Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli

Digital

Autores:
Peña-Niño, Juan Diego
Tipo de recurso:
Trabajo de grado de pregrado
Fecha de publicación:
2023
Institución:
Universidad de Santander
Repositorio:
Repositorio Universidad de Santander
Idioma:
spa
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oai:repositorio.udes.edu.co:001/10459
Acceso en línea:
https://repositorio.udes.edu.co
https://repositorio.udes.edu.co/handle/001/10459
Palabra clave:
Escherichia coli
Péptidos Antimicrobianos
Mecanismos de Acción
Resistencia Antimicrobiana
Docking Molecular
Ensayo de Cambio de la Movilidad Electroforética
Escherichia coli
Antimicrobial Peptides
Mechanisms Of Action
Antimicrobial Resistance
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id RUDES2_0fbdd522d36f292afcdedea35448b3ba
oai_identifier_str oai:repositorio.udes.edu.co:001/10459
network_acronym_str RUDES2
network_name_str Repositorio Universidad de Santander
repository_id_str
dc.title.spa.fl_str_mv Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
dc.title.translated.none.fl_str_mv Evaluation of the Interaction of Ib-M1 and Ib-M2 Peptides With Escherichia coli Nucleic Acids
title Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
spellingShingle Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
Escherichia coli
Péptidos Antimicrobianos
Mecanismos de Acción
Resistencia Antimicrobiana
Docking Molecular
Ensayo de Cambio de la Movilidad Electroforética
Escherichia coli
Antimicrobial Peptides
Mechanisms Of Action
Antimicrobial Resistance
title_short Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
title_full Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
title_fullStr Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
title_full_unstemmed Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
title_sort Evaluación de la Interacción de los Péptidos Ib-M1 e Ib-M2 con los Ácidos Nucleicos de Escherichia coli
dc.creator.fl_str_mv Peña-Niño, Juan Diego
dc.contributor.advisor.none.fl_str_mv Farfán-Garcia, Ana Elvira
Alarcon-Aldana, Juan Sebastian
dc.contributor.author.none.fl_str_mv Peña-Niño, Juan Diego
dc.contributor.jury.none.fl_str_mv Arias-Guerrero, Monica Yurley
Osorio-Alvarado, Carlos Enrique
dc.contributor.researchgroup.none.fl_str_mv Nova Ciencia
dc.subject.proposal.spa.fl_str_mv Escherichia coli
Péptidos Antimicrobianos
Mecanismos de Acción
