Caracterización de pomelo "Paraná" mediante marcadores moleculares

Tesis de maestría para obtener el grado de Magister en Producción Vegetal presentada en la Universidad Nacional del Nordeste, Facultad de Ciencias Agrarias, en 2018El género Citrus al igual que los géneros Fortunella (kumquat) y Poncirus (naranjo trifolio) pertenecen a la familia Rutacea (subfamilia Aurantoidea, tribu Citreae) subtribu Citrinae, y son los tres géneros de importancia comercial en la familia. La gran diversidad de especies y variedades en el género Citrus, se debe principalmente a la ocurrencia de mutaciones y la frecuente aparición de semillas poliembriónicas. El pomelo ‘Paraná’ (Citrus sp.) como la mayoría de las especies cítricas, surgió en el Sudoeste asiático, en contraste con los demás pomelos (C. paradisi) cuyo centro de origen fue en las islas Barbados, en el Caribe. Tal como los citrus diploides, pomelo ‘Paraná’ cuenta con número cromosómico 2n= 18. Además de presentar características industriales mejoradas, como la calidad de frutos y buen comportamiento a campo, el pomelo ‘Paraná’ ha demostrado alta resistencia cuantitativa a la cancrosis de los cítricos, enfermedad bacteriana causada por Xanthomonas citri subsp citri. Esta enfermedad es endémica en la zona del NEA, afecta a todos los cítricos, pero en particular todas las variedades de pomelo son muy susceptibles. En la actualidad, las investigaciones en cuanto a sanidad, principalmente están dirigidas a la obtención de nuevas variedades cítricas para contrarrestar las pérdidas producidas por una enfermedad nueva en la zona, cuyo agente causal es la bacteria Candidatus Liberibacter y provoca el “greening” o HLB (huanglongbing).The genus Citrus as well as the genera Fortunella (kumquat) and Poncirus (orange trifolio) belong to the family Rutacea (subfamily Aurantoidea, tribe Citreae) subtribe Citrinae, and are the three genera of commercial importance in the family. The great diversity of species and varieties in the genus Citrus, is mainly due to the occurrence of mutations and the frequent appearance of polyembryonic seeds. The grapefruit ‘Paraná’ (Citrus sp.) like the majority of the citric species, arose in the Asian Southwest, in contrast with the other grapefruit (C. paradisi) whose center of origin was in the Barbados Islands in the Caribbean. As citrus diploids, ‘Paraná’ grapefruit has chromosome number 2n = 18. In addition to presenting improved industrial characteristics, such as fruit quality and good field behavior, grapefruit ‘Paraná’ has shown high quantitative resistance to canker citrus, a bacterial disease caused by Xanthomonas citri subsp citri. This disease is endemic in the NEA zone, it affects all citrus fruits, but in particular all cultivars of grapefruit are very susceptible. Currently, research in terms of health, mainly are aimed at obtaining new citrus varieties to offset the losses caused by a new disease in the area, whose causative agent is the bacterium Candidatus Liberibacter and causes "greening" or HLB (huanglongbing).Fil: Lezcano, Cecilia Carolina. INTA. Estación Experimental Agropecuaria Bella Vista; ArgentinaEstación Experimental Agropecuaria Bella Vist

Regulación de la contractilidad miocárdica por vías de señalización EPAC-dependientes

La estimulación beta-adrenérgica es un modulador importantísimo de la contractilidad cardíaca. Clásicamente los efectos de la estimulación del receptor beta1, se atribuyen al aumento del AMPc, que se produce cuando el agonista se une al receptor y activa a la AC a través de la proteína Gs. Inicialmente los efectos del AMPc se atribuyeron a la activación de la PKA, pero recientemente se conoce que el AMPc puede activar directamente a las proteínas Epac (proteínas que intercambian nucleótidos de guanina en las proteínas Rap 1 y 2). En el corazón, el rol de Epac es poco conocido y todavía existen muchos interrogantes respecto a su función. Se ha visto que la estimulación de Epac por el análogo del AMPc específico 8-(4-clorofenilo)-2´-O-metiladenosina-3´-5´-monofosfato cíclico (8-CPT) genera hipertrofia en miocitos neonatales de rata. Y que la expresión de la proteína aumenta en varios modelos de hipertrofia en animales e incluso en humanos. La estimulación de Epac ha mostrado diferentes efectos en la regulación del Ca2+ intracelular en miocitos aislados, provocando tanto un aumento como una disminución del transitorio de Ca2+ (CaT).Facultad de Ciencias Médica

