Identificação e validação funcional de novos alvos das PKCs em célula tronco embrionária / Identification and functional validation of new targets of PKC in embryonic stem cell

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCßI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCßI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCßI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a ß-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCßI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e ß-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na ß-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCßI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCßI em CTE indiferenciadas

Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCßI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCßI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCßI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and ß-tubulin, as a phosphorylation site of α-tubulin by PKCßI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and ß-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in ß-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCßI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCßI in undifferentiated ESC

Principales marcadores moleculares utilizados para la identificación de Babesia bovis y Babesia bigemina / Principal molecular markers used to identify Babesia bovis and Babesia bigemina

Este artículo describe los principales marcadores moleculares utilizados para la identificación de B. bovis y B. bigemina reportados en la literatura científica. Para ello se diseñó una revisión sistemática a partir de la aplicación de la estrategia metodológica PICO modificada con el objetivo de definir las secuencias nucleotídicas detectadas en los diferentes sitios geográficos y su utilidad diagnóstica. Se realizó una búsqueda avanzada con los términos “Babesia bovis” y “DNA” y “Babesia bigemina” y “DNA” en las bases de datos ScienceDirect, SpringerLink y PubMed que después de ser filtradas permitieron obtener un resultado total de 68 artículos originales. Tanto los artículos incluidos como los excluidos fueron almacenados en tablas, en las cuales se presenta la justificación de su condición dentro del estudio. A los 68 artículos seleccionados se les aplicó una evaluación con criterios de inclusión y exclusión previamente definidos, de este modo, 21 artículos originales cumplieron con los criterios de inclusión y se incluyeron en el estudio. Se describe la utilidad de los marcadores moleculares referenciados en la literatura científica desde 1995 hasta el 2010: la subunidad pequeña RNAr, el gen citocromo b, gen msa-1 and msa-2c, el gen Bv, el factor de elongación alfa (EF-1α), el gen de la beta-Tubulina, SBP 1-2-3, y los RAP; su aplicación diagnóstica y su utilización en los diferentes sitios geográficos. Los marcadores moleculares utilizados para la detección de las babesias bovinas varían dependiendo de la región geográfica, grado de conservación genética y resultados de estudios previos que concluyen su utilidad diagnóstica.

Protozoarios en aguas superficiales y muestras fecales de individuos de poblaciones rurales del municipio Montes, estado Sucre, Venezuela / Protozoans in superficial waters and faecal samples of individuals of rural populations of the Montes municipality, Sucre state, Venezuela

En el estado Sucre, el Río Manzanares se ve amenazado por actividades domésticas, agrícolas e industriales originadas por el hombre, convirtiéndose en factor de riesgo ambiental para sus habitantes. En este sentido se planteó evaluar la presencia de protozoarios en aguas superficiales de afluentes del Río Manzanares (Río Orinoco, Quebrada Seca, Río San Juan), municipio Montes, estado Sucre, Venezuela, así como también el análisis de muestras fecales de los habitantes de poblados aledaños. Se recolectaron muestras de aguas superficiales de los afluentes en estudio y fecales entre mayo 2006-abril 2007. Las muestras de aguas superficiales se procesaron con sedimentación por centrifugación, floculación y tinciones de Kinyoun y tricrómica; las muestras fecales se sometieron a examen directo con solución salina fisiológica y lugol, Ritchie modificado y las coloraciones antes mencionadas. Los protozoarios observados con mayor frecuencia en las aguas superficiales en los afluentes fueron: Amebas, Blastocystis sp., Endolimax sp., Chilomastix sp. y Giardia sp. Mientras que Blastocystis hominis, Endolimax nana y Entaomeba coli fueron los de mayor frecuencia observada en las muestras fecales. Los habitantes de Orinoco La Peña resultaron ser los más afectados por las infecciones parasitarias (77,60%), seguido de Río San Juan con 46,63%, y Quebrada Seca con 39,49%. La presencia de protozoarios patógenos y no patógenos en las aguas superficiales demuestra la contaminación fecal de los afluentes evaluados, por lo que representa un foco de infección permanente para los individuos que viven en las cercanías de estas aguas, esto se refleja por la observación de los mismos parásitos en ambas muestras.