Resistencia Antimicrobiana
Docking Molecular
Ensayo de Cambio de la Movilidad Electroforética
topic Escherichia coli
Péptidos Antimicrobianos
Mecanismos de Acción
Resistencia Antimicrobiana
Docking Molecular
Ensayo de Cambio de la Movilidad Electroforética
Escherichia coli
Antimicrobial Peptides
Mechanisms Of Action
Antimicrobial Resistance
dc.subject.proposal.eng.fl_str_mv Escherichia coli
Antimicrobial Peptides
Mechanisms Of Action
Antimicrobial Resistance
description Digital
publishDate 2023
dc.date.issued.none.fl_str_mv 2023-11-08
dc.date.accessioned.none.fl_str_mv 2024-05-29T20:41:43Z
dc.date.available.none.fl_str_mv 2024-05-29T20:41:43Z
2025-11-15
dc.type.none.fl_str_mv Trabajo de grado - Pregrado
dc.type.coar.none.fl_str_mv http://purl.org/coar/resource_type/c_7a1f
dc.type.coarversion.none.fl_str_mv http://purl.org/coar/version/c_71e4c1898caa6e32
dc.type.content.none.fl_str_mv Text
dc.type.driver.none.fl_str_mv info:eu-repo/semantics/bachelorThesis
dc.type.version.none.fl_str_mv info:eu-repo/semantics/submittedVersion
format http://purl.org/coar/resource_type/c_7a1f
status_str submittedVersion
dc.identifier.instname.none.fl_str_mv Universidad de Santander
dc.identifier.local.none.fl_str_mv T 17.23 P261e
dc.identifier.reponame.none.fl_str_mv Repositorio Digital Universidad de Santander
dc.identifier.repourl.none.fl_str_mv https://repositorio.udes.edu.co
dc.identifier.uri.none.fl_str_mv https://repositorio.udes.edu.co/handle/001/10459
identifier_str_mv Universidad de Santander
T 17.23 P261e
Repositorio Digital Universidad de Santander
url https://repositorio.udes.edu.co
https://repositorio.udes.edu.co/handle/001/10459
dc.language.iso.none.fl_str_mv spa
language spa
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spelling Farfán-Garcia, Ana Elviraf8af4974-ba16-439c-8b0b-9a876f9b4330-1Alarcon-Aldana, Juan Sebastiand6e7f697-4d9e-49b1-ae04-2d893e99cf25-1Peña-Niño, Juan Diegoa1c46f40-c8cd-47d1-b8e6-7e5a0fa578a5-1Arias-Guerrero, Monica Yurley220d3f91-dab8-4bfe-966e-89587451af41-1Osorio-Alvarado, Carlos Enrique957fcf19-3936-442e-8af3-42aa16b3e1d9-1Nova Ciencia2024-05-29T20:41:43Z2025-11-152024-05-29T20:41:43Z2023-11-08DigitalIntroducción: En los últimos años, se ha visto un rápido aumento de la resistencia a los antimicrobianos, lo que ha generado demoras en tratamientos hospitalarios, aumento de la mortalidad y el desarrollo de superbacterias resistente a muchos antibióticos, por la cual se ha incrementado la búsqueda de nuevas alternativas terapéuticas para disminuir o eliminar este problema. En este sentido, los péptidos antimicrobianos, son moléculas formadas por residuos de aminoácidos, que tiene la capacidad de interactuar con la membrana lipídica de las bacterias y algunos tiene la capacidad de interactuar con mecanismos intracelulares. Metodología: Se realizó la extracción de los ácidos nucleicos (ADN y ARN) de E. coli ATCCC25922, mediante kits comerciales. Para los ensayos de cambio en la movilidad electroforética (EMSA) se usaron concentraciones de los péptidos en el orden μM y fijas de ADN o ARN. La interacción entre péptidos y ácidos nucleicos se realizo en tres tiempos de exposición (30, 60 y 90 minutos). Se usó el péptido Buforina II como control positivo. Luego del tratamiento, las interacciones se visualizaron mediante electroforesis en geles de agarosa. Para estimar los sitios de interacción, entre el material genético y los péptidos se realizó un docking molecular in silico en diferentes plataformas bioinformáticas. Conclusiones: Se observó retardo en la movilidad electroforética del ADN y ARN en los tiempo evaluados y esta interacción fue dosis-dependiente comparada con el control Buforina II. In silico, los péptidos interaccionan en común con los aminoácidos Arginina, Glicina, sin embargo el péptido Ib-M2, genera otras interacciones con los aminoácidos Triptófano, Glutamina generando un mayor desempeño a comparación del péptido Ib-M1.Introduction: In recent years, there has been a quick increase in antimicrobial resistance, leading to delays in hospital's treatments, increased mortality, and the development of superbugs resistant to many antibiotics. This has prompted the search for new therapeutic alternatives to reduce or eliminate this problem. One potential candidate is antimicrobial peptides, which are molecules composed of amino acid residues that have the ability to interact with the lipid membrane of bacteria, and some can also interact with intracellular mechanisms. Methodology: The genetic material (DNA/RNA) of E. coli ATCCC25922 was extracted using commercial kits. It was then used to interact with the antimicrobial peptides Ib-M1 and Ib-M2 through an electrophoretic mobility shift assay (EMSA) at the in vitro