Efectos de la microcistina sobre el estado de fosforilación de proteínas e inducción de apoptosis en diferentes tejidos

La microcistina-LR (MC-LR) es una toxina que se encuentra en agua de consumo humano. La intoxicación con bajas dosis tiene un efecto crónico y acumulativo a nivel hepático. MC-LR es un potente inhibidor de fosfatasas y aumenta el estado de fosforilación de proteínas involucradas en diversas rutas metabólicas incluídas las que conducen a la apoptosis. Nuestro objetivo fue estudiar el efecto de la administración crónica de MC-LR sobre la fosforilación de proteínas involucradas en la transducción de señales y el grado de apoptosis en distintos tejidos.Facultad de Ciencias Médica

Señalización pro-apoptótica mediada por microcistina-LR en ratones intoxicados subcrónicamente

La microcistina-LR (MC-LR) es una toxina producida por cianobacterias de agua fresca capaz de generar graves intoxicaciones tanto en animales como en humanos. Se sugiere que la acción de MC-LR se debe a un aumento del estrés oxidativo y a su capacidad de inhibir proteínas fosfatasas tipo 1 y 2A (PP1 y PP2A), lo cual deriva en hiperfosforilación proteica, desestabilización del citoesqueleto, activación de cascadas apoptóticas y muerte celular. Estos efectos han sido generalmente estudiados en forma aguda, en células expuestas a dosis máximas de MC-LR, y poco se sabe acerca de las consecuencias de la exposición crónica a dosis subletales de la toxina en el animal entero, exposición similar a la que estaría sometido el hombre o animal que consume agua contaminada con MC-LR.Facultad de Ciencias Médica

El HLB de los cítricos, una amenaza para la citricultura del NEA

El Huanglongbing (HLB) es considerado mundialmente como la enfermedad más destructiva de los cítricos. Detectado originalmente en China, el HLB se ha expandido por la mayoría de los países citrícolas de Asia, Africa y América. Desde su aparición en el hemisferio sur, en el año 2004, ha mostrado un preocupante avance sobre nuestro continente. En Argentina, fue detectado por primera vez en el año 2012, provocando perjuicios para toda la cadena de producción citrícola. Hasta el momento no se conoce ninguna especie cítrica resistente a esta enfermedad. El control se basa en la eliminación plantas enfermas y hospedadoras de la bacteria causal utilizando diagnóstico molecular temprano, control del vector por métodos químicos y biológicos, y la utilización de plantas sanas de origen conocido, de viveros certificados.EEA Bella VistaFil: Gochez, Alberto Martin. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina. Asociación Argentina de Fitopatólogos. Capítulo NEA; ArgentinaFil: Chelotti, Maria Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Aranda, Maria Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Lezcano, Cecilia Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; ArgentinaFil: Canteros, Blanca Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina. Asociación Argentina de Fitopatólogos. Capítulo NEA; Argentin

Loss of 5-hydroxymethylcytosine correlates with increasing morphologic dysplasia in melanocytic tumors

DNA methylation is the most well studied epigenetic modification in cancer biology. 5-hydroxymethylcytosine is an epigenetic mark that can be converted from 5-methylcytosine by the ten-eleven translocation gene family. We recently reported the loss of 5-hydroxymethylcytosine in melanoma compared to benign nevi and suggested that loss of this epigenetic marker is correlated with tumor virulence based on its association with a worse prognosis. In this study we further characterize the immunoreactivity patterns of 5-hydroxymethylcytosine in the full spectrum of melanocytic lesions to further validate the potential practical application of this epigenetic marker. 175 cases were evaluated: 18 benign nevi, 20 dysplastic nevi (10 low-grade and 10 high-grade lesions), 10 atypical Spitz nevi, 20 borderline tumors, 5 melanomas arising within nevi, and 102 primary melanomas. Progressive loss of 5-hydroxymethylcytosine from benign dermal nevi to high-grade dysplastic nevi to borderline melanocytic neoplasms to melanoma was observed. In addition, an analysis of the relationship of nuclear diameter to 5-hydroxymethylcytosine staining intensity within lesional cells revealed a significant correlation between larger nuclear diameter and decreased levels of 5-hydroxymethylcytosine. Furthermore, borderline lesions uniquely exhibited a diverse spectrum of staining of each individual case. This study further substantiates the association of 5-hydroxymethylcytosine loss with dysplastic cytomorphologic features and tumor progression and supports the classification of borderline lesions as a biologically distinct category of melanocytic lesions

