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dc.contributor.advisorRengifo Castillo, Aura Caterinespa
dc.contributor.authorMontaño Rojas, Leidy Nataliaspa
dc.contributor.otherCoy Barrera, Ericsson Davidspa
dc.contributor.otherRivera Rivera, Jorge Alonsospa
dc.contributor.otherGrupo Morfología celular, Instituto Nacional de saludspa
dc.contributor.otherGrupo InQuiBio, Universidad Militar Nueva Granadaspa
dc.coverage.spatialCampus UMNGspa
dc.date.accessioned2019-12-13T17:00:43Z
dc.date.accessioned2019-12-26T21:09:17Z
dc.date.available2019-12-13T17:00:43Z
dc.date.available2019-12-26T21:09:17Z
dc.date.issued2019-10-15
dc.identifier.urihttp://hdl.handle.net/10654/32654
dc.description.abstractEl virus del dengue presenta cuatro serotipos y provoca la enfermedad conocida con el mismo nombre del virus. Este virus pertenece al género Flavivirus y se transmite al hombre por medio de la picadura de los mosquitos del género Aedes (albopictus y aegypti). Esta enfermedad aún no tiene un cuadro clínico definido, debido a que las manifestaciones en la mayoría de los casos son asintomáticas. Las principales células diana de la infección por DENV son los monocitos, los macrófagos y los linfocitos CD4+ y CD8+. Además, In vitro se ha reportado la infección de células del endotelio, varias líneas celulares hepáticas, fibroblásticas y neuronales. Algunos reportes indican que el DENV puede presentar un carácter neurotrópico, posiblemente asociado al genotipo viral o a las condiciones del hospedero, sin embargo, aún no existe un consenso en la comunidad científica sobre aceptar esta condición a pesar del creciente número de casos en donde se ha evidenciado la presencia del virus en el SNC acompañado de manifestaciones neurológicas, tales como cefalea severa, vomito en niños, alteraciones en la conciencia, convulsiones, síndrome de Guillain-Barre y mielitis transversa. Distintos receptores han sido planteados como blancos de entrada viral tales como DC-SIGN, heparán sulfato, integrinas, entre otros, sin embargo, se desconoce en gran manera los receptores que podrían participar en el ingreso y replicación del virus dengue en el SNC. Los receptores de glutamato tipo NMDA podrían contribuir en la neuroinfección por DENV, porque se ha demostrado que el uso de antagonistas del receptor tales como MK-801 y ketamina, inhiben la replicación viral por lo que resulta necesario plantear modelos de estudio para dar respuesta a la compresión del sistema glutamatérgico durante la forma encefálica del dengue asociada a la presencia viral. En este trabajo se evaluó el efecto de un virus DENV-IV neuroadaptado, sobre la expresión de la subunidad GLUN2A del receptor NMDA y la inmunorreactividad del neurotransmisor glutamato en la corteza de ratones BALB/c infectados por dos vías distintas. Además, se evaluó el acoplamiento molecular y la dinámica de interacción entre la subunidad GLUN2A-NMDA y la proteína de envoltura E del virus dengue mediante ensayos in silico. Los resultados indicaron que durante la infección por DENV no se afecta la inmunorreactividad del neurotransmisor glutamato, pero ocurre una desregulación de la expresión subunidad GLUN2A y un alto acoplamiento entre esta subunidad GluN2A y la proteína E de envoltura que se puede mantener en el tiempo. Este es el primer reporte del papel de estos elementos del sistema glutamatérgico durante la neuroinfección por dengue y de aproximaciones a la dinámica de interacción molecular entre proteínas virales y los receptores NMDA. Los resultados de este trabajo se presentan divididos de la siguiente manera: Marco teórico (artículo de revisión), objetivos, resultados de ensayos en forma de dos artículos científicos, uno de ensayos in silico y otro de ensayos in vivo. Al final del documento aparecerán las conclusiones, como respuesta a cada uno de los objetivos planteados. Lo anterior con el fin de cumplir con lo estipulado por la Facultad de Ciencias Básicas y Aplicadas de la Universidad Militar Nueva Granada en pro de fomentar la generación de publicaciones científicas.spa
dc.description.sponsorshipInstituto Nacional de Saludspa