In Sucre state, the Manzanares river is threatened by domestic, agricultural and industrial activities, becoming an environmental risk factor for its inhabitants. In this sense, the presence of protozoans in superficial waters of tributaries of the Manzanares river (Orinoco river, Quebrada Seca, San Juan river), Montes municipality, Sucre state, as well as the analysis of faecal samples from inhabitants of towns bordering these tributaries were evaluated. We collected faecal and water samples from may 2006 through april 2007. The superficial water samples were processed after centrifugation by the direct examination and floculation, using lugol, modified Kinyoun and trichromic colorations. Fecal samples where analyzed by direct examination with physiological saline solution and the modified Ritchie concentration method and using the other colorations techniques above mentioned. The most frequently observed protozoans in superficial waters in the three tributaries were: Amoebas, Blastocystis sp, Endolimax sp., Chilomastix sp. and Giardia sp. Whereas in faecal samples, Blastocystis hominis, Endolimax nana and Entaomeba coli had the greatest frequencies in the three communities. The inhabitants of Orinoco La Peña turned out to be most susceptible to these parasitic infections (77.60%), followed by San Juan River (46.63%) and Quebrada Seca (39.49%). The presence of pathogenic and nonpathogenic protozoans in superficial waters demonstrates the faecal contamination of the tributaries, representing a constant focus of infection for their inhabitants, inferred by the observation of the same species in both types of samples.

[Protozoans in superficial waters and faecal samples of individuals of rural populations of the Montes municipality, Sucre state, Venezuela]. / Protozoarios en aguas superficiales y muestras fecales de individuos de poblaciones rurales del municipio Montes, estado Sucre, Venezuela.

In Sucre state, the Manzanares river is threatened by domestic, agricultural and industrial activities, becoming an environmental risk factor for its inhabitants. In this sense, the presence of protozoans in superficial waters of tributaries of the Manzanares river (Orinoco river, Quebrada Seca, San Juan river), Montes municipality, Sucre state, as well as the analysis of faecal samples from inhabitants of towns bordering these tributaries were evaluated. We collected faecal and water samples from may 2006 through april 2007. The superficial water samples were processed after centrifugation by the direct examination and floculation, using lugol, modified Kinyoun and trichromic colorations. Fecal samples where analyzed by direct examination with physiological saline solution and the modified Ritchie concentration method and using the other colorations techniques above mentioned. The most frequently observed protozoans in superficial waters in the three tributaries were: Amoebas, Blastocystis sp, Endolimax sp., Chilomastix sp. and Giardia sp. Whereas in faecal samples, Blastocystis hominis, Endolimax nana and Entaomeba coli had the greatest frequencies in the three communities. The inhabitants of Orinoco La Peña turned out to be most susceptible to these parasitic infections (77.60%), followed by San Juan River (46.63%) and Quebrada Seca (39.49%). The presence of pathogenic and nonpathogenic protozoans in superficial waters demonstrates the faecal contamination of the tributaries, representing a constant focus of infection for their inhabitants, inferred by the observation of the same species in both types of samples.

[Transmission of enteropasitosis through currency notes]. / Transmissão de enteroparasitoses através do papel-moeda.

PURPOSE: There are several ways to propagation of parasitary diseases and how there aren't many bibliographic work about this theme, it was realized a study near to the Natal-RN population, to verify the enteroparasites transmission through the paper money. METHODS: In the Laboratório Parasitologia Clínica was analysed 500 bills of several worths gotten in shops and, concomitantly it was examined samples of a water got from the washed hands of the people who held the money. The methods to realize the parasitologic exams were: Ritchie and Faust et al. RESULTS: It was observed the following parasites prevalent in 48 (9.6%) examined bills: Endolimax nana 31 (6.2%), Entamoeba coli 06 (1.2%), Entamoeba histolytica 01 (0.2%), Ascaris lumbricoides 10 (2.0%). In the water, it vas observed that 40 (40.0%) of them had the same kinds of parasites in their hands. CONCLUSIONS: Finally, it is postulated that the paper money is important to the enteroparasites transmission and it is suggested that new researches must be done in this area.

Transmissão de enteroparasitoses através do papel-moeda / Transmission of enteropasitosis through currency notes

PURPOSE: There are several ways to propagation of parasitary diseases and how there aren't many bibliographic work about this theme, it was realized a study near to the Natal-RN population, to verify the enteroparasites transmission through the paper money. METHODS: In the Laboratório Parasitologia Clínica was analysed 500 bills of several worths gotten in shops and, concomitantly it was examined samples of a water got from the washed hands of the people who held the money. The methods to realize the parasitologic exams were: Ritchie and Faust et al. RESULTS: It was observed the following parasites prevalent in 48 (9.6%) examined bills: Endolimax nana 31 (6.2%), Entamoeba coli 06 (1.2%), Entamoeba histolytica 01 (0.2%), Ascaris lumbricoides 10 (2.0%). In the water, it vas observed that 40 (40.0%) of them had the same kinds of parasites in their hands. CONCLUSIONS: Finally, it is postulated that the paper money is important to the enteroparasites transmission and it is suggested that new researches must be done in this area.