Early effects of Epac depend on the fine-tuning of the sarcoplasmic reticulum Ca2+ handling in cardiomyocytes

In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca2+]o (0.5 mM), no changes at 1 mM [Ca2+]o and a negative inotropic effect when [Ca2+]o was increased to 1.8 mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca2+ content. At all [Ca2+]o studied, 8-CPT induced an increase in Ca2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca2+]o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca2+ handling (uptake vs. leak) in the different situations.Centro de Investigaciones Cardiovasculare

ABCB5-Targeted chemoresistance reversal inhibits merkel cell carcinoma growth

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with profound but poorly understood resistance to chemotherapy, which poses a significant barrier to clinical MCC treatment. Here we show that ATP-binding cassette member B5 (ABCB5) confers resistance to standard-of-care MCC chemotherapeutic agents and provide proof-of-principle that ABCB5 blockade can inhibit human MCC tumor growth through sensitization to drug-induced cell cytotoxicity. ABCB5 expression was detected in both established MCC lines and clinical MCC specimens at levels significantly higher than those in normal skin. Carboplatin- and etoposide-resistant MCC cell lines exhibited increased expression of ABCB5, along with enhanced ABCB1 and ABCC3 transcript expression. ABCB5-expressing MCC cells in heterogeneous cancers preferentially survived treatment with carboplatin and etoposide in vitro and in human MCC xenograft-bearing mice in vivo. Moreover, patients with MCC also exhibited enhanced ABCB5 positivity after carboplatin- and etoposide-based chemotherapy, pointing to clinical significance of this chemoresistance mechanism. Importantly, ABCB5 blockade reversed MCC drug resistance and impaired tumor growth in xenotransplantation models in vivo. Our results establish ABCB5 as a chemoresistance mechanism in MCC and suggest utility of this molecular target for improved MCC therapy. © 2016 The Author

A Genome-Wide Pharmacogenetic Study of Growth Hormone Responsiveness

Individual patients vary in their response to growth hormone (GH). No large-scale genome-wide studies have looked for genetic predictors of GH responsiveness. To identify genetic variants associated with GH responsiveness. Genome-wide association study (GWAS). Cohorts from multiple academic centers and a clinical trial. A total of 614 individuals from 5 short stature cohorts receiving GH: 297 with idiopathic short stature, 276 with isolated GH deficiency, and 65 born small for gestational age. Association of more than 2 million variants was tested. Primary analysis: individual single nucleotide polymorphism (SNP) association with first-year change in height standard deviation scores. Secondary analyses: SNP associations in clinical subgroups adjusted for clinical variables; association of polygenic score calculated from 697 genome-wide significant height SNPs with GH responsiveness. No common variant associations reached genome-wide significance in the primary analysis. The strongest suggestive signals were found near the B4GALT4 and TBCE genes. After meta-analysis including replication data, signals at several loci reached or retained genome-wide significance in secondary analyses, including variants near ST3GAL6. There was no significant association with variants previously reported to be associated with GH response nor with a polygenic predicted height score. We performed the largest GWAS of GH responsiveness to date. We identified 2 loci with a suggestive effect on GH responsiveness in our primary analysis and several genome-wide significant associations in secondary analyses that require further replication. Our results are consistent with a polygenic component to GH responsiveness, likely distinct from the genetic regulators of adult height

Nestin depletion induces melanoma matrix metalloproteinases and invasion

Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. Development of 3-dimensionsal melanospheres that in vitro partially recapitulate non-invasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase (pFAK) and increased melanoma cell responsiveness to transforming growth factor-beta (TGF-β), both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence