Under the experimental conditions presented here, these results demonstrate the potential of the urease inhibitor NBPT in abating NH3, N2O and NO emissions from arable soils fertilized with urea, slowing urea hydrolysis and releasing lower concentrations of NH4 + to the upper soil layer. Drip irrigation combined with split application of N fertilizer dissolved in the irrigation water (i.e. drip fertigation) is commonly considered best management practice for water and nutrient efficiency. Some of the main factors (WFPS, NH4 + and NO3 -) regulating the emissions of GHGs (i.e. N2O, carbon dioxide (CO2) and methane (CH4)) and NO can easily be manipulated by drip fertigation without yield penalties. In this study, we tested management options to reduce these emissions in a field experiment with a melon (Cucumis melo L.) crop. Treatments included drip irrigation frequency (weekly/daily) and type of N fertilizer (urea/calcium nitrate) applied by fertigation. Crop yield, environmental parameters, soil mineral N concentrations, N2O, NO, CH4, and CO2 fluxes were measured during the growing season. Fertigation with urea instead of calcium nitrate increased N2O and NO emissions by a factor of 2.4 and 2.9, respectively (P < 0.005). Daily irrigation reduced NO emissions by 42% (P < 0.005) but increased CO2 emissions by 21% (P < 0.05) compared with weekly irrigation. Based on yield-scaled Global Warming Potential as well as NO emission factors, we conclude that weekly fertigation with a NO3 --based fertilizer is the best option to combine agronomic productivity with environmental sustainability. Agricultural soils in semiarid Mediterranean areas are characterized by low organic matter contents and low fertility levels. Application of crop residues and/or manures as amendments is a cost-effective and sustainable alternative to overcome this problem. However, these management practices may induce important changes in the nitrogen oxide emissions from these agroecosystems, with additional impacts on CO2 emissions. In this context, a field experiment was carried out with a barley (Hordeum vulgare L.) crop under Mediterranean conditions to evaluate the effect of combining maize (Zea mays L.) residues and N fertilizer inputs (organic and/or mineral) on these emissions. Crop yield and N uptake, soil mineral N concentrations, dissolved organic carbon (DOC), denitrification capacity, N2O, NO and CO2 fluxes were measured during the growing season. The incorporation of maize stover increased N2O emissions during the experimental period by c. 105 %. Conversely, NO emissions were significantly reduced in the plots amended with crop residues. The partial substitution of urea by pig slurry reduced net N2O emissions by 46 and 39 %, with and without the incorporation of crop residues respectively. Net emissions of NO were reduced 38 and 17 % for the same treatments. Molar DOC:NO3 - ratio was found to be a robust predictor of N2O and NO fluxes. The main effect of the interaction between crop residue and N fertilizer application occurred in the medium term (4-6 month after application), enhancing N2O emissions and decreasing NO emissions as consequence of residue incorporation. The substitution of urea by pig slurry can be considered a good management strategy since N2O and NO emissions were reduced by the use of the organic residue. Grassland ecosystems worldwide provide many important ecosystem services but they also function as a major source of N2O, especially in response to N deposition by grazing animals. In order to explore the role of plants as mediators of these emissions, we tested whether and how N2O emissions are dependent on grass species richness and/or specific grass species composition in the absence and presence of urine deposition. We hypothesized that: 1) N2O emissions relate negatively to plant productivity; 2) four-species mixtures have lower emissions than monocultures (as they are expected to be more productive); 3) emissions are lowest in combinations of species with diverging root morphology and high root biomass; and 4) the identity of the key species that reduce N2O emissions is dependent on urine deposition. We established monocultures and two- and four-species mixtures of common grass species with diverging functional traits: Lolium perenne L. (Lp), Festuca arundinacea Schreb. (Fa), Phleum pratense L. (Php) and Poa trivialis L. (Pt), and quantified N2O emissions for 42 days. We found no relation between plant species richness and N2O emissions. However, N2O emissions were significantly reduced in specific plant species combinations. In the absence of urine, plant communities of Fa+Php acted as a sink for N2O, whereas the monocultures of these species constituted a N2O source. With urine application Lp+Pt plant communities reduced (P < 0.001) N2O emissions by 44% compared to monocultures of Lp. Reductions in N2O emissions by species mixtures could be explained by total biomass productivity and by complementarity in root morphology. Our study shows that plant species composition is a key component underlying N2O emissions from grassland ecosystems. Selection of specific grass species combinations in the context of the expected nitrogen deposition regimes may therefore provide a key management practice for mitigation of N2O emissions.