dc.description.tableofcontentsCONTENIDO Resumen Pág. 4 Capítulo I 1. Neurotropismo del virus del dengue y posible papel de los receptores NMDA en la replicación de virus neurotrópicos…………….................. 6 1.1. Introducción ………………………………………………………………...... 7 1.2. Biología del virus Dengue…………………………………………………...... 8 1.3. Proteínas de fusión de membrana……………………………………………... 9 1.4. Posibles formas de anclaje de la proteína E del DENV a la célula huésped...10 1.5. Epidemiologia del virus Dengue (DENV)…………………………….............. 11 1.6. Patogénesis y neuropatogénia del DENV……………………………............... 12 1.6.1. La amplificación dependiente de anticuerpos ADE o hipótesis de Halstead…………………………………………………………………………….. 13 1.6.2. Respuesta inmune aberrante………………….…………………...........13 1.6.3. Virulencia viral…………………….…………………………............... 13 1.7. Blancos celulares del DENV………………………………………………....... 14 1.8. Neuroinvasión y neurotropismo del DENV…………………………………… 16 1.8.1. Corteza cerebral ……………………………………………………...... 19 1.9. Principales sistemas de neurotransmisión del sistema nervioso ……….........19 1.10. Receptores NMDA y virus neurotrópicos..…………………………………... 22 1.10.1. Expresión, distribución, y función de los NMDAR ………………….. 23 1.10.2. Expresión de la subunidad GluN1 ………………………………......... 24 1.10.3. Expresión de la subunidad GluN2 ………………………………......... 24 1.11. Referencias ………………………………………………………………....... 26 Capitulo II 2. Evaluación in silico de la interacción de la subunidad GluN2A del receptor NMDA con la proteína E de envoltura del virus dengue…………………………... 34 2.1. Introducción……………………………………………………………………. 35 2.2. Materiales y métodos…………………………………………………………... 37 2.2.1. Obtención archivos de trabajo de las proteínas………………………….37 2.2.2. Acoplamiento molecular proteína-ligando…………………………...…. 38 2.2.3. Acoplamiento molecular proteína – proteína………………………….... 38 2.2.3.1. Selección de los residuos activos y pasivos………………………....38 2.2.4. Simulación de dinámica molecular……………………………………... 39 2.3. Resultados y Discusión……………………………………………………….... 40 2.3.1. Selección de los residuos activos y pasivos para acoplamiento……… 40 2.3.2. Acoplamiento molecular proteína-proteína, primera metodología…… 41 2.3.3. Acoplamiento molecular proteína-proteína, segunda y tercera metodología………………………………………………………………………….......42 2.3.4. Simulación de dinámica…………………………………………………. 46 2.4. Conclusión…………………………………………………………………….. 50 2.5. Referencias…………………………………………………………………….. 50 Capitulo III 3. Análisis del efecto de la infección por virus dengue neuroadaptado sobre la inmunorreactividad de glutamato y la expresión de la subunidad GluN2A del receptor de NMDA ……………………………………………………………….... 55 2.1. Introducción……………………………………………………………………. 56 2.2. Materiales y métodos…………………………………………………………... 58 3.2.1. Manejo de animales…………………………...……………………….... 58 3.2.2. Inmunohistoquímica…………………………………………………….. 58 3.2.2.1. Procedimiento para la obtención de tejido cerebral………………....58 3.2.2.2. Obtención de los cortes de tejido cerebral y definición del área de estudio…………………………..…………………………………………………... 59 3.2.2.3. Protocolo de inmunohistoquímica en flotación……………………... 60 3.2.2.4. Análisis cuantitativo………………………………………………… 60 3.2.3. Expresión de la subunidad GluN2A.NMDA por RT.PCR……………… 60 3.2.3.1. Extracción del ARN de las cortezas cerebrales…………………….60 3.2.3.2. Cuantificación de ARN…………………………………………….. 61 3.2.3.3. Determinación de la presencia o ausencia de virus DENV-4 en las muestras de tejido cerebral…………………………………………………………. 61 3.2.4. Evaluación de la expresión diferencial de la subunidad GluN2A en la corteza cerebral……………………………………………………………………... 62 3.2.4.1. Evaluación de la eficiencia de la PCR……………………………... 62 3.2.5. Expresión diferencial……………………………………………………. 64 3.3. Resultados y Discusión………………………………………………………… 64 3.3.1. Inmunohistoquímica…………………………………………………….. 64 3.3.1.1. Ratones inoculados con DENV por dos vías distintas de infección64 3.3.1.2. Inmunorreactividad del glutamato en la corteza motora de ratones infectados con DENV-4……………………………………………………………. 65 3.3.1.3. Inmunorreactividad del glutamato en la corteza somatosensorial de ratones infectados con DENV-4……………………………………………………. 66 3.3.1.4. Relación de la distribución de glutamato en la corteza motora y somatosensorial por las dos vías de inoculación…………………………………… 67 3.3.2 Expresión diferencial de la subunidad GluN2A en la corteza cerebral…..73 3.3.2.1. Presencia o ausencia de virus DENV-4 en las muestras de tejido cerebral……………………………………………………………………………... 73 3.3.2.2. Eficiencia de la PCR-validación……………………………………. 73 3.3.2.3. Expresión diferencial……………………………………………….. 73 3.4. Conclusión…………………………………………………………………….. 76 3.5. Referencias…………………………………………………………………….. 77spa
dc.format.mimetypeapplication/pdfspa
dc.language.isospaspa
dc.rightsDerechos Reservados - Universidad Militar Nueva Granada, 2019spa
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/2.5/co/spa
dc.titleAnálisis del efecto de la infección por virus dengue neuroadaptado sobre la inmunorreactividad de glutamato y la expresión de la subunidad GLUN2A del receptor de NMDAspa
