{"id":114501,"date":"2013-12-07T00:00:00","date_gmt":"2013-12-07T00:00:00","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/caracterizacion-funcional-de-los-receptores-de-tnf-de-pez-cebra\/"},"modified":"2013-12-07T00:00:00","modified_gmt":"2013-12-07T00:00:00","slug":"caracterizacion-funcional-de-los-receptores-de-tnf-de-pez-cebra","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/embriologia\/caracterizacion-funcional-de-los-receptores-de-tnf-de-pez-cebra\/","title":{"rendered":"Caracterizaci\u00f3n funcional de los receptores de tnf de pez cebra."},"content":{"rendered":"<h2>Tesis doctoral de <strong> Raquel Esp\u00edn Palaz\u00f3n <\/strong><\/h2>\n<p>Caracterizaci\u00f3n funcional de los receptores de tnf de pez cebra objetivos 1. Caracterizaci\u00f3n de los receptores de tnf (tnfr1 y tnfr2) en la homeostasis vascular durante el desarrollo en pez cebra. 2. Caracterizaci\u00f3n de las rutas de se\u00f1alizaci\u00f3n de tnfr1 y tnfr2 involucradas en el desarrollo de las c\u00e9lulas endoteliales y su homeostasis. 3. Caracterizaci\u00f3n del papel que juegan tnfrs y sus ligandos (tnfa y lta) en la ola primitiva de hematopoyesis en el pez cebra. 4. Caracterizaci\u00f3n de los tnfrs y sus ligandos (tnfa y lta) en la especificaci\u00f3n y mantenimiento de las hscs en el embri\u00f3n de pez cebra. metodolog\u00eda \tpara la presente tesis doctoral, se ha utilizado el pez cebra (danio rerio) como modelo de experimentaci\u00f3n animal. Adem\u00e1s, para la experimentaci\u00f3n in vitro se han usado varias l\u00edneas celulares y t\u00e9cnicas de cultivo celulares. En cuanto a las t\u00e9cnicas de biolog\u00eda molecular, podemos destacar la microinyecci\u00f3n en estadio de huevo de rnam, morfolinos y dna; rt-qpcr e hibridaci\u00f3n in situ. Tambi\u00e9n se han usado t\u00e9cnicas de microscop\u00eda tales como la microscop\u00eda confocal y de fluorescencia; t\u00e9cnicas inmunohistoqu\u00edmicas y tunel para la detecci\u00f3n de apoptosis; y t\u00e9cnicas de citometr\u00eda y aislamiento f\u00edsico de c\u00e9lulas mediante fluorescencia (facs). resultados \tdurante la presente tesis doctoral, hemos caracterizado el papel que desempe\u00f1an el factor de necrosis tumoral (tnfa) y sus receptores (receptores del factor de necrosis tumoral, tnfrs) en el desarrollo de las c\u00e9lulas endoteliales y del sistema hematopoy\u00e9tico. Para ello hemos usado el pez cebra (danio rerio), como modelo animal de vertebrados. \tpor un lado, nuestros resultados nos han llevado a concluir que la deficiencia en el gen de tnfr2 en embriones de pez cebra conlleva la inducci\u00f3n en c\u00e9lulas endoteliales de un programa apopt\u00f3tico que depende de caspasa-8, caspasa-2 y p53, pero no caspasa-3. Adem\u00e1s, la deficiencia simult\u00e1nea de tnfr1 o la activaci\u00f3n de nf-?B, rescata la apoptosis de las c\u00e9lulas endoteliales, lo que indica que debe haber un equilibrio entre los dos receptores de tnf para la correcta homeostasis vascular de este tipo celular. De forma similar, el tnfa promueve la apoptosis de las c\u00e9lulas endoteliales humanas a trav\u00e9s de tnfr1, desencadenando la activaci\u00f3n de caspasa-2 y p53. \tpor otro lado, en este trabajo demostramos que la se\u00f1alizaci\u00f3n de tnfa a trav\u00e9s de tnfr2 se necesita de forma intr\u00ednseca por las c\u00e9lulas madre hematopoy\u00e9ticas (hscs) para su mantenimiento y expansi\u00f3n. La deficiencia gen\u00e9tica de tnfa o tnfr2, pero no de linfotoxina (lta) o tnfr1, conlleva la apoptosis de las hscs cuando se especifican en el embri\u00f3n. conclusiones 1. El silenciamiento g\u00e9nico dirigido contra tnfr2 resulta en la inducci\u00f3n de un programa apopt\u00f3tico dependiente de caspasa-8 en c\u00e9lulas endoteliales. Esta apoptosis se rescata mediante la eliminaci\u00f3n de tnfr1, lo que indica que se requiere un balance adecuado entre los tnfrs para la integridad de las c\u00e9lulas endoteliales y la homeostasis vascular. 2. En las c\u00e9lulas endoteliales, el tnfr1 se\u00f1aliza apoptosis a trav\u00e9s de la formaci\u00f3n del complejo ii y la consecuente activaci\u00f3n de caspasa-8 y caspasa-2, mientras que tnfr2 se\u00f1aliza supervivencia mediante el comple i y la activaci\u00f3n de nf-?B. 3. El programa apopt\u00f3tico inducido por tnfr1, en el cual participa caspasa-8, caspasa-2 y p53, se encuentra conservado evolutivamente en c\u00e9lulas endoteliales humanas. 4. La se\u00f1alizaci\u00f3n de tnfrs es dispensable para la hematopoyesis primitiva en el embri\u00f3n de pez cebra. 5. La inhibici\u00f3n g\u00e9nica de tnfa o tnfr2, pero no tnfr1 o lta, resulta en la apoptosis de las hscs cuando \u00e9stas emergen de la aorta dorsal. 6. La se\u00f1alizaci\u00f3n tnfa\/tnfr2 se requiere de forma intr\u00ednseca para la supervivencia de las hscs a partir del endotelio hemog\u00e9nico.         functional characterization of tnf receptors in zebrafish objectives 1. Characterization of tnfa receptors (tnfr1 and tnfr2) in vascular homeostasis during the zebrafish embryo development. 