Análisis funcional del gen aquaporina1 en respuesta a estrés oxidativo y antimonio en Leishmania (Viannia) brazilienis

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dc.contributor.advisorTellez Meneses, Jair Alexander
dc.contributor.advisorContreras Rodriguez, Luis Ernesto
dc.contributor.authorRiaño Cocunubo, Leydi Johana
dc.contributor.researchgroupZajuna jwa samu (Semilla del conocimiento) del Cesarspa
dc.date.accessioned2025-03-17T20:07:41Z
dc.date.available2025-03-17T20:07:41Z
dc.date.issued2025-03-12
dc.descriptionilustraciones, diagramas, fotografías, tablasspa
dc.description.abstractLa leishmaniasis es una enfermedad parasitaria causada por protozoarios pertenecientes al género Leishmania. El tratamiento de primera línea para esta patología incluye el uso de antimoniales, ante los cuales el parásito ha desarrollado mecanismos de resistencia, en los que participa la aquaporina 1 (AQP1). En consecuencia, la implementación de estrategias robustas de edición génica para el análisis funcional de genes asociados con fenómenos de resistencia como AQP1 resulta necesario, permitiendo iniciar el desarrollo de potenciales nuevas estrategias de control. En este trabajo se analizó la participación del gen multicopia AQP1 en respuesta ante el estrés oxidativo y el antimonio trivalente en promastigotes de Leishmania (Viannia) braziliensis editados genéticamente por CRISPR/Cas9. La edición génica consistió en el diseño de plantillas para obtener un ARN guía (sgARN) contra el gen AQP1 y casetes de reparación con los marcadores mCherry y mNeonGreen, con el propósito de generar parásitos Knockout (KO). Las plantillas obtenidas mediante PCR se transfectaron por electroporación en promastigotes de L. braziliensis que expresan constitutivamente las proteínas Cas9 de Streptococcus pyogenes (SpCas9) y T7 ARN polimerasa (LbCas9/T7). La edición exitosa se evaluó por PCR diagnóstico y secuenciación de ADN, confirmándose la edición específica de todas las copias del gen AQP1 y, por lo tanto, la generación de parásitos KO-AQP1, usando el marcador mNeonGreen. El análisis de las curvas de crecimiento de los promastigotes editados KO-AQP1 no evidenció diferencias significativas en comparación con los parásitos control LbCas9/T7. Adicionalmente, los parásitos KO-AQP1 indicaron valores mayores de IC50 ante el peróxido de hidrógeno y el antimonio trivalente. De esta manera, la implementación de estrategias de edición génica robustas como CRISPR/Cas9 en parásitos de Leishmania, constituye un aporte novedoso para Colombia, permitiendo enfocar el análisis de genes multicopia relacionados con fenómenos de resistencia desde una perspectiva de edición de ADN dirigida, lo cual potenciaría el desarrollo de nuevas estrategias terapéuticas para el tratamiento de la leishmaniasis (Texto tomado de la fuente)spa
dc.description.abstractLeishmaniasis is a parasitic disease caused by protozoa belonging to the genus Leishmania. The first-line treatment for this pathology includes the use of antimonials, against which the parasite has developed resistance mechanisms, in which aquaporin 1 (AQP1) participates. Consequently, the implementation of robust gene editing strategies for the functional analysis of genes associated with resistance phenomena such as AQP1 is necessary to initiate the development of potential new control strategies. In this work we analyzed the involvement of the multicopy AQP1 gene in response to oxidative stress and trivalent antimony in Leishmania (Viannia) braziliensis promastigotes gene-edited by CRISPR/Cas9. Gene editing consisted in the design of templates to obtain a guide RNA (sgRNA) against the AQP1 gene and repair cassettes with the mCherry and mNeonGreen markers, with the purpose of generating knockout (KO) parasites. PCR-derived templates were transfected by electroporation into L. braziliensis