Estudio comparado de métodos de diagnóstico de la Leishmaniasis y caracterización molecular de los agentes etiológicos en la región La Libertad / Comparative study of diagnosis methods for Leishmaniasis and molecular characterization of aetiologic agents in La Libertad Region

Objetivo: Comparar el rendimiento del PCR frente al cultivo y examen directo en el diagnóstico de leishmaniasis. Material y métodos: Estudio analítico transversal, se estudió un total de 69 pacientes con antecedente de vivir en zonas endémicas de leishmaniasis en la Región La Libertad. Se procedió a tomar muestras de bordes de las lesiones por raspado profundo que abarcaba tejido, para el posterior análisis directo, cultivo en medio bifásico y PCR. Resultados: La positividad del PCR alcanzó el 93 por ciento; el examen directo 85 por ciento y el cultivo, el 72 por ciento. La existencia de más de una especie además de L. peruviana es una probabilidad respaldada por el hallazgo de dos nuevos perfiles genéticos en el examen del DNA en casos de leishmaniosis de pacientes del distrito de Salpo provincia de Otuzco. No se ha podido establecer, por limitaciones de número de pacientes y marcadores moleculares, la relación entre especies y variedades clínicas. Conclusiones: El PCR presenta un mejor rendimiento como prueba diagnóstica. Este estudio preliminar muestra que la Leishmania que circula en las zonas endémicas tiene distintos perfiles de ADN.

Separación de proteínas del disco ventral de Giardia intestinalis / Separation of proteins from the ventral disk of Giardia intestinalis

Mediante separación isoeléctrica y reisoelectroenfoque se obtuvieron las proteínas de citoesqueleto de Giardia intestinalis con características de giardinas y tubulinas, constituyentes principales del disco ventral. El peso molecular fue 30 a 45 kDa así como las de sus variantes determinadas por la diferencia de pH. Además de las mismas se obtuvieron predominantemente polipéptidos de peso molecular de 97 y 115 kDa. Por inmunocitoquímica se demostró que estas proteínas de peso molecular elevado se localizaron sobre la membrana del trofozoíto, y en menor proporción se observaron proteínas con pesos moleculares semejantes a los de giardinas y tubulinas

Colchicine binding to tubulin from brain homogenates in the presence of the sugars, glycols and metal ions: effect of nickel ions on the tubulin solubility

The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions A1(III), Co(II), Ni(II), while compounds structurally related to glycero (glucose and sucrose) did not inhibition it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and NI (II)] favored tubulin sedimentation when these latterwere centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion madeinsoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and streteched structures, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein (AU)

Colchicine binding to tubulin from brain homogenates in the presence of the sugars, glycols and metal ions: effect of nickel ions on the tubulin solubility

The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions A1(III), Co(II), Ni(II), while compounds structurally related to glycero (glucose and sucrose) did not inhibition it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and NI (II)] favored tubulin sedimentation when these latterwere centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion madeinsoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and streteched structures, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein

Effect of low body temperature of the microtubles of toad brain and testes studied with a morphometric and a biochemical method

El análisis morfométrico aplicado a fibras preterminales de cerebro y a células de Sertoli de testículo del sapo Bufo arenarum mostraron que los microtúbos desaparecen en un 75% y 92% respectivamente cuando los animales son enfriados a 1-2-C por 90 minuots. Las estimaciones hechas en cuerpos neuronales de cerebro y en otras células del testículo mostraron los mismos resultados. Trabajos previos de los autores (López y Bertini, 1982) mostraron que el grado de polimerización de tubulina (porcentaje de tubulina insoluble) en sapos enfriados, solo decreció en un 40%. Se postula que el elevado porcentaje de tubulina insolubre en animales enfriados es decreció en un 40%. Se postula que el elevado porcentaje de tubulina insoluble en animales enfriados es debido a la existencia de un pool de tubulina no estructurada en microtúbulos, unida a estructuras membranosas en sitios de alta afinidad y/o a oligómeros de tubulina no reconocible como microtúbulos por microscopía electrónica

Effect of low body temperature of the microtubles of toad brain and testes studied with a morphometric and a biochemical method

El análisis morfométrico aplicado a fibras preterminales de cerebro y a células de Sertoli de testículo del sapo Bufo arenarum mostraron que los microtúbos desaparecen en un 75% y 92% respectivamente cuando los animales son enfriados a 1-2-C por 90 minuots. Las estimaciones hechas en cuerpos neuronales de cerebro y en otras células del testículo mostraron los mismos resultados. Trabajos previos de los autores (López y Bertini, 1982) mostraron que el grado de polimerización de tubulina (porcentaje de tubulina insoluble) en sapos enfriados, solo decreció en un 40%. Se postula que el elevado porcentaje de tubulina insolubre en animales enfriados es decreció en un 40%. Se postula que el elevado porcentaje de tubulina insoluble en animales enfriados es debido a la existencia de un pool de tubulina no estructurada en microtúbulos, unida a estructuras membranosas en sitios de alta afinidad y/o a oligómeros de tubulina no reconocible como microtúbulos por microscopía electrónica (AU)