���Estudio de los mecanismos moleculares involucrados en la inmunosupresi��n observada durante la infecci��n con Trypanosoma cruzi: Rol de la v��a PD-1/PD1-L y E3 ubiquitina ligasas���.

Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, un problema de salud importante en Am��rica Latina, as�� como tambi��n en Am��rica Central, ya que causa infecci��n cr��nica afectando a millones de personas [1]. Durante esta enfermedad se han descripto varias alteraciones de la respuesta inmune, entre ellas una severa inmunosupresi��n durante la etapa aguda de la infecci��n, tanto en humanos como en ratones. C��lulas T provenientes de ratones infectados activadas in vitro, muestran reducci��n en la respuesta proliferativa a mit��genos, caracter��stica de un estado de inmunosupresi��n [2-4]. La falla del sistema inmune durante estadios tempranos de la infecci��n probablemente colabore con la diseminaci��n y el establecimiento del par��sito. Un gran n��mero de estudios se han focalizado en la identificaci��n de mecanismos moleculares responsables del fen��meno de inmunosupresi��n, entre los mecanismos citados se ha demostrado presencia de c��lulas supresoras [5-9], factores inmunosupresores presentes en el par��sito [2, 3, 10-13], producci��n excesiva de ��xido nítrico [14], disminuida producci��n de IL-2 y reducida expresi��n del receptor de IL2 en c��lulas de bazo de animales infectados [9, 15-17]. Muchos de estos mecanismos han sido exhaustivamente investigados, sin embargo no est�� del todo claro si existen mecanismos adicionales involucrados en la inmunosupresi��n de la c��lula T. Adicionalmente, en los ��ltimos a��os nuevas mol��culas que median la regulaci��n negativa de la c��lula T, entre las cuales est��n PD-1/PD1-L [18], arginasa [19] y E3 ubiquitina ligasas [20-22], han sido reportadas durante inmunosupresi��n en diversas infecciones.

Identificaci��n de aspectos inmunol��gicos en la infecci��n placentaria por Trypanosoma cruzi en modelos in vitro e in vivo.

El Trypanosoma cruzi, agente causal del Chagas, atraviesa la placenta, pudiendo infectar el feto y causando la enfermedad de Chagas cong��nita. Hay evidencias de que la competencia inmunol��gica de la placenta juega un rol en la transmisi��n cong��nita. El proceso de infecci��n placentario puede verse modificado por el juego entre factores delet��reos para T. cruzi, como el ��xido nítrico (NO), estr��s nitrosativo-oxidativo (EO) y la cantidad de par��sitos y capacidad de la c��lula parasitaria de resistir, invadir y proliferar dentro del tejido placentario. El factor inhibitorio de la migraci��n de macr��fagos (MIF) es una citoquina proinflamatoria que juega un importante rol inmuno-regulatorio que estimula la producci��n de NO. Por ello postulamos como hip��tesis que T. cruzi incrementa la producci��n de MIF en placenta, con aumento de citoquinas proinflamatorias, ��xido nítrico e incremento del estr��s nitrosativo, participando en la infecci��n de la placenta y el mecanismo de transmisi��n cong��nita de la enfermedad. Los objetivos espec��ficos son: a)Analizar el sistema MIF - ICAM1 - NO en la infecci��n de explantos de vellosidades placentarias por formas trypomastigotes del T. cruzi en dise��os experimentales in vitro. b)Verificar estado de estr��s oxidativo-nitrosativo y alteraciones de la barrera placentaria inducido por ��xido nítrico en explantos placentarios en presencia de T. cruzi in vitro. c)Analizar la expresi��n de MIF e ICAM-1 en placentas en un modelo de Chagas cong��nito en ratones infectados con T. cruzi de la cepa Tulahuen con dieta normoproteica y normocalorica. d)Verificar nivel de transmisi��n cong��nita en las cr��as de ratones infectados por T. cruzi seg��n expresi��n de MIF e ICAM1 con dietas normales para estos animales. Los dise��os experimentales ser��n in Vitro mediante empleo de explantos de placentas en cultivo en interacci��n con formas infectivas de T. cruzi y dise��os en ratones en un modelo de transmisi��n cong��nita. Las t��cnicas a emplear ser��n cultivos de tejidos, t��cnicas inmunohistoqu��micas, western blot, PCR y qPCR, RT-qPCR, mediciones anal��ticas en medios de cultivos y plasma y suero de ratones. Esperamos encontrar una respuesta inflamatoria exacerbada, como ya ha sido descripta en embarazadas que produjeron transmisi��n cong��nita de la enfermedad de Chagas a sus hijos, el incremento de citoquinas pro-inflamatorias y del estr��s nitrosativo como el observado in vitro inducido por T. cruzi, (resultados preliminares) que podr��an da��ar la barrera placentaria y favorecer la transmisi��n cong��nita de la enfermedad de Chagas mediada por MIF. Como MIF se puede detectar en circulaci��n sangu��nea de la embarazada que en parte es aportado por la placenta (Cardalopolis y col., 2012), podr��a emplearse como un indicador de la probabilidad de transmisi��n cong��nita de la enfermedad.

Sildenafil na hipertens��o pulmonar persistente do rec��m-nascido associada a h��rnia diafragm��tica cong��nita

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Micropart��culas de quitosana estruturadas com aerosil��: estabilidade, adsor����o, encapsula����o e libera����o de subst��ncias ativas

Biodegradable microspheres used as controlled release systems are important in pharmaceutics. Chitosan biopolymer represents an attractive biomaterial alternative because of its physicochemical and biological characteristics. Chitosan microspheres are expected to become promising carrier systems for drug and vaccine delivery, especially for non-invasive ways oral, mucosal and transdermal routes. Controlling the swelling rate and swelling capacity of the hydrogel and improving the fragile nature of microspheres under acidic conditions are the key challenges that need to be overcomed in order to enable the exploration of the full pharmaceutical potential use of these microparticles. Many studies have focused on the modification of chitosan microsphere structures with cross-linkers, various polymers blends and new organic-inorganic hybrid systems in order to obtain improved properties. In this work, microspheres made of chitosan and nanosized hydrophobic silica (Aerosil R972) were produced by a method consisting of two steps. First, a preparation of a macroscopically homogeneous chitosan-hydrophobic silica dispersion was prepared followed by spray drying. FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry, thermal gravimetric analysis, scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (TEM) were used to characterize the microspheres. Also, the were conducted acid stability, moisture sorption capacity, release properties and biological assays. The chitosan-hydrophobic silica composite microspheres showed improved thermal degradation, lower water affinity, better acid stability and ability to retard rifampicin and propranolol hydrochloride (drug models) release under simulated physiological conditions. In vitro biocompatibility studies indicated low cytotoxicity and low capacity to activate cell production of the pro-inflammatory mediator nitric oxide. The results show here encourage further studies on the use of the new chitosan-hydrophobic silica composite microspheres as drug carrier systems via oral or nasal routes.

Xilanas de sabugo de milho como agente antioxidante, citot��xico, anticoagulante e imunomodulador

Prospecting pharmacological active polysaccharides from agricultural byproducts, such as corncobs, is an underexplored practice in the scientific community. Thus, this work aims to expand knowledge about pharmacological activities of polysaccharides extracted from corncobs. From corn cob flour a extract was obtained by ultrasound waves in an alkaline medium, and the end of the process the product was termed PECC (polysaccharidic extract from corncobs). This extract was physicochemical characterized and evaluated by in vitro assays as an antioxidant, cytotoxic, anticoagulant and imunomodulator agent. Results indicated significant activity metal chelating by PECC, and the use of PECC in cell culture cells showed no toxic effects to normal cell lines, but toxic action against HeLa tumor cells due promoting cell death by apoptosis. In addition, other pharmacological effects were observed, the PECC decreased nitric oxide (NO) production by activated macrophages, and prolonged blood clotting time through APTT assay. Then methanolic, ethanolic and ketone fractions were obtained from fractionation of PECC polysaccharides. Five methanolic fractions, six ethanolic fractions and two ketones were obtained; and all fractions were evaluated for antioxidant, cytotoxic, anticoagulant, immunomodulatory activities. E1.4 fraction exhibited significant metal chelating effect, a toxic action to induce apoptosis in HeLa cells, decreased NO production by activated macrophages, and extended blood clotting time. These results showed that the PECC pharmacological active polysaccharides would be present in the fraction E1.4. From fractionation of E1.4 polysaccharide six subfractions with different sizes were obtained: <3; 3-10; 10-30; 30-50; 50-100 and >100 KDa. About 80% of E1.4 polysaccharides had lower size to 10 KDa, and all the subfractions showed over 61% sugar in their chemical compositions. These subfractions exhibited different monosaccharide compositions, but xylose was presented in all of them. The subfractions exhibited distinct pharmacological effects in in vitro assays. Smaller subfractions (<30 KDa) had highest metal chelating activity and greater toxic action in tumor cells. The intermediate fractions (between 30-100 KDa) decreased more NO production of activated macrophages, for other side, the larger size (>100 KDa) modulated a greater number of inflammatory cytokines, and the had greatest anticoagulant effect. Therefore, when analyzing all the results together it is evident that the PECC pharmacological polysaccharides are heteroxylans, and were concentrated in E1.4 fraction, and heteroxilanas pharmacological effects depends on their molecular size. Thus, corncobs could be used as source from molecules with biotechnology potential

Efeito imunomodulador e atividade antimicrobiana de heterofucanas de Sargassum filipendula

Fucans, sulphated polysaccharides that contain L-fucose in its constitution, obtained from species of Phaeophyceae of the Sargassum kind, display several biological activities. Heterofucans from Sargassum filipendula are bioactive molecules that contain strong antiproliferative and antioxidant activity. However, their immunomodulatory and antimicrobial activities have not yet been examined. In this context, the aim of this research was to evaluate the heterofucans as for their immunomodulatory capacity and antimicrobial action against Leishmania infantum, Trichomonas vaginalis, Staphylococcus epidermidis and Klebsiella pneumonia (KPC). The five heterofucans obtained from S. filipendula show activities that are distant as stimulants of the immune system and microbial agent. The SF0.5V, SF0.7V amd SF1.0V heterofucans were capable of acting in the activation of murine and human macrophages. In addition to that, SF0.5V has shown antibiofilm activity of S. epidermides and SF0.7V and 1.0V almost completely inhibited the survival of the protozoan T. vaginalis. Results such as this one, reflect the broad range of action of the sulphated polysaccharides obtained from seaweeds, especially from the species S.filipendula

Extra����o, caracteriza����o e prospec����o de atividades farmacol��gicas de polissacar��deos sulfatados da macroalga verde Caulerpa prolifera