promastigotes constitutively expressing Streptococcus pyogenes Cas9 (SpCas9) and T7 RNA polymerase (LbCas9/T7) proteins. Successful editing was assessed by diagnostic PCR and DNA sequencing, confirming specific editing of all copies of the AQP1 gene and, therefore, generation of KOAQP1 parasites, using the mNeonGreen marker. Analysis of the growth curves of the KOAQP1-edited promastigotes showed no significant differences compared to the LbCas9/T7 control parasites. Additionally, KO-AQP1 parasites showed higher IC50 values against hydrogen peroxide and trivalent antimony. Thus, the implementation of robust gene editing strategies such as CRISPR/Cas9 in Leishmania parasites constitutes a novel contribution for Colombia, allowing to focus the analysis of multicopy genes related to resistance phenomena from a perspective of targeted DNA editing, which would enhance the development of new therapeutic strategies for the treatment of leishmaniasis.eng
dc.description.degreelevelMaestríaspa
dc.description.degreenameMagíster en Ciencias - Microbiologíaspa
dc.description.researchareaEnfermedades infecciosasspa
dc.format.extentxv, 137 páginasspa
dc.format.mimetypeapplication/pdfspa
dc.identifier.instnameUniversidad Nacional de Colombiaspa
dc.identifier.reponameRepositorio Institucional Universidad Nacional de Colombiaspa
dc.identifier.repourlhttps://repositorio.unal.edu.co/spa
dc.identifier.urihttps://repositorio.unal.edu.co/handle/unal/87679
dc.language.isospaspa
dc.publisherUniversidad Nacional de Colombiaspa
dc.publisher.branchUniversidad Nacional de Colombia - Sede Bogotáspa
dc.publisher.facultyFacultad de Cienciasspa
dc.publisher.placeBogotá, Colombiaspa
dc.publisher.programBogotá - Ciencias - Maestría en Ciencias - Microbiologíaspa
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dc.relation.referencesZambrano, P. (2020). DOCUMENTO ELABORADO POR. 04, 16.spa
dc.rights.accessrightsinfo:eu-repo/semantics/openAccessspa
dc.rights.licenseReconocimiento 4.0 Internacionalspa
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/spa
dc.subject.ddc570 - Biología::576 - Genética y evoluciónspa
dc.subject.decsAntimoniospa
dc.subject.decsAcuaporina 1spa
dc.subject.decsAquaporin 1eng
dc.subject.decsEstrés Oxidativospa
dc.subject.decsOxidative Stresseng
dc.subject.proposalLeishmaniasisspa
dc.subject.proposalLeishmania braziliensisspa
dc.subject.proposalEdición génicaspa
dc.subject.proposalAQP1spa
dc.subject.proposalCRISPRCas9spa
dc.subject.proposalAntimonio trivalentespa
dc.subject.proposalEstrés oxidativospa
dc.subject.proposalGene editingeng
dc.subject.proposalAntimonyeng
dc.subject.proposalOxidative stresseng
dc.titleAnálisis funcional del gen aquaporina1 en respuesta a estrés oxidativo y antimonio en Leishmania (Viannia) brazilienisspa
dc.title.translatedFunctional analysis of the aquaporin1 gene in response to oxidative stress and antimony in Leishmania (Viannia) brazilieniseng
dc.typeTrabajo de grado - Maestríaspa
dc.type.coarhttp://purl.org/coar/resource_type/c_bdccspa
dc.type.coarversionhttp://purl.org/coar/version/c_ab4af688f83e57aaspa
dc.type.contentTextspa
dc.type.driverinfo:eu-repo/semantics/masterThesisspa
dc.type.redcolhttp://purl.org/redcol/resource_type/TMspa
dc.type.versioninfo:eu-repo/semantics/acceptedVersionspa
dcterms.audience.professionaldevelopmentEstudiantesspa
dcterms.audience.professionaldevelopmentInvestigadoresspa
dcterms.audience.professionaldevelopmentMaestrosspa
dcterms.audience.professionaldevelopmentPersonal de apoyo escolarspa
dcterms.audience.professionaldevelopmentProveedores de ayuda financiera para estudiantesspa
dcterms.audience.professionaldevelopmentPúblico generalspa
dcterms.audience.professionaldevelopmentReceptores de fondos federales y solicitantesspa
oaire.accessrightshttp://purl.org/coar/access_right/c_abf2spa

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