Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer

Ovarian cancer is the fifth leading cause of cancer‐related deaths. It causes approximately 125,000 deaths per year worldwide; its diagnosis is made in advanced stages resulting in a high mortality rate. The objective of the study was optimizing the isolation of cells obtained from the solid tumor a...

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Autores:
Gutiérrez Castañeda, Luz D.
Tovar Parra, David
Quintero, Gloria
Amezquita, Lorena
Guerrero, Carlos
Sanabria, Daniel
Tipo de recurso:
Article of journal
Fecha de publicación:
2019
Institución:
Fundación Universitaria de Ciencias de la Salud - FUCS
Repositorio:
Repositorio Digital Institucional ReDi
Idioma:
eng
OAI Identifier:
oai:repositorio.fucsalud.edu.co:001/2683
Acceso en línea:
https://repositorio.fucsalud.edu.co/handle/001/2683
Palabra clave:
Epithelial
Mesenchyma
Ovarian cancer
Tumor
Neoplasias ovarianas
Mesodermo
Epitelial
Rights
openAccess
License
Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)
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dc.title.eng.fl_str_mv Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
title Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
spellingShingle Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
Epithelial
Mesenchyma
Ovarian cancer
Tumor
Neoplasias ovarianas
Mesodermo
Epitelial
title_short Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
title_full Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
title_fullStr Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
title_full_unstemmed Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
title_sort Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer
dc.creator.fl_str_mv Gutiérrez Castañeda, Luz D.
Tovar Parra, David
Quintero, Gloria
Amezquita, Lorena
Guerrero, Carlos
Sanabria, Daniel
dc.contributor.author.none.fl_str_mv Gutiérrez Castañeda, Luz D.
Tovar Parra, David
Quintero, Gloria
Amezquita, Lorena
Guerrero, Carlos
Sanabria, Daniel
dc.subject.proposal.eng.fl_str_mv Epithelial
Mesenchyma
Ovarian cancer
Tumor
topic Epithelial
Mesenchyma
Ovarian cancer
Tumor
Neoplasias ovarianas
Mesodermo
Epitelial
dc.subject.decs.none.fl_str_mv Neoplasias ovarianas
Mesodermo
Epitelial
description Ovarian cancer is the fifth leading cause of cancer‐related deaths. It causes approximately 125,000 deaths per year worldwide; its diagnosis is made in advanced stages resulting in a high mortality rate. The objective of the study was optimizing the isolation of cells obtained from the solid tumor and ascitic fluid of patients with ovarian cancer and the phenotype with markers related to the epithelial–mesenchymal transition. For this, the solid tumor tissue was disaggregated and cultivated with different methodologies. As a result, cell growth was obtained and epi‐immunofluorescence was performed using antibodies against E‐cadherin, EpCAM, N‐cadherin, vimentin, CD133, and CD44. The primary culture from the solid tumor was obtained using Dispase II and DMEM/F12. Finally, heterogeneity was detected in terms of the expression of mesenchymal and epithelial type markers in the two types of isolated cells. Additionally, CD133 and CD44
publishDate 2019
dc.date.issued.none.fl_str_mv 2019-09
dc.date.accessioned.none.fl_str_mv 2022-04-05T20:09:27Z
dc.date.available.none.fl_str_mv 2022-04-05T20:09:27Z
dc.type.spa.fl_str_mv Artículo de revista
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identifier_str_mv 0021-9541
1097-4652
url https://repositorio.fucsalud.edu.co/handle/001/2683
dc.language.iso.spa.fl_str_mv eng
language eng
dc.relation.ispartof.none.fl_str_mv Journal of Cellular Physiology
dc.relation.citationendpage.spa.fl_str_mv 3328
dc.relation.citationissue.spa.fl_str_mv 4
dc.relation.citationstartpage.spa.fl_str_mv 3320
dc.relation.citationvolume.spa.fl_str_mv 235
dc.relation.ispartofjournal.spa.fl_str_mv Journal of Cellular Physiology
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rights_invalid_str_mv Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)
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dc.format.extent.spa.fl_str_mv 9 p.
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dc.publisher.spa.fl_str_mv John Wiley and Sons, Inc
dc.publisher.place.spa.fl_str_mv Estados Unidos
dc.source.spa.fl_str_mv https://onlinelibrary.wiley.com/doi/10.1002/jcp.29220
institution Fundación Universitaria de Ciencias de la Salud - FUCS
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spelling Gutiérrez Castañeda, Luz D.79a204ebe069de6c5446265a10f8aef2Tovar Parra, David58f644fe94e4991c6daa054cbafeff94Quintero, Gloria2507f4a0a4e0bb5e9c2f0e4972d118a2Amezquita, Lorena0e1fe33e7bbaa853e0d9923ab0584edeGuerrero, Carlosfd864ef6bdf86d108cd266e07c046929Sanabria, Daniel0efd98b0e27206da402a46157767ca512022-04-05T20:09:27Z2019-092022-04-05T20:09:27ZJohn Wiley and Sons, IncEstados UnidosOvarian cancer is the fifth leading cause of cancer‐related deaths. It causes approximately 125,000 deaths per year worldwide; its diagnosis is made in advanced stages resulting in a high mortality rate. The objective of the study was optimizing the isolation of cells obtained from the solid tumor and ascitic fluid of patients with ovarian cancer and the phenotype with markers related to the epithelial–mesenchymal transition. For this, the solid tumor tissue was disaggregated and cultivated with different methodologies. As a result, cell growth was obtained and epi‐immunofluorescence was performed using antibodies against E‐cadherin, EpCAM, N‐cadherin, vimentin, CD133, and CD44. The primary culture from the solid tumor was obtained using Dispase II and DMEM/F12. Finally, heterogeneity was detected in terms of the expression of mesenchymal and epithelial type markers in the two types of isolated cells. 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yYXRhbWllbnRvIHNlYSBvIG5vIGF1dG9tYXRpemFkbyB5IHBhcmEgdXNhcmxvcyBjb25mb3JtZSBhIGxvIGV4cHJlc2FkbyBlbiBlbCBwcmVzZW50ZSBkb2N1bWVudG8sIGRlIGFjdWVyZG8gY29uIGxhIHBvbMOtdGljYSBpbnRlcm5hIGRlIHByb3RlY2Npw7NuIGRlIGRhdG9zLgpFbCBVc3VhcmlvIGVudGllbmRlIHkgYWNlcHRhIHF1ZSBsYSBGVUNTIHBvZHLDoSBlZmVjdHVhciBlbCB0cmF0YW1pZW50byBkZSBsb3MgZGF0b3MgZGVsIHVzdWFyaW8sIGRlIG1hbmVyYSBkaXJlY3RhIG8gYSB0cmF2w6lzIGRlIHRlcmNlcm9zIGV4cHJlc2FtZW50ZSBkZXNpZ25hZG9zIHBvciBlbGxhLCBxdWllbmVzIGFjdHVhcsOhbiBjb21vIGVuY2FyZ2Fkb3MgZGVsIHRyYXRhbWllbnRvIGRlIHN1IGluZm9ybWFjacOzbiBwZXJzb25hbCwgY29uIGxhcyBvYmxpZ2FjaW9uZXMgcXVlIGxhIGxleSBjb250ZW1wbGUuCkxhIGluZm9ybWFjacOzbiBkZSByZWdpc3RybyBxdWUgc2UgbGUgc29saWNpdGEgYWwgdXN1YXJpbyBlcyBsYSBtw61uaW1hIHBhcmEgZGFyIGN1bXBsaW1pZW50byBhIGxhIHBvbMOtdGljYSBkZSB0cmF0YW1pZW50byB5IHByb3RlY2Npw7NuIGRlIGRhdG9zIHBlcnNvbmFsZXMgZGUgbGEgRlVDUyB5IGFsYXMgbm9ybWFzIGxlZ2FsZXMgdmlnZW50ZXMgZW4gQ29sb21iaWEuCkVuIGVsIHByb2Nlc28gZGUgcmVnaXN0cm8sIHNlIGxlIGFkdmVydGlyw6EgYWwgdXN1YXJpbyBjb24gdW4gYXN0ZXJpc2NvICgqKSBxdcOpIGluZm9ybWFjacOzbiBkZWJlIHNlciBzdW1pbmlzdHJhZGEgb2JsaWdhdG9yaWFtZW50ZSB5IGN1w6FsIGVzIG9wY2lvbmFsLgpFbCB1c3VhcmlvIGRlIGVzdGUgc2l0aW8gd2ViIGVudGllbmRlIHkgYWNlcHRhIHF1ZSBzdXMgZGF0b3MgcGVyc29uYWxlcyBzZSByZWNvcGlsYW4gY29uIGVsIGZpbiBkZSBwcm9jdXJhcmxlIHVuIHNlcnZpY2lvIGVmaWNpZW50ZSB5IHBlcnNvbmFsaXphZG8sIHkgcXVlIHBvZHLDoW4gc2VyIHVzYWRvcyBjb24gZmluZXMgYWNhZMOpbWljb3MsIGludmVzdGlnYXRpdm9zIHkgY29tZXJjaWFsZXMsIHBvciBsbyBjdWFsIGF1dG9yaXphIGV4cHJlc2FtZW50ZSBxdWUgc2VhbiB1c2Fkb3MgcGFyYSByZW1pdGlyIGluZm9ybWFjacOzbiBkZSBzdXMgcHJvZ3JhbWFzIGFjYWTDqW1pY29zIGZvcm1hbGVzIGUgaW5mb3JtYWxlcywgcGFyYSBkYXIgY3VtcGxpbWllbnRvIGEgc3VzIG9ibGlnYWNpb25lcyBjb250cmFjdHVhbGVzIG8gbGVnYWxlcywgcGFyYSBldmFsdWFyIGxhIGNhbGlkYWQgZGVsIHNlcnZpY2lvLCBhc8OtIGNvbW8gcGFyYSByZW1pdGlyIGluZm9ybWFjacOzbiBkZSBzdXMgcHJveWVjdG9zLCBwbGFuZXMgeSBkZW3DoXMgaW5mb3JtYWNpw7NuIGluc3RpdHVjaW9uYWwgZGUgbGEgRlVDUy4gRW4gdG9kbyBjYXNvLCBlc3RhIGluZm9ybWFjacOzbiBzZXLDoSB1dGlsaXphZGEgZW4gZGVzYXJyb2xsbyBkZSBsYXMgZnVuY2lvbmVzIHByb3BpYXMgZGUgbGEgRlVDUy4KMy4gRGl2dWxnYWNpw7NuIGRlIGxhIGluZm9ybWFjacOzbiBkZSByZWdpc3RybzoKIApMYSBpbmZvcm1hY2nDs24gZGUgcmVnaXN0cm8gZGUgY2FkYSB1c3VhcmlvIGVzIGNvbmZpZGVuY2lhbCwgeSBubyBzZSBjb21wYXJ0aXLDoSBjb24gb3RyYXMgcGVyc29uYXMgbyBlbnRpZGFkZXMsIHNhbHZvIGN1YW5kbyBsYSBGVUNTIGNvbnNpZGVyZSBxdWUgc2UgcHJlc2VudGVuIGxvcyBzaWd1aWVudGVzIGNhc29zOgogCkN1YW5kbyBsYSBhdXRvcml6YWNpw7NuIGRlbCB0aXR1bGFyIG5vIHNlYSBvYmxpZ2F0b3JpYSAoTnVtZXJhbCA4LiDigJxBdXRvcml6YWNpw7NuIGRlbCBUaXR1bGFy4oCdIOKAkyBQb2zDrXRpY2EgZGUgVHJhdGFtaWVudG8geSBQcm90ZWNjacOzbiBkZSBEYXRvcyBQZXJzb25hbGVzIGRlIGxhIEZVQ1MpClBvciBsYSBleGlzdGVuY2lhIGRlIHVuIG1hbmRhdG8gbGVnYWwsIGRlYmVyIGVzdGF0dXRhcmlvIG8ganVkaWNpYWwgcXVlIG9ibGlndWUgYSBzdW1pbmlzdHJhcmxhLgpDdWFscXVpZXIgb3RyYSBleGNlcGNpw7NuIGRlZmluaWRhIGVuIGxhcyBsZXllcyBhcGxpY2FibGVzCkVuIHRvZG8gY2FzbywgZWwgVXN1YXJpbyBlbnRpZW5kZSB5IGFjZXB0YSBxdWUgc3VzIGRhdG9zIHBlcnNvbmFsZXMgZXZlbnR1YWxtZW50ZSBzZSBjb21wYXJ0aXLDoW4gY29uIG90cmFzIGVudGlkYWRlcyB1IG9yZ2FuaXphY2lvbmVzIHByaXZhZGFzIG8gZXN0YXRhbGVzLCBjb24gZWwgZmluIGRlIGN1bXBsaXIgY29uIGxvcyBkZWJlcmVzIHkgb2JsaWdhY2lvbmVzIHF1ZSBsZWdhbG1lbnRlIHRpZW5lIGNvbW8gSW5zdGl0dWNpw7NuIGRlIEVkdWNhY2nDs24gU3VwZXJpb3IuCiAKNC4gT2JsaWdhY2lvbmVzIHkgZGViZXJlcyBkZWwgdXN1YXJpbzoKIApFbCB1c3VhcmlvIGVzdMOhIGRlIGFjdWVyZG8gZW4gcHJvcG9yY2lvbmFyIGEgbGEgRlVDUywgaW5mb3JtYWNpw7NuIHZlcmRhZGVyYSwgcHJlY2lzYSwgYWN0dWFsaXphZGEgeSBjb21wbGV0YSwgc2Vnw7puIHNlIGluZGlxdWUgZW4gZWwgZm9ybXVsYXJpbyBkZSByZWdpc3Ryby4gSWd1YWxtZW50ZSwgZWwgdXN1YXJpbyBlc3TDoSBkZSBhY3VlcmRvIGVuIGNvbGFib3JhciBjb24gbGEgRlVDUywgcGFyYSBtYW50ZW5lciBlc3RhIGluZm9ybWFjacOzbiBhY3R1YWxpemFkYSB5IGNvbXBsZXRhLgogCkVsIHVzdWFyaW8gc2Ugb2JsaWdhIGEgbm8gcmVwcm9kdWNpciwgZHVwbGljYXIsIGNvcGlhciwgdmVuZGVyLCByZXZlbmRlciBvIGV4cGxvdGFyIHBhcmEgZmluZXMgY29tZXJjaWFsZXMsIGN1YWxxdWllciBzZWNjacOzbiBkZWwgc2VydmljaW8sIHVzbyBvIGFjY2VzbyBhbCBtaXNtby4gVGFudG8gZWwgc2VydmljaW8gY29tbyB0ZXJjZXJvcyBwb2Ryw6FuIHByb3BvcmNpb25hciB2w61uY3Vsb3MgYSBvdHJvcyBzaXRpb3MgbyByZWN1cnNvcyBkZSBsYSByZWQgbXVuZGlhbC4gRGViaWRvIGEgcXVlIGxhIEZVQ1Mgbm8gdGllbmUgY29udHJvbCBhbGd1bm8gc29icmUgZGljaG9zIHNpdGlvcyBvIHJlY3Vyc29zLCBlbCB1c3VhcmlvIHJlY29ub2NlIHkgYWNlcHRhIHF1ZSBsYSBGVUNTIG5vIHNlcsOhIHJlc3BvbnNhYmxlIHBvciBsYSBkaXNwb25pYmlsaWRhZCBkZSBsb3Mgc2l0aW9zIG8gcmVjdXJzb3MgZXh0ZXJub3MgbmkgcG9yIGN1YWxxdWllciBjb250ZW5pZG8sIHB1YmxpY2lkYWQsIHByb2R1Y3Rvcywgc2VydmljaW9zIHUgb3RybyB0aXBvIGRlIG1hdGVyaWFsIGNvbnRlbmlkbyDDsyBhIGRpc3Bvc2ljacOzbiBlbiB0YWxlcyBzaXRpb3MgbyByZWN1cnNvcy4KIApFbCB1c3VhcmlvIHJlY29ub2NlIHkgYWNlcHRhIHF1ZSBsYSBGVUNTIG5vIHNlcsOhIHJlc3BvbnNhYmxlLCBkaXJlY3RhIG8gaW5kaXJlY3RhbWVudGUsIHBvciBjdWFscXVpZXIgZGHDsW8gbyBwZXJqdWljaW8gY2F1c2FkbyBvIHF1ZSBzZSBwcmVzdW1hIHF1ZSBzZWEgY2F1c2FkbyBwb3IgdGFsZXMgY29udGVuaWRvcywgcHJvZHVjdG9zIG8gc2VydmljaW9zIGRpc3BvbmlibGVzIGVuIGRpY2hvcyBzaXRpb3MgbyByZWN1cnNvcyBleHRlcm5vcywgbyBwb3IgbGEgdXRpbGl6YWNpw7NuIG8gY29uZmlhbnphIGRlcG9zaXRhZGEgcG9yIGVsIHVzdWFyaW8gZW4gdGFsZXMgY29udGVuaWRvcywgcHJvZHVjdG9zIG8gc2VydmljaW9zLgogCkVsIHVzdWFyaW8gc2Ugb2JsaWdhIGEgTk8gdXRpbGl6YXIgZWwgcG9ydGFsIHdlYiBuaSBlc3RlIFNlcnZpY2lvIGNvbiBlbCBmaW4gZGU6CiAKUG9uZXIgYSBkaXNwb3NpY2nDs24gZGUgbG9zIGRlbcOhcywgZW52aWFyIHBvciBlLW1haWwgbywgZGUgY3VhbHF1aWVyIG1vZG8sIHRyYW5zbWl0aXIgY29udGVuaWRvcyBpbGVnYWxlcywgZGHDsWlub3MsIG1vbGVzdG9zLCBhbWVuYXphZG9yZXMsIGFidXNpdm9zLCB0b3J0dW9zb3MsIGRpZmFtYXRvcmlvcywgdnVsZ2FyZXMsIG9ic2Nlbm9zLCBpbnZhc29yZXMgZGUgbGEgaW50aW1pZGFkIGRlIHRlcmNlcm9zLCBvZGlvc29zLCB4ZW7Ds2ZvYm9zLCByYWNpc3RhcywgaWxlZ2FsZXMgbywgZGUgYWxnw7puIG1vZG8sIGluYWNlcHRhYmxlczsKSGFjZXJzZSBwYXNhciBvIGZpbmdpciBzZXIgY3VhbHF1aWVyIG90cmEgcGVyc29uYSBvIGVudGlkYWQsIGluY2x1eWVuZG8sIHNpbiBsaW1pdGFjacOzbiwgdW4gcmVwcmVzZW50YW50ZSBkZSBsYSBGVUNTLCBmdW5kYWRvciwgZ3XDrWEgbyBhbmZpdHJpw7NuIGRlIGZvcnVtcywgbyBkZSBjdWFscXVpZXIgb3RybyBtb2RvIG1lbnRpciBvIGZpbmdpciBzb2JyZSBzdSByZWxhY2nDs24gY29uIGN1YWxxdWllciBvdHJhIHBlcnNvbmEgbyBhZmlsaWFjacOzbiBhIGN1YWxxdWllciBlbnRpZGFkOwpQb25lciBhIGRpc3Bvc2ljacOzbiBkZSBsb3MgZGVtw6FzIHVzdWFyaW9zLCBlbnZpYXIgcG9yIGUtbWFpbCBvLCBkZSBhbGfDum4gbW9kbyB0cmFuc21pdGlyLCBjdWFscXVpZXIgY29udGVuaWRvIHF1ZSwgZGUgYWN1ZXJkbyBjb24gbGFzIGxleWVzIGFwbGljYWJsZXMgbyByZWxhY2lvbmVzIGNvbnRyYWN0dWFsZXMgZXhpc3RlbnRlcywgZWwgdXN1YXJpbyBubyBlc3TDoSBhdXRvcml6YWRvIGEgdHJhbnNtaXRpciAodGFsZXMgY29tbyBpbmZvcm1hY2nDs24gcHJpdmlsZWdpYWRhLCBpbmZvcm1hY2nDs24gcHJvdGVnaWRhIHBvciBkZXJlY2hvcyBkZSBwcm9waWVkYWQgaW5kdXN0cmlhbCBvIGludGVsZWN0dWFsIG8gaW5mb3JtYWNpw7NuIHNvYnJlIGxhIGN1YWwgdGllbmUgdW4gZGViZXIgZGUgY29uZmlkZW5jaWFsaWRhZCkuClBvbmVyIGEgZGlzcG9zaWNpw7NuIGRlIGxvcyBkZW3DoXMgdXN1YXJpb3MsIGVudmlhciBwb3IgZS1tYWlsIG8sIGRlIGFsZ8O6biBtb2RvLCB0cmFuc21pdGlyIGNvbnRlbmlkbyBhbGd1bm8gc3VzY2VwdGlibGUgZGUgaW5mcmluZ2lyIHBhdGVudGVzLCBtYXJjYXMsIHNlY3JldG9zIGNvbWVyY2lhbGVzLCBkZXJlY2hvcyBkZSBhdXRvciB1IG90cm9zIGRlcmVjaG9zIGRlIHRlcmNlcm9zICgiRGVyZWNob3MiKTsKUG9uZXIgYSBkaXNwb3NpY2nDs24gZGUgbG9zIGRlbcOhcyB1c3VhcmlvcywgZW52aWFyIHBvciBlLW1haWwgbywgZGUgYWxnw7puIG1vZG8sIHRyYW5zbWl0aXIgcHVibGljaWRhZCBubyBzb2xpY2l0YWRhIG8gYXV0b3JpemFkYSwgbWF0ZXJpYWwgcHVibGljaXRhcmlvLCAiY29ycmVvIGJhc3VyYSIsICJjYXJ0YXMgZW4gY2FkZW5hIiwgImVzdHJ1Y3R1cmFzIHBpcmFtaWRhbGVzIiwgbyBjdWFscXVpZXIgb3RyYSBmb3JtYSBkZSBzb2xpY2l0dWQ7ClBvbmVyIGEgZGlzcG9zaWNpw7NuIGRlIGxvcyBkZW3DoXMgdXN1YXJpb3MsIGVudmlhciBwb3IgZS1tYWlsIG8sIGRlIGFsZ8O6biBtb2RvLCB0cmFuc21pdGlyIG1hdGVyaWFsIGFsZ3VubyBxdWUgc2VhIHBvcnRhZG9yIGRlIHZpcnVzIG8gY3VhbHF1aWVyIG90cm8gY8OzZGlnbyBpbmZvcm3DoXRpY28sIGFyY2hpdm9zIG8gcHJvZ3JhbWFzIGRpc2XDsWFkb3MgcGFyYSBpbnRlcnJ1bXBpciwgZGVzdHJ1aXIgbyBsaW1pdGFyIGVsIGZ1bmNpb25hbWllbnRvIGRlIGN1YWxxdWllciBzb2Z0d2FyZSwgaGFyZHdhcmUgbyBlcXVpcG8gZGUgdGVsZWNvbXVuaWNhY2lvbmVzOwpQb25lciBhIGRpc3Bvc2ljacOzbiBkZSBsb3MgZGVtw6FzIHVzdWFyaW9zLCBlbnZpYXIgcG9yIGUtbWFpbCBvLCBkZSBhbGfDum4gbW9kbyB0cmFuc21pdGlyLCBjdWFscXVpZXIgY29udGVuaWRvIHF1ZSwgZGUgYWN1ZXJkbyBjb24gbGFzIGxleWVzIGFwbGljYWJsZXMsIHNlYSBkZSBjYXLDoWN0ZXIgaWxlZ2FsIG8gaW1wbGlxdWUsIGF5dWRlIG8gY29hZHl1dmUgbGEgY29taXNpw7NuIGRlIGNvbmR1Y3RhcyBkZWxpY3R1YWxlcyBvIGlsZWdhbGVzIGRlIGN1YWxxdWllciBtYW5lcmEuCkludGVycnVtcGlyIGVsIGN1cnNvIG5vcm1hbCBkZSBsYXMgY29udmVyc2FjaW9uZXMsIHByb3ZvY2FyIHF1ZSBsYSBwYW50YWxsYSBkZWwgb3JkZW5hZG9yIGFkcXVpZXJhIG1heW9yIHZlbG9jaWRhZCBkZSBsYSBub3JtYWwgY29uIGxhIHF1ZSBsb3MgdXN1YXJpb3MgcHVlZGVuIHBhcnRpY2lwYXIgZW4gZWwgU2VydmljaW8sIG8gZGUgYWxnw7puIG1vZG8gYWN0dWFyIGRlIG1hbmVyYSBxdWUgYWZlY3RlIGRlIGZvcm1hIG5lZ2F0aXZhIGEgbGEgcG9zaWJpbGlkYWQgZGUgY29tdW5pY2Fyc2UgZW4gdGllbXBvIHJlYWw7CkludGVyZmVyaXIgbyBpbnRlcnJ1bXBpciBlbCBTZXJ2aWNpbywgc2Vydmlkb3JlcyBvIHJlZGVzIGNvbmVjdGFkb3MgYWwgU2VydmljaW8sIG8gaW5jdW1wbGlyIGxvcyByZXF1aXNpdG9zLCBwcm9jZWRpbWllbnRvcyB5IHJlZ3VsYWNpb25lcyBkZSBsYSBwb2zDrXRpY2EgZGUgcmVkZXMgY29uZWN0YWRhcyBhbCBzZXJ2aWNpby4KNS4gTGltaXRhY2lvbmVzIGRlIGxhIFJlc3BvbnNhYmlsaWRhZDoKIApDb24gdG9kYSBsYSBleHRlbnNpw7NuIHBlcm1pdGlkYSBwb3IgbGFzIGxleWVzIG5hY2lvbmFsZXMgYXBsaWNhYmxlcywgbGEgRlVDUyBubyBhc3VtZSByZXNwb25zYWJpbGlkYWQgY29udHJhY3R1YWwgbyBleHRyYWNvbnRyYWN0dWFsIGFsZ3VuYSBkZSBsb3MgZGHDsW9zIHkgcGVyanVpY2lvcyBkaXJlY3RvcywgaW5kaXJlY3RvcywgaW5jaWRlbnRhbGVzLCBlc3BlY2lhbGVzIG8gZW1lcmdlbnRlcyAoaW5jbHVzbyBzaSBsYSBGdW5kYWNpw7NuIFVuaXZlcnNpdGFyaWEgZGUgQ2llbmNpYXMgZGUgbGEgU2FsdWQgaHViaWVyYSBzaWRvIGFkdmVydGlkbyBkZSBsYSBwb3NpYmlsaWRhZCBkZSBsb3MgbWlzbW9zKSBxdWUgdHJhaWdhbiBjYXVzYSBkZWw6IGVsIHVzbyBvIGRlIGxhIGltcG9zaWJpbGlkYWQgZGUgdXNhciBlbCBzZXJ2aWNpbzsgZWwgZGVsIGFjY2VzbyBubyBhdXRvcml6YWRvIMOzIGFsdGVyYWNpw7NuIGRlIHN1cyB0cmFuc21pc2lvbmVzIGRlIGRhdG9zOyBlbCBkZWNsYXJhY2lvbmVzIGRlIGNvbmR1Y3RhIGRlIHRlcmNlcm9zIGEgdHJhdsOpcyBkZWwgc2VydmljaW8uCiAKRWwgdXN1YXJpbyBhY3VlcmRhIGFkZW3DoXMgcXVlIGxhIEZ1bmRhY2nDs24gVW5pdmVyc2l0YXJpYSBkZSBDaWVuY2lhcyBkZSBsYSBTYWx1ZCBubyBzZXLDoSByZXNwb25zYWJsZSBkZSBuaW5nw7puIGRhw7FvIG8gcGVyanVpY2lvIGRlcml2YWRvIGRlIGxhIGludGVycnVwY2nDs24sIHN1c3BlbnNpw7NuIG8gdGVybWluYWNpw7NuIGRlbCBzZXJ2aWNpbywgaW5jbHV5ZW5kbywgcGVybyBubyBsaW1pdMOhbmRvc2UsIGEgZGHDsW9zLCBsdWNybyBjZXNhbnRlLCB1c28sIGRhdG9zIHUgb3Ryb3MgYmllbmVzIGludGFuZ2libGVzLCBvIHBlcmp1aWNpb3MgZGlyZWN0b3MsIGluZGlyZWN0b3MsIGluY2lkZW50YWxlcywgZXNwZWNpYWxlcywgY29uc2VjdWVudGVzLCBvIGVqZW1wbGFyZXMsIHlhIHNlYSBxdWUgdGFsIGludGVycnVwY2nDs24sIHN1c3BlbnNpw7NuIG8gdGVybWluYWNpw7NuIHNlYSBqdXN0aWZpY2FkYSBvIG5vLCBuZWdsaWdlbnRlIMOzIGludGVuY2lvbmFsLCBhZHZlcnRpZGEgw7MgaW5hZHZlcnRpZGEuCiAKTGEgRlVDUywgc2UgcmVzZXJ2YSBlbCBkZXJlY2hvIGEgcmV0aXJhciBzaW4gcHJldmlhIG5vdGlmaWNhY2nDs24gYSBjdWFscXVpZXIgdXN1YXJpbyBxdWUgc2UgY29uc2lkZXJlIG9mZW5zaXZvIGVuIGxhcyBjb252ZXJzYWNpb25lcyBvIGRpw6Fsb2dvcyBkZSBsb3MgdXN1YXJpb3MgbyB2aXNpdGFudGVzIGRlbCBzaXRpbyB3ZWIgZW4gZm9yb3MgbyBjaGF0cy4KIApMYSBGVUNTIG5vIGVzIHJlc3BvbnNhYmxlIGNvbiByZXNwZWN0byBhIGxhIHByaXZhY2lkYWQgZGUgbGEgaW5mb3JtYWNpw7NuIHBlcnNvbmFsIHF1ZSBlbCB1c3VhcmlvIGRpdnVsZ3VlIG8gcmVtaXRhIGVuIGZvcm9zLCBzZXNpb25lcyBkZSBjaGF0IGRlbCBzaXRpbyB3ZWIsIGVuIGNvcnJlb3MgZWxlY3Ryw7NuaWNvcyBvIGVuIGN1YWxxdWllciBvdHJvIHNpdGlvIHdlYi4gRWwgdXN1YXJpbyBlcyBleGNsdXNpdm8gcmVzcG9uc2FibGUgZGUgbGEgcHJpdmFjaWRhZCB5IGN1c3RvZGlhIGRlIGN1YWxxdWllciBkYXRvIHBlcnNvbmFsIHF1ZSBlbnRyZWd1ZSBhIHVuYSBwZXJzb25hIG8gZW1wcmVzYSBkaWZlcmVudGUgRlVDUy4KIAo2LiBEZXJlY2hvcyBkZSBQcm9waWVkYWQgSW50ZWxlY3R1YWw6CiAKRWwgVXN1YXJpbyByZWNvbm9jZSB5IGFjZXB0YSBxdWUgZWwgc2VydmljaW8geSBjdWFscXVpZXIgc29mdHdhcmUgdXNhZG8gZW4gcmVsYWNpw7NuIGNvbiBlbCBzZXJ2aWNpbyBxdWUgY29udGllbmUgaW5mb3JtYWNpw7NuIGNvbmZpZGVuY2lhbCB5IGRlIHByb3BpZWRhZCBhamVuYSBwcm90ZWdpZGEgcG9yIGxhIGxlZ2lzbGFjacOzbiBkZSBwcm9waWVkYWQgaW50ZWxlY3R1YWwgeSBvdHJhcyBkaXNwb3NpY2lvbmVzIGxlZ2FsZXMuIEFkZW3DoXMgZWwgdXN1YXJpbyByZWNvbm9jZSB5IGFjZXB0YSBxdWUgZWwgY29udGVuaWRvIGluY2x1eWVuZG8sIHBlcm8gbm8gbGltaXTDoW5kb3NlLCB0ZXh0b3MsIHByb2dyYW1hcyAoYXBwbGV0cyksIGZvdG9ncmFmw61hcywgZ3LDoWZpY29zLCB2aWRlb3MgdSBvdHJvIG1hdGVyaWFsIGNvbnRlbmlkbyBlbiBlbCBzZXJ2aWNpbywgYXPDrSBjb21vIHRhbWJpw6luIGluZm9ybWFjaW9uZXMgZGl2dWxnYWRhcyBhbCB1c3VhcmlvIGEgdHJhdsOpcyBkZWwgc2VydmljaW8sIGVzdMOhIHByb3RlZ2lkbyBwb3IgZGVyZWNob3MgZGUgYXV0b3IsIG1hcmNhcyBjb21lcmNpYWxlcywgcGF0ZW50ZXMgeSBvdHJvcyBkZXJlY2hvcyBkZSBwcm9waWVkYWQgaW50ZWxlY3R1YWwsIGRlIGFjdWVyZG8gY29uIGxhIGxlZ2lzbGFjacOzbiBjb2xvbWJpYW5hLCBjb211bml0YXJpYSBlIGludGVybmFjaW9uYWwgYXBsaWNhYmxlLgogCkVsIFVzdWFyaW8gcmVjb25vY2UgeSBhY3VlcmRhIHF1ZSBzb2xhbWVudGUgcG9kcsOhIHV0aWxpemFyIHRhbGVzIG1hdGVyaWFsZXMgZSBpbmZvcm1hY2lvbmVzIHNlZ8O6biBsbyBleHByZXNhbWVudGUgYXV0b3JpemFkbyBwb3IgbGEgRlVDUywgeSBubyBwb2Ryw6EgY29waWFyLCByZXByb2R1Y2lyLCB0cmFuc21pdGlyLCBkaXN0cmlidWlyIG8gY3JlYXIgb2JyYXMgZGVyaXZhZGFzIGEgcGFydGlyIGRlIHRhbGVzIG1hdGVyaWFsZXMgbyBpbmZvcm1hY2lvbmVzIHNpbiBsYSBleHByZXNhIGF1dG9yaXphY2nDs24gZGVsIHJlc3BlY3Rpdm8gcHJvcGlldGFyaW8uCiAKRWwgVXN1YXJpbyBzZSBvYmxpZ2EgYSBubyBtb2RpZmljYXIgZWwgc29mdHdhcmUgaW52b2x1Y3JhZG8gZW4gZWwgcG9ydGFsIHdlYiBkZSBuaW5ndW5hIG1hbmVyYSwgbyB1dGlsaXphciB2ZXJzaW9uZXMgZGUgc29mdHdhcmUgbW9kaWZpY2FkYXMgY29uIGVsIGZpbiwgZW50cmUgb3Ryb3MsIGRlIG9idGVuZXIgYWNjZXNvIG5vIGF1dG9yaXphZG8gYWwgU2VydmljaW8uIEVsIFVzdWFyaW8gc2Ugb2JsaWdhIGEgbm8gYWNjZWRlciBhbCBTZXJ2aWNpbyBwb3Igb3Ryb3MgbWVkaW9zIHF1ZSBubyBzZWFuIGEgdHJhdsOpcyBkZSBsYSBpbnRlcmZheiBxdWUgbGEgRlVDUyBwcm9wb3JjaW9uYSBwYXJhIGFjY2VkZXIgYWwgU2VydmljaW8uCiAKNy4gR2FyYW50w61hIExpbWl0YWRhOgogCkVsIHVzbyBkZWwgc2VydmljaW8gc2Vyw6EgYmFqbyBleGNsdXNpdm8gcmllc2dvIHkgcmVzcG9uc2FiaWxpZGFkIGRlbCB1c3VhcmlvLiBFbCBzZXJ2aWNpbyBlcyBzdW1pbmlzdHJhZG8gZ3JhdHVpdGFtZW50ZSB5IGRlcGVuZGUgZGUgbGEgZnVuY2lvbmFsaWRhZCBkZSB2YXJpb3MgZmFjdG9yZXMsIGNvbW8gbGEgaW50ZXJhY2Npw7NuIGRlIHNlcnZpZG9yZXMgeSBzZXJ2aWNpb3MgZGUgdGVsZWNvbXVuaWNhY2lvbmVzIGRlIHRlcmNlcm9zLCBsYSBhZGVjdWFjacOzbiBkZSBsb3MgZXF1aXBvcyBkZSB1c3VhcmlvIHkgZGVzdGluYXRhcmlvIGRlbCBtZW5zYWplLiBDb25zaWRlcmFuZG8gdGFsZXMgZmFjdG9yZXMsIGxhIEZVQ1MgZW1wbGVhcsOhIHN1cyBtZWpvcmVzIGVzZnVlcnpvcyBwYXJhIHF1ZSBsb3MgbWVuc2FqZXMgZGUgZS1tYWlsIGVudmlhZG9zIGEgdHJhdsOpcyBkZWwgc2VydmljaW8sIHNlYW4gcmVjaWJpZG9zIHBvciBlbCBkZXN0aW5hdGFyaW8gZW4gZm9ybWEgY29ycmVjdGEgeSBzaW4gaW50ZXJmZXJlbmNpYXMuIFNpbiBlbWJhcmdvLCBwb3IgZXN0YXMgbWlzbWFzIHJhem9uZXMsIGxhIEZVQ1Mgbm8gcHVlZGUgZ2FyYW50aXphciBxdWUgbG9zIG1lbnNhamVzIHNlcsOhbiBlbnRyZWdhZG9zIGFsIGRlc3RpbmF0YXJpbyBjb3JyZWN0bywgZW4gdW4gcGxhem8gYWRlY3VhZG8sIG8gbm8gc3Vmcmlyw6FuIGV4dHJhdsOtbywgZGl2dWxnYWNpw7NuIG8gdmlvbGFjacOzbiBwb3IgcGFydGVzIGRlIHRlcmNlcm9zIG5vIGF1dG9yaXphZG9zLCBjb21vIHBvciBlamVtcGxvcyAiaGFja2VycyIuCiAKU2UgYWNvbnNlamEgYWwgdXN1YXJpbyBubyBjb25maWFyIGV4Y2x1c2l2YW1lbnRlIGVuIGVsIHNlcnZpY2lvIHBhcmEgZW52aWFyIGluZm9ybWFjaW9uZXMgaW1wb3J0YW50ZXMgbyBjb25maWRlbmNpYWxlcyB5IGxhIEZ1bmRhY2nDs24gVW5pdmVyc2l0YXJpYSBkZSBDaWVuY2lhcyBkZSBsYSBTYWx1ZCBubyBzZSByZXNwb25zYWJpbGl6YXLDoSBwb3IgbmluZ3VuYSBmYWxsYSByZXN1bHRhbnRlIGRlbCBlbnbDrW8gZGUgbWVuc2FqZXMgcG9yIHBhcnRlIGRlbCB1c3VhcmlvLgogCkxhIEZVQ1Mgbm8gZ2FyYW50aXphIHF1ZSBlbCBzZXJ2aWNpbyBzZSBwcmVzdGFyw6EgZGUgbWFuZXJhIGluaW50ZXJydW1waWRhLCBzZWd1cmEgbyBsaWJyZSBkZSBlcnJvci4gTGEgRlVDUyB0YW1wb2NvIG90b3JnYSBuaW5ndW5hIGdhcmFudMOtYSBlbiBjdWFudG8gYSBsb3MgcmVzdWx0YWRvcyBxdWUgc2UgcHVlZGFuIG9idGVuZXIgZGVsIHVzbyBkZWwgc2VydmljaW8sIG5pIHF1ZSBsb3MgZGVmZWN0b3MgZW4gbG9zIHByb2dyYW1hcyBzZXLDoW4gY29ycmVnaWRvcy4KIApFbCB1c3VhcmlvIHJlY29ub2NlIHkgYWN1ZXJkYSBxdWUgY3VhbHF1aWVyIG1hdGVyaWFsIHkvbyBpbmZvcm1hY2nDs24gb2J0ZW5pZGEgYSB0cmF2w6lzIGRlbCBzZXJ2aWNpbyBvIGRlIHN1IHV0aWxpemFjacOzbiwgZXN0YXLDoSBzdWpldGEgYSBzdSBlbnRlcm8gY3JpdGVyaW8geSByaWVzZ287IHkgcXVlIHNlcsOhIGVsIMO6bmljbyByZXNwb25zYWJsZSBkZSBjdWFscXVpZXIgZGHDsW8gb2N1cnJpZG8gZW4gc3UgY29tcHV0YWRvcmEgbyBww6lyZGlkYSBkZSBkYXRvcyBxdWUgcHVlZGEgcmVzdWx0YXIgZGUgcmVjaWJpciB0YWwgbWF0ZXJpYWwuCiAKTm90YTogc2kgcmVjaWJlIHVuIG1lbnNhamUgZGUgY29ycmVvIGVsZWN0csOzbmljbyBubyBzb2xpY2l0YWRvLCBvIGN1YWxxdWllciBtZW5zYWplIGFtZW5hemFkb3IgdSBvZmVuc2l2bywgbyBjb25zaWRlcmEgcXVlIG90cm8gdXN1YXJpbyBvIGN1YWxxdWllciBwZXJzb25hIGxlIGVzdMOhIHZ1bG5lcmFuZG8gdW4gZGVyZWNobyBjb25mb3JtZSBhIGVzdGUgZG9jdW1lbnRvLCByZW3DrXRhbm9zIHVuYSBjb3BpYSBjb21wbGV0YSB5IHNpbiBtb2RpZmljYXIgZGVsIG1lbnNhamUgZGUgY29ycmVvIGVsZWN0csOzbmljbyBjb24gbG9zIGVuY2FiZXphZG9zIGNvbXBsZXRvcyBhIGxhIHNpZ3VpZW50ZSBkaXJlY2Npw7NuIGVsZWN0csOzbmljYTogeWx0b3JvQGZ1Y3NhbHVkLmVkdS5jbwoKTGFzIHByZWd1bnRhcyBxdWUgZWwgdXN1YXJpbyBvIGN1YWxxdWllciBwZXJzb25hIGRlc2VlIHJlYWxpemFyIHNvYnJlIGxvIGV4cHJlc2FkbyBlbiBlc3RlIGRvY3VtZW50bywgbGFzIHB1ZWRlIGZvcm11bGFyIGEgbGEgc2lndWllbnRlIGRpcmVjY2nDs24gZWxlY3Ryw7NuaWNhOiB5bHRvcm9AZnVjc2FsdWQuZWR1LmNvCg==

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