dc.typeinfo:eu-repo/semantics/bachelorThesisspa
dc.rights.accessrightsinfo:eu-repo/semantics/openAccessspa
dc.subject.lembDENGUE - PREVENCION Y CONTROLspa
dc.subject.lembINFECCIONESspa
dc.subject.lembGLUTAMATOspa
dc.publisher.departmentFacultad de Ciencias Básicasspa
dc.type.localTrabajo de gradospa
dc.description.abstractenglishDengue virus has four serotypes and causes the known disease with the same name as the virus. This virus belongs to the Flavivirus genus and is transmitted to man through the bite of Aedes mosquitoes (albopictus and aegypti). This disease does not yet have a defined clinical picture, because the manifestations in most cases are asymptomatic. The main target cells of DENV infection are monocytes, macrophages and CD4 + and CD8 + lymphocytes. In addition, in vitro infection of endothelial cells, several hepatic, fibroblastic and neuronal cell lines have been reported. Some reports indicate that DENV may have a neurotropic character, possibly associated with the viral genotype or host conditions, however, there is still no consensus in the scientific community about accepting this condition despite the growing number of cases where it has been evidenced the presence of the virus in the CNS accompanied by neurological manifestations, such as severe headache, vomiting in children, alterations in consciousness, seizures, Guillain-Barre syndrome and transverse myelitis. Different receptors have been postulated as viral entry targets such as DC-SIGN, heparan sulfate, integrins, among others, however, the receptors that could participate in the entry and replication of dengue virus in the CNS are largely unknown. NMDA type glutamate receptors could contribute to DENV neuroinfection, because it has been shown that the use of receptor antagonists such as MK-801 and ketamine inhibit viral replication, so it is necessary to propose study models to respond to understanding of the glutamatergic system during the encephalic form of dengue associated with viral presence. In this work, the effect of a neuroadapted DENV-IV virus on the expression of the GLUN2A subunit of the NMDA receptor and the immunoreactivity of the glutamate neurotransmitter in the cortex of BALB / c mice infected by two different pathways was evaluated. In addition, the molecular coupling and interaction dynamics between the GluN2A-NMDA subunit and the envelope protein E of the dengue virus were evaluated by in silico assays. The results indicated that during the DENV infection the immunoreactivity of the glutamate neurotransmitter is not affected, but there is a deregulation of the GLUN2A subunit expression and a high coupling between this GluN2A subunit and the envelope protein E that can be maintained over time. This is the first report of the role of these elements of the glutamatergic system during dengue neuroinfection and approaches to the dynamics of molecular interaction between viral proteins and NMDA receptors. The results of this work are presented divided as follows: Theoretical framework (review article), objectives, assays result in the form of two scientific articles, one in silico assays and another in vivo assays. The conclusions will appear at the end of the document, in response to each of the objectives set. The foregoing in order to comply with the provisions of the Faculty of Basic and Applied Sciences of the University Militar Nueva Granada to promote the generation of scientific publications.eng
dc.title.translatedAnalysis of the effect of neuroadapted dengue virus infection on glutamate immuno-performance and expression of the GLUN2A subunity of the NMDA receptorspa
dc.subject.keywordsDenguespa
dc.subject.keywordsinfectiónspa
dc.subject.keywordsneurotropicspa
dc.subject.keywordsserotypesspa
dc.subject.keywordspathogenesisspa
dc.subject.keywordsglutamatespa
dc.subject.keywordsimmunoreactivityspa
dc.publisher.programBiología Aplicadaspa
dc.creator.degreenameBiólogospa
dc.description.degreelevelPregradospa
dc.publisher.facultyCiencias Básicas - Biología Aplicadaspa
dc.type.dcmi-type-vocabularyTextspa
dc.type.versioninfo:eu-repo/semantics/acceptedVersionspa
dc.rights.creativecommonsAtribución-NoComercial-SinDerivadasspa
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dc.subject.proposalDenguespa
dc.subject.proposalinfecciónspa
dc.subject.proposalneurotrópicospa
dc.subject.proposalserotiposspa
dc.subject.proposalpatogénesisspa
dc.subject.proposalInmunorreactividadspa
dc.subject.proposalglutamatospa
dc.publisher.grantorUniversidad Militar Nueva Granadaspa


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