level. To estimate the interaction sites between the genetic material and peptides, a molecular docking was performed in silico through various bioinformatics platforms. Conclusions: A considerable delay was observed in the electrophoretic mobility of DNA and RNA at the evaluated times, and this interaction was dose-dependent compared to the Buforina II control. In silico the peptides commonly interact with the amino acids Arginine, Glycine. However, the Ib-M2 peptide generates other interactions with the amino acids Tryptophan, Glutamine, resulting in better performance compared to the Ib-M1 peptide.PregradoBacteriólogo(a) y Laboratorista ClínicoIntroducción 23 Planteamiento del Problema 28 Justificación 34 Objetivos 36 Objetivo General 36 Objetivos Específicos 36 Marco Referencial 37 Estado del Arte 37 Marco Conceptual 41 Escherichia coli 41 Resistencia Antibacteriana 44 Péptidos Antimicrobianos (AMPs) 47 Fuentes de los AMPs 47 Clasificación de los AMPs 50 Mecanismos de Acción de los AMPs 55 Mecanismos de Acción de AMPs no Líticos 57 Docking Molecular Péptido-DNA, Péptido-RNA 62 Metodología 65 Tipo de Estudio 65 Área de Estudio 65 Variables e Hipótesis 65 Procedimientos 66 Material Biológico 66 Péptidos y Reactivos 67 Extracción de ADN 68 Extracción de RNA 69 Ensayo de Cambio de la Movilidad Electroforética (Electrophoretic Mobility Shift Assay -EMSA) de los Ácidos Nucleicos (ADN y ARN) Tratados con Péptidos Ib-M 70 EMSA del ADN Genómico Bacteriano 70 EMSA del ARN Bacteriano 71 Docking Molecular 71 Análisis de Datos 76 Aspectos Éticos 76 Resultados 78 Extracción de ADN Genómico de E. coli 78 EMSA del ADN Genómico de E. coli ATCC 25922 Tratado con Péptidos Ib-M1 e Ib-M2 79 Extracción de ARN 81 EMSA del ARN de E. coli ATCC 25922 y los Péptidos Ib-M1 e Ib-M2. 82 Docking Molecular (Acople Molecular) 86 Modelamiento del Péptido Ib-M1 86 Modelamiento del Péptido Ib-M2 88 Acople Molecular del Péptido Ib-M1 con la Estructura de un B-DNA Dodecámero 90 Acople Molecular Entre Ib-M2 con la Estructura de un B-DNA Dodecámero 92 Acople Molecular Entre el Péptido Ib-M1 con Subunidad Ribosómica 5 S de E. coli 94 Interacción del Péptido Ib-M2 con la Subunidad Ribosómica 5 S de E. coli 96 Discusión 99 Conclusión 106 Recomendaciones 108 Referencias Bibliografía 109 Apéndices 129134 papplication/pdfapplication/mswordUniversidad de SantanderT 17.23 P261eRepositorio Digital Universidad de Santanderhttps://repositorio.udes.edu.cohttps://repositorio.udes.edu.co/handle/001/10459spaUniversidad de SantanderBucaramangaFacultad de Ciencias Médicas y de la SaludBucaramanga, ColombiaBacteriología y Laboratorio ClínicoPulingam T, Parumasivam T, Gazzali AM, Sulaiman AM, Chee JY, Lakshmanan M, Chin CF, Sudesh K. 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GF1dG9yKSBwYXJhIGVqZXJjZXIgZXN0b3MgZGVyZWNob3Mgc29icmUgbGEgT2JyYSB0YWwgeSBjb21vIHNlIGluZGljYSBhIGNvbnRpbnVhY2nDs246PC9wPgogICAgPG9sIHR5cGU9ImEiPgogICAgICA8bGk+UmVwcm9kdWNpciBsYSBPYnJhLCBpbmNvcnBvcmFyIGxhIE9icmEgZW4gdW5hIG8gbcOhcyBPYnJhcyBDb2xlY3RpdmFzLCB5IHJlcHJvZHVjaXIgbGEgT2JyYSBpbmNvcnBvcmFkYSBlbiBsYXMgT2JyYXMgQ29sZWN0aXZhcy48L2xpPgogICAgICA8bGk+RGlzdHJpYnVpciBjb3BpYXMgbyBmb25vZ3JhbWFzIGRlIGxhcyBPYnJhcywgZXhoaWJpcmxhcyBww7pibGljYW1lbnRlLCBlamVjdXRhcmxhcyBww7pibGljYW1lbnRlIHkvbyBwb25lcmxhcyBhIGRpc3Bvc2ljacOzbiBww7pibGljYSwgaW5jbHV5w6luZG9sYXMgY29tbyBpbmNvcnBvcmFkYXMgZW4gT2JyYXMgQ29sZWN0aXZhcywgc2Vnw7puIGNvcnJlc3BvbmRhLjwvbGk+CiAgICAgIDxsaT5EaXN0cmlidWlyIGNvcGlhcyBkZSBsYXMgT2JyYXMgRGVyaXZhZGFzIHF1ZSBzZSBnZW5lcmVuLCBleGhpYmlybGFzIHDDumJsaWNhbWVudGUsIGVqZWN1dGFybGFzIHDDumJsaWNhbWVudGUgeS9vIHBvbmVybGFzIGEgZGlzcG9zaWNpw7NuIHDDumJsaWNhLjwvbGk+CiAgICA8L29sPgogICAgPHA+TG9zIGRlcmVjaG9zIG1lbmNpb25hZG9zIGFudGVyaW9ybWVudGUgcHVlZGVuIHNlciBlamVyY2lkb3MgZW4gdG9kb3MgbG9zIG1lZGlvcyB5IGZvcm1hdG9zLCBhY3R1YWxtZW50ZSBjb25vY2lkb3MgbyBxdWUgc2UgaW52ZW50ZW4gZW4gZWwgZnV0dXJvLiBMb3MgZGVyZWNob3MgYW50ZXMgbWVuY2lvbmFkb3MgaW5jbHV5ZW4gZWwgZGVyZWNobyBhIHJlYWxpemFyIGRpY2hhcyBtb2RpZmljYWNpb25lcyBlbiBsYSBtZWRpZGEgcXVlIHNlYW4gdMOpY25pY2FtZW50ZSBuZWNlc2FyaWFzIHBhcmEgZWplcmNlciBsb3MgZGVyZWNob3MgZW4gb3RybyBtZWRpbyBvIGZvcm1hdG9zLCBwZXJvIGRlIG90cmEgbWFuZXJhIHVzdGVkIG5vIGVzdMOhIGF1dG9yaXphZG8gcGFyYSByZWFsaXphciBvYnJhcyBkZXJpdmFkYXMuIFRvZG9zIGxvcyBkZXJlY2hvcyBubyBvdG9yZ2Fkb3MgZXhwcmVzYW1lbnRlIHBvciBlbCBMaWNlbmNpYW50ZSBxdWVkYW4gcG9yIGVzdGUgbWVkaW8gcmVzZXJ2YWRvcywgaW5jbHV5ZW5kbyBwZXJvIHNpbiBsaW1pdGFyc2UgYSBhcXVlbGxvcyBxdWUgc2UgbWVuY2lvbmFuIGVuIGxhcyBzZWNjaW9uZXMgNChkKSB5IDQoZSkuPC9wPgogIDwvbGk+CiAgPGJyLz4KICA8bGk+CiAgICBSZXN0cmljY2lvbmVzLgogICAgPHA+TGEgbGljZW5jaWEgb3RvcmdhZGEgZW4gbGEgYW50ZXJpb3IgU2VjY2nDs24gMyBlc3TDoSBleHByZXNhbWVudGUgc3VqZXRhIHkgbGltaXRhZGEgcG9yIGxhcyBzaWd1aWVudGVzIHJlc3RyaWNjaW9uZXM6PC9wPgogICAgPG9sIHR5cGU9ImEiPgogICAgICA8bGk+VXN0ZWQgcHVlZGUgZGlzdHJpYnVpciwgZXhoaWJpciBww7pibGljYW1lbnRlLCBlamVjdXRhciBww7pibGljYW1lbnRlLCBvIHBvbmVyIGEgZGlzcG9zaWNpw7NuIHDDumJsaWNhIGxhIE9icmEgc8OzbG8gYmFqbyBsYXMgY29uZGljaW9uZXMgZGUgZXN0YSBMaWNlbmNpYSwgeSBVc3RlZCBkZWJlIGluY2x1aXIgdW5hIGNvcGlhIGRlIGVzdGEgbGljZW5jaWEgbyBkZWwgSWRlbnRpZmljYWRvciBVbml2ZXJzYWwgZGUgUmVjdXJzb3MgZGUgbGEgbWlzbWEgY29uIGNhZGEgY29waWEgZGUgbGEgT2JyYSBxdWUgZGlzdHJpYnV5YSwgZXhoaWJhIHDDumJsaWNhbWVudGUsIGVqZWN1dGUgcMO6YmxpY2FtZW50ZSBvIHBvbmdhIGEgZGlzcG9zaWNpw7NuIHDDumJsaWNhLiBObyBlcyBwb3NpYmxlIG9mcmVjZXIgbyBpbXBvbmVyIG5pbmd1bmEgY29uZGljacOzbiBzb2JyZSBsYSBPYnJhIHF1ZSBhbHRlcmUgbyBsaW1pdGUgbGFzIGNvbmRpY2lvbmVzIGRlIGVzdGEgTGljZW5jaWEgbyBlbCBlamVyY2ljaW8gZGUgbG9zIGRlcmVjaG9zIGRlIGxvcyBkZXN0aW5hdGFyaW9zIG90b3JnYWRvcyBlbiBlc3RlIGRvY3VtZW50by4gTm8gZXMgcG9zaWJsZSBzdWJsaWNlbmNpYXIgbGEgT2JyYS4gVXN0ZWQgZGViZSBtYW50ZW5lciBpbnRhY3RvcyB0b2RvcyBsb3MgYXZpc29zIHF1ZSBoYWdhbiByZWZlcmVuY2lhIGEgZXN0YSBMaWNlbmNpYSB5IGEgbGEgY2zDoXVzdWxhIGRlIGxpbWl0YWNpw7NuIGRlIGdhcmFudMOtYXMuIFVzdGVkIG5vIHB1ZWRlIGRpc3RyaWJ1aXIsIGV4aGliaXIgcMO6YmxpY2FtZW50ZSwgZWplY3V0YXIgcMO6YmxpY2FtZW50ZSwgbyBwb25lciBhIGRpc3Bvc2ljacOzbiBww7pibGljYSBsYSBPYnJhIGNvbiBhbGd1bmEgbWVkaWRhIHRlY25vbMOzZ2ljYSBxdWUgY29udHJvbGUgZWwgYWNjZXNvIG8gbGEgdXRpbGl6YWNpw7NuIGRlIGVsbGEgZGUgdW5hIGZvcm1hIHF1ZSBzZWEgaW5jb25zaXN0ZW50ZSBjb24gbGFzIGNvbmRpY2lvbmVzIGRlIGVzdGEgTGljZW5jaWEuIExvIGFudGVyaW9yIHNlIGFwbGljYSBhIGxhIE9icmEgaW5jb3Jwb3JhZGEgYSB1bmEgT2JyYSBDb2xlY3RpdmEsIHBlcm8gZXN0byBubyBleGlnZSBxdWUgbGEgT2JyYSBDb2xlY3RpdmEgYXBhcnRlIGRlIGxhIG9icmEgbWlzbWEgcXVlZGUgc3VqZXRhIGEgbGFzIGNvbmRpY2lvbmVzIGRlIGVzdGEgTGljZW5jaWEuIFNpIFVzdGVkIGNyZWEgdW5hIE9icmEgQ29sZWN0aXZhLCBwcmV2aW8gYXZpc28gZGUgY3VhbHF1aWVyIExpY2VuY2lhbnRlIGRlYmUsIGVuIGxhIG1lZGlkYSBkZSBsbyBwb3NpYmxlLCBlbGltaW5hciBkZSBsYSBPYnJhIENvbGVjdGl2YSBjdWFscXVpZXIgcmVmZXJlbmNpYSBhIGRpY2hvIExpY2VuY2lhbnRlIG8gYWwgQXV0b3IgT3JpZ2luYWwsIHNlZ8O6biBsbyBzb2xpY2l0YWRvIHBvciBlbCBMaWNlbmNpYW50ZSB5IGNvbmZvcm1lIGxvIGV4aWdlIGxhIGNsw6F1c3VsYSA0KGMpLjwvbGk+CiAgICAgIDxsaT5Vc3RlZCBubyBwdWVkZSBlamVyY2VyIG5pbmd1bm8gZGUgbG9zIGRlcmVjaG9zIHF1ZSBsZSBoYW4gc2lkbyBvdG9yZ2Fkb3MgZW4gbGEgU2VjY2nDs24gMyBwcmVjZWRlbnRlIGRlIG1vZG8gcXVlIGVzdMOpbiBwcmluY2lwYWxtZW50ZSBkZXN0aW5hZG9zIG8gZGlyZWN0YW1lbnRlIGRpcmlnaWRvcyBhIGNvbnNlZ3VpciB1biBwcm92ZWNobyBjb21lcmNpYWwgbyB1bmEgY29tcGVuc2FjacOzbiBtb25ldGFyaWEgcHJpdmFkYS4gRWwgaW50ZXJjYW1iaW8gZGUgbGEgT2JyYSBwb3Igb3RyYXMgb2JyYXMgcHJvdGVnaWRhcyBwb3IgZGVyZWNob3MgZGUgYXV0