This study aimed to extract, characterize and conduct a prospective analysis of pharmacological activities of sulfated polysaccharides from green seaweed Caulerpa prolifera. Seven fractions (CP-0.3/CP-0.5/CP-0.7/CP-0.9/CP-1.1/CP-1.5/CP-2.0) were obtained from C. prolifera by alkaline proteolysis followed by sequential precipitation in acetone. The physicochemical analyzes indicated that C. prolifera synthesizes a homogalactan (CP-0.9) and different populations of sulfated heteropolysaccharides. In the analysis of anticoagulant activity, all fractions except CP-0.3, influenced the intrinsic coagulation pathway. All fractions showed antioxidant activity in six different assays being more pronounced in hydrogen peroxide scavenging assay, especially CP-0.3, CP-0.7 and CP-0.9 (which obtained 61% of hydrogen peroxide scavenging), in ferric chelation assay (especially CP-0.9 with 56% chelation) and cupric chelation assay (especially CP-2.0 with 78% chelation). With respect to immunomodulatory activity, the presence of CP-0.3, CP-0.7 and CP-0.9 showed an immunogenic potential, increasing the production of nitric oxide (NO) by 48, 142 and 163 times, respectively. Conversely, the NO synthesis fell 73% after the activation of macrophages by LPS, incubated concurrently with CP-2.0. The anti-adipogenic activity of the fractions was also evaluated and CP-1.5 was able to reduce the differentiation of pre-adipocytes (3T3-L1) into adipocytes by 60%, without affecting the cell viability. The fractions CP-0.3, CP-0.5 and CP-0.9 reduced the viability of the HeLa cells (human cervical adenocarcinoma) by 55% and CP-1.5 reduced the viability of the 786-0 cells (human renal adenocarcinoma) by 75%. Leishmanicidal activity and microbicide effect against Carbapenem-resistant Klebsiella pneumoniae (KPC) have not been identified. However, the viability of Staphylococcus epidermidis was reduced by 23.8% in the presence of CP -1.5. All fractions were able to change the formation of calcium oxalate crystals. CP-0.3, CP-0.5 and CP-1.1 only promoted the formation of COD type crystals with a very small size (1 ��m). Confocal microscopy and zeta potential data of crystals formed in the presence of the samples showed that the polysaccharides present in the fractions must interact with calcium ions present throughout the crystal lattice, affecting the growth and morphology of crystals The results described herein indicate that the fractions rich in polysaccharides obtained from the green seaweed C. prolifera present a multi therapeutic potential, and subsequent purification steps, as well as research on the mechanisms of action by which these polymers act should be investigated.

Avalia����o das atividades farmacol��gicas de fucoidans obtidos do extrato bruto de fucoidan comercial de Fucus vesiculosus

Fucoidan is a term used to define heteropolysaccharides that are composed of less than 90% L-fucose. The exception to this rule is the homofucoidan obtained from the seaweed Fucus vesiculosus. This fucoidan can be purchased from SIGMA Co. and have been used in various research for evaluation of their pharmacological activities. However, it is not a pure molecule. In fact, it is a mix of several fucoidan molecules. In this work, were obtained, from acetone precipitation, and biochemically characterized, four fucoidan molecules from SIGMA-ALDRICH Co. fucoidan to evaluate their anticoagulant, antioxidant, antiadipogenic, immunomodulatory and antiurolithiatic activities. In anticoagulant activity, evaluated by aPTT assay, fucoidans F0.9, F1.1 and F2.0 increased eightfold the coagulation time, compared to the control, when a mass of 10 ��g was used. To PT test, only fucoidan F0.9 was capable of increase the coagulation time, compared to control. In the total antioxidant capacity assay (TAC), the fucoidan F2.0 showed 400 ascorbic acid equivalents, while fucoidan F0.5, the lest effective, 38 equivalents. In respect to the effect on pre-adipocyte cell lines (3T3-L1) adipogenesis, was observed that fucoidan F1.1 and F2.0 reduced the adipogenesis and this effect was associated to the reduction in the expression of regulatoy proteins C/EBP��, C/EBP�� and PPAR��. On the other hand, fucoidans F0.5 and F0.9 induced increased expression of these regulatory proteins. Furthermore, fucoidan F2.0 induced hydrolysis of triglycerides present in the interior of adipocytes. The immunomodulatory effect was evaluated and observed that the presence of fucoidans F0.5 , F1.1 and F2.0 significantly reduced the production of nitric oxide by activated macrophages with LPS specially fucoidan F2.0 that in 100 ��g/mL, reduced about 55% the effect caused by LPS. Relative to the effect upon the formation of calcium oxalate crystals, fucoidan F0.5 was more effective in reduce the aggregation of the crystals and this effect it was not significantly different regarding the effect caused by the crude. Besides, fucoidan F0.5 only promoted the formation of COD type crystals, while fucoidans F1.1 and F2.0 did not influence the formation of crystals compared with the control. The results described in this study indicate that the commercial crude fucoidan of Fucus vesiculosus it���s a mix of several fucoidan which, in turn, have different chemical compositions besides having different pharmacological activities. The use of these fucoidans it��s indicated according the pharmacological activity to be evaluated.

Avalia����o das atividades farmacol��gicas de fucoidans obtidos do extrato bruto de fucoidan comercial de Fucus vesiculosus

Fucoidan is a term used to define heteropolysaccharides that are composed of less than 90% L-fucose. The exception to this rule is the homofucoidan obtained from the seaweed Fucus vesiculosus. This fucoidan can be purchased from SIGMA Co. and have been used in various research for evaluation of their pharmacological activities. However, it is not a pure molecule. In fact, it is a mix of several fucoidan molecules. In this work, were obtained, from acetone precipitation, and biochemically characterized, four fucoidan molecules from SIGMA-ALDRICH Co. fucoidan to evaluate their anticoagulant, antioxidant, antiadipogenic, immunomodulatory and antiurolithiatic activities. In anticoagulant activity, evaluated by aPTT assay, fucoidans F0.9, F1.1 and F2.0 increased eightfold the coagulation time, compared to the control, when a mass of 10 ��g was used. To PT test, only fucoidan F0.9 was capable of increase the coagulation time, compared to control. In the total antioxidant capacity assay (TAC), the fucoidan F2.0 showed 400 ascorbic acid equivalents, while fucoidan F0.5, the lest effective, 38 equivalents. In respect to the effect on pre-adipocyte cell lines (3T3-L1) adipogenesis, was observed that fucoidan F1.1 and F2.0 reduced the adipogenesis and this effect was associated to the reduction in the expression of regulatoy proteins C/EBP��, C/EBP�� and PPAR��. On the other hand, fucoidans F0.5 and F0.9 induced increased expression of these regulatory proteins. Furthermore, fucoidan F2.0 induced hydrolysis of triglycerides present in the interior of adipocytes. The immunomodulatory effect was evaluated and observed that the presence of fucoidans F0.5 , F1.1 and F2.0 significantly reduced the production of nitric oxide by activated macrophages with LPS specially fucoidan F2.0 that in 100 ��g/mL, reduced about 55% the effect caused by LPS. Relative to the effect upon the formation of calcium oxalate crystals, fucoidan F0.5 was more effective in reduce the aggregation of the crystals and this effect it was not significantly different regarding the effect caused by the crude. Besides, fucoidan F0.5 only promoted the formation of COD type crystals, while fucoidans F1.1 and F2.0 did not influence the formation of crystals compared with the control. The results described in this study indicate that the commercial crude fucoidan of Fucus vesiculosus it���s a mix of several fucoidan which, in turn, have different chemical compositions besides having different pharmacological activities. The use of these fucoidans it��s indicated according the pharmacological activity to be evaluated.

Condicionantes nutricionales que influyen sobre los factores de riesgo de s��ndrome metab��lico en ni��os de la Comunidad de Madrid