2. Characterization of the tnfr1 and tnfr2 signaling pathways involved in endothelial cell development and homeostasis. 3. Characterization of the role played by tnfrs and their ligands (tnfa and lta) in the primitive wave of hematopoiesis in the zebrafish embryo. 4. Characterization of tnfrs and their ligands (tnfa and lta) in hscs specification and maintenance in the zebrafish embryo. methodology in this doctoral thesis, zebrafish (danio rerio) has been utilized as research animal model. In addition, several cell lines and tissue culture techniques have been used for the in vitro experimentation. Related to the molecular biology techniques utilized, the injection at one-cell stage with mrna, morpholinos and dna; rt-qpcr and in situ hybridization can be emphasized. Moreover, other techniques used have been used such as confocal microscopy and fluorescence microscopy; immunohystochemistry techniques and tunel for apoptosis detection; as well as cytometry-related assays as fluorescence activated cell sorting (facs). results during this doctoral thesis, we have characterized the role of tnfa and tnf receptors (tnfrs) during endothelial and hematopoiesis development in the embryo using zebrafish (danio rerio) as a vertebrate model. \ttargeted gene knockdown of tnfr2 in zebrafish embryos results in the induction of a caspase-8, caspase-2 and p53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of tnfr1 or the activation of nf-?B rescue endothelial cell apoptosis, indicating that a signaling balance between both tnfrs is required for endothelial cell integrity. Similarly, tnfa promotes the apoptosis of human endothelial cells through tnfr1 and triggers caspase-2 and p53 activation. \ton the other hand, we show that tnfa signaling through tnfr2 is intrinsically required for hematopoietic stem cells (hscs) maintenance and expansion. The genetic inhibition of tnfa or tnfr2, but not of lymphotoxin ? (Lta) or tnfr1, results in the apoptosis of hscs soon after their emergence. conclusions 1. Target gene silencing of tnfr2 results in the induction of a caspase-8-dependent apoptotic program in endothelial cells. This apoptosis can be rescued by depletion of tnfr1, indicating that an appropriate signaling balance between both tnfrs is required for endothelial cell integrity and vascular homeostasis. 2. In endothelial cells, tnfr1 signals apoptosis through complex ii formation and caspase-8 and caspase-2 activation, while tnfr2 signals survival via complex i and nf-?B activation. 3. The apoptotic program induced by tnfr1, which involves caspase-8, caspase-2 and p53, is evolutionary conserved in human endothelial cells. 4. Tnfrs signaling is dispensable for primitive hematopoiesis in the zebrafish embryo. 5. Genetic inhibition of tnfa or tnfr2, but not of tnfr1 or lta, results in hsc apoptosis soon after their emergence from the floor of the dorsal aorta. 6. Tnfa\/tnfr2 signaling is intrinsically required for hsc survival after their emergence from the hemogenic endothelium.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Caracterizaci\u00f3n funcional de los receptores de tnf de pez cebra.<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Caracterizaci\u00f3n funcional de los receptores de tnf de pez cebra. <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Raquel Esp\u00edn Palaz\u00f3n <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 12\/07\/2013<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Victoriano Francisco Mulero Mendez<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: alfonsa Garcia ayala <\/li>\n<li>susana Gonz\u00e1lez l\u00f3pez (vocal)<\/li>\n<li>david Sancho Madrid (vocal)<\/li>\n<li>encarnacion Mu\u00f1oz delgado (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Raquel Esp\u00edn Palaz\u00f3n Caracterizaci\u00f3n funcional de los receptores de tnf de pez cebra objetivos 1. Caracterizaci\u00f3n de [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"footnotes":""},"categories":[350,1737,8235],"tags":[132351,77512,53156,226944,226945,93770],"class_list":["post-114501","post","type-post","status-publish","format-standard","hentry","category-embriologia","category-inmunologia","category-murcia","tag-alfonsa-garcia-ayala","tag-david-sancho-madrid","tag-encarnacion-munoz-delgado","tag-raquel-espin-palazon","tag-susana-gonzalez-lopez","tag-victoriano-francisco-mulero-mendez"],"_links":{"self":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/posts\/114501","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/comments?post=114501"}],"version-history":[{"count":0,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/posts\/114501\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/media?parent=114501"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/categories?post=114501"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.deberes.net\/tesis\/wp-json\/wp\/v2\/tags?post=114501"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}