b3IsIHlhIHNlYSBhIHRyYXbDqXMgZGUgdW4gc2lzdGVtYSBwYXJhIGNvbXBhcnRpciBhcmNoaXZvcyBkaWdpdGFsZXMgKGRpZ2l0YWwgZmlsZS1zaGFyaW5nKSBvIGRlIGN1YWxxdWllciBvdHJhIG1hbmVyYSBubyBzZXLDoSBjb25zaWRlcmFkbyBjb21vIGVzdGFyIGRlc3RpbmFkbyBwcmluY2lwYWxtZW50ZSBvIGRpcmlnaWRvIGRpcmVjdGFtZW50ZSBhIGNvbnNlZ3VpciB1biBwcm92ZWNobyBjb21lcmNpYWwgbyB1bmEgY29tcGVuc2FjacOzbiBtb25ldGFyaWEgcHJpdmFkYSwgc2llbXByZSBxdWUgbm8gc2UgcmVhbGljZSB1biBwYWdvIG1lZGlhbnRlIHVuYSBjb21wZW5zYWNpw7NuIG1vbmV0YXJpYSBlbiByZWxhY2nDs24gY29uIGVsIGludGVyY2FtYmlvIGRlIG9icmFzIHByb3RlZ2lkYXMgcG9yIGVsIGRlcmVjaG8gZGUgYXV0b3IuPC9saT4KICAgICAgPGxpPlNpIHVzdGVkIGRpc3RyaWJ1eWUsIGV4aGliZSBww7pibGljYW1lbnRlLCBlamVjdXRhIHDDumJsaWNhbWVudGUgbyBlamVjdXRhIHDDumJsaWNhbWVudGUgZW4gZm9ybWEgZGlnaXRhbCBsYSBPYnJhIG8gY3VhbHF1aWVyIE9icmEgRGVyaXZhZGEgdSBPYnJhIENvbGVjdGl2YSwgVXN0ZWQgZGViZSBtYW50ZW5lciBpbnRhY3RhIHRvZGEgbGEgaW5mb3JtYWNpw7NuIGRlIGRlcmVjaG8gZGUgYXV0b3IgZGUgbGEgT2JyYSB5IHByb3BvcmNpb25hciwgZGUgZm9ybWEgcmF6b25hYmxlIHNlZ8O6biBlbCBtZWRpbyBvIG1hbmVyYSBxdWUgVXN0ZWQgZXN0w6kgdXRpbGl6YW5kbzogKGkpIGVsIG5vbWJyZSBkZWwgQXV0b3IgT3JpZ2luYWwgc2kgZXN0w6EgcHJvdmlzdG8gKG8gc2V1ZMOzbmltbywgc2kgZnVlcmUgYXBsaWNhYmxlKSwgeS9vIChpaSkgZWwgbm9tYnJlIGRlIGxhIHBhcnRlIG8gbGFzIHBhcnRlcyBxdWUgZWwgQXV0b3IgT3JpZ2luYWwgeS9vIGVsIExpY2VuY2lhbnRlIGh1YmllcmVuIGRlc2lnbmFkbyBwYXJhIGxhIGF0cmlidWNpw7NuICh2LmcuLCB1biBpbnN0aXR1dG8gcGF0cm9jaW5hZG9yLCBlZGl0b3JpYWwsIHB1YmxpY2FjacOzbikgZW4gbGEgaW5mb3JtYWNpw7NuIGRlIGxvcyBkZXJlY2hvcyBkZSBhdXRvciBkZWwgTGljZW5jaWFudGUsIHTDqXJtaW5vcyBkZSBzZXJ2aWNpb3MgbyBkZSBvdHJhcyBmb3JtYXMgcmF6b25hYmxlczsgZWwgdMOtdHVsbyBkZSBsYSBPYnJhIHNpIGVzdMOhIHByb3Zpc3RvOyBlbiBsYSBtZWRpZGEgZGUgbG8gcmF6b25hYmxlbWVudGUgZmFjdGlibGUgeSwgc2kgZXN0w6EgcHJvdmlzdG8sIGVsIElkZW50aWZpY2Fkb3IgVW5pZm9ybWUgZGUgUmVjdXJzb3MgKFVuaWZvcm0gUmVzb3VyY2UgSWRlbnRpZmllcikgcXVlIGVsIExpY2VuY2lhbnRlIGVzcGVjaWZpY2EgcGFyYSBzZXIgYXNvY2lhZG8gY29uIGxhIE9icmEsIHNhbHZvIHF1ZSB0YWwgVVJJIG5vIHNlIHJlZmllcmEgYSBsYSBub3RhIHNvYnJlIGxvcyBkZXJlY2hvcyBkZSBhdXRvciBvIGEgbGEgaW5mb3JtYWNpw7NuIHNvYnJlIGVsIGxpY2VuY2lhbWllbnRvIGRlIGxhIE9icmE7IHkgZW4gZWwgY2FzbyBkZSB1bmEgT2JyYSBEZXJpdmFkYSwgYXRyaWJ1aXIgZWwgY3LDqWRpdG8gaWRlbnRpZmljYW5kbyBlbCB1c28gZGUgbGEgT2JyYSBlbiBsYSBPYnJhIERlcml2YWRhICh2LmcuLCAiVHJhZHVjY2nDs24gRnJhbmNlc2EgZGUgbGEgT2JyYSBkZWwgQXV0b3IgT3JpZ2luYWwsIiBvICJHdWnDs24gQ2luZW1hdG9ncsOhZmljbyBiYXNhZG8gZW4gbGEgT2JyYSBvcmlnaW5hbCBkZWwgQXV0b3IgT3JpZ2luYWwiKS4gVGFsIGNyw6lkaXRvIHB1ZWRlIHNlciBpbXBsZW1lbnRhZG8gZGUgY3VhbHF1aWVyIGZvcm1hIHJhem9uYWJsZTsgZW4gZWwgY2Fzbywgc2luIGVtYmFyZ28sIGRlIE9icmFzIERlcml2YWRhcyB1IE9icmFzIENvbGVjdGl2YXMsIHRhbCBjcsOpZGl0byBhcGFyZWNlcsOhLCBjb21vIG3DrW5pbW8sIGRvbmRlIGFwYXJlY2UgZWwgY3LDqWRpdG8gZGUgY3VhbHF1aWVyIG90cm8gYXV0b3IgY29tcGFyYWJsZSB5IGRlIHVuYSBtYW5lcmEsIGFsIG1lbm9zLCB0YW4gZGVzdGFjYWRhIGNvbW8gZWwgY3LDqWRpdG8gZGUgb3RybyBhdXRvciBjb21wYXJhYmxlLjwvbGk+CiAgICAgIDxsaT4KICAgICAgICBQYXJhIGV2aXRhciB0b2RhIGNvbmZ1c2nDs24sIGVsIExpY2VuY2lhbnRlIGFjbGFyYSBxdWUsIGN1YW5kbyBsYSBvYnJhIGVzIHVuYSBjb21wb3NpY2nDs24gbXVzaWNhbDoKICAgICAgICA8b2wgdHlwZT0iaSI+CiAgICAgICAgICA8bGk+UmVnYWzDrWFzIHBvciBpbnRlcnByZXRhY2nDs24geSBlamVjdWNpw7NuIGJham8gbGljZW5jaWFzIGdlbmVyYWxlcy4gRWwgTGljZW5jaWFudGUgc2UgcmVzZXJ2YSBlbCBkZXJlY2hvIGV4Y2x1c2l2byBkZSBhdXRvcml6YXIgbGEgZWplY3VjacOzbiBww7pibGljYSBvIGxhIGVqZWN1Y2nDs24gcMO6YmxpY2EgZGlnaXRhbCBkZSBsYSBvYnJhIHkgZGUgcmVjb2xlY3Rhciwgc2VhIGluZGl2aWR1YWxtZW50ZSBvIGEgdHJhdsOpcyBkZSB1bmEgc29jaWVkYWQgZGUgZ2VzdGnDs24gY29sZWN0aXZhIGRlIGRlcmVjaG9zIGRlIGF1dG9yIHkgZGVyZWNob3MgY29uZXhvcyAocG9yIGVqZW1wbG8sIFNBWUNPKSwgbGFzIHJlZ2Fsw61hcyBwb3IgbGEgZWplY3VjacOzbiBww7pibGljYSBvIHBvciBsYSBlamVjdWNpw7NuIHDDumJsaWNhIGRpZ2l0YWwgZGUgbGEgb2JyYSAocG9yIGVqZW1wbG8gV2ViY2FzdCkgbGljZW5jaWFkYSBiYWpvIGxpY2VuY2lhcyBnZW5lcmFsZXMsIHNpIGxhIGludGVycHJldGFjacOzbiBvIGVqZWN1Y2nDs24gZGUgbGEgb2JyYSBlc3TDoSBwcmltb3JkaWFsbWVudGUgb3JpZW50YWRhIHBvciBvIGRpcmlnaWRhIGEgbGEgb2J0ZW5jacOzbiBkZSB1bmEgdmVudGFqYSBjb21lcmNpYWwgbyB1bmEgY29tcGVuc2FjacOzbiBtb25ldGFyaWEgcHJpdmFkYS48L2xpPgogICAgICAgICAgPGxpPlJlZ2Fsw61hcyBwb3IgRm9ub2dyYW1hcy4gRWwgTGljZW5jaWFudGUgc2UgcmVzZXJ2YSBlbCBkZXJlY2hvIGV4Y2x1c2l2byBkZSByZWNvbGVjdGFyLCBpbmRpdmlkdWFsbWVudGUgbyBhIHRyYXbDqXMgZGUgdW5hIHNvY2llZGFkIGRlIGdlc3Rpw7NuIGNvbGVjdGl2YSBkZSBkZXJlY2hvcyBkZSBhdXRvciB5IGRlcmVjaG9zIGNvbmV4b3MgKHBvciBlamVtcGxvLCBsb3MgY29uc2FncmFkb3MgcG9yIGxhIFNBWUNPKSwgdW5hIGFnZW5jaWEgZGUgZGVyZWNob3MgbXVzaWNhbGVzIG8gYWxnw7puIGFnZW50ZSBkZXNpZ25hZG8sIGxhcyByZWdhbMOtYXMgcG9yIGN1YWxxdWllciBmb25vZ3JhbWEgcXVlIFVzdGVkIGNyZWUgYSBwYXJ0aXIgZGUgbGEgb2JyYSAo4oCcdmVyc2nDs24gY292ZXLigJ0pIHkgZGlzdHJpYnV5YSwgZW4gbG9zIHTDqXJtaW5vcyBkZWwgcsOpZ2ltZW4gZGUgZGVyZWNob3MgZGUgYXV0b3IsIHNpIGxhIGNyZWFjacOzbiBvIGRpc3RyaWJ1Y2nDs24gZGUgZXNhIHZlcnNpw7NuIGNvdmVyIGVzdMOhIHByaW1vcmRpYWxtZW50ZSBkZXN0aW5hZGEgbyBkaXJpZ2lkYSBhIG9idGVuZXIgdW5hIHZlbnRhamEgY29tZXJjaWFsIG8gdW5hIGNvbXBlbnNhY2nDs24gbW9uZXRhcmlhIHByaXZhZGEuPC9saT4KICAgICAgICA8L