El S��ndrome Metab��lico (SM) es un conjunto de factores de riesgo asociados con el incremento del riesgo de enfermedad cardiovascular y diabetes mellitus de tipo 2 (DM2). Dichos factores son obesidad (OB), resistencia a la Insulina (RI), hiperglucemia, dislipidemia e hipertensi��n (HTA) e interact��an entre s�� sobre las anomal��as vasculares, el estr��s oxidativo, la grasa visceral, la inflamaci��n y el cortisol en un entorno de OB y RI y bajo la influencia de las predisposiciones gen��ticas y condiciones ambientales, tales como h��bitos de alimentaci��n y actividad f��sica. Seg��n una hip��tesis, la RI y la OB son los factores que m��s contribuyen en la manifestaci��n de anormalidades metab��licas y las manifestaciones m��s tempranas del desarrollo de SM en ni��os. Mientras el p��ncreas compensa adecuadamente la RI mediante una mayor secreci��n de insulina, las concentraciones de glucosa en sangre se mantienen normales. Sin embargo, en algunos pacientes la capacidad de las c��lulas �� del p��ncreas disminuye con el tiempo, lo que conduce al desarrollo de DM2. Otro factor a tener en cuenta en el diagn��stico del SM es la dislipidemia, caracterizada por el aumento de los triglic��ridos (TG) y del VLDL���colesterol, bajas cifras de HDL���colesterol, as�� como por la presencia de part��culas de LDL���colesterol m��s peque��as y densas de lo normal. Otro factor de riesgo de padecimiento de SM es la presi��n arterial elevada, que suele aparecer ligada a la presencia de OB, ya que el aumento de algunas adipoquinas que en esta se produce, entre las que se encuentran el AGE, PAI���1, IL���6, TNF����� y leptina, pueden llevar a disfunci��n endotelial a trav��s de la ruta del ��xido nítrico. Debido a que el sobrepeso y el incremento de los valores de insulina plasm��tica son componentes clave del SM, es importante tener en cuenta los h��bitos alimentarios y otros estilos de vida que influyen en los mismos...

Estudio prospectivo de morbimortalidad en pacientes en hemodi��lisis cr��nica: papel de la dimetilarginina asim��trica (ADMA)

Programa de doctorado: Avances en Medicina Interna, Bienio 2008/10. La fecha de publicaci��n es la fecha de lectura

Avalia����o do extrato do fungo caripia montagnei e de agonistas de PPAR - &#945; no processo inflamat��rio

The mushrooms have been object of intense research in view of its potential raising of application in different sectors of the pharmacology and alimentary industry. Among diverse bioactive composites of polyssacharides nature that exist in the fungus the glucans are much searched. These are polymers of glucose and classified as the type of glicosidic linking [&#945;, &#946;]. Peroxisome proliferator-activated receptors (PPARs), ranscription factors belonging to the family of nuclear receptors that bind themselves o specific agonists, have shown their importance in controlling the inflammatory process. The aim of this study was to perform a chemical characterization of extract rom the mushroom Caripia montagnei, assess its antiinflammatory and antibacterial effect and determine if this effect occurs via PPAR. This mushroom is composed of carbohydrates (63.3��4.1%), lipids (21.4l��0.9%) and proteins (2.2�� 0.3%). The aqueous solution resulting from the fractionation contained carbohydrates (98.7��3.3%) and protein (1.3��0.25%). Analyses of infrared spectrophotometry and of nuclear magnetic esonance demonstrated that the extract of mushroom C. montagnei is rich in &#946;-glucans. In hioglycolate-induced peritonitis, the C. montagnei glucans (50 mg/kg) educed the inflammatory process in 65.5��5.2% and agonists, pharmacological igands, for PPAR: Wy-14643 (49.3��6.1%), PFOA (48.9��3.8%) and clofibrate in 45.2��3.2%. Sodium diclofenac showed a reduction of 81.65��0.6%. In the plantar edema, the glucans from C. montagnei (50 mg/kg) and L-NAME reduced the edema to a similar degree 91.4��0.3% and 92.8��0,5 %, respectively. In all the groups tested, nitric oxide (NO), an inflammation mediator, showed a significant reduction in the nitrate/nitrite levels when compared to the positive control (P<0.001). The C. montagnei glucans did not show cytotoxicity in the concentrations tested (2.5, 5.0, 10.0, 20.0 and 40.0 ��g/100 ��L). Antibacterial activity demonstrated that, unlike total extract, there was no inhibition of bacterial growth. The C. montagnei glucans show great potential for antiinflammatory applications. This effect suggests that it is mediated by PPAR activation and by COX and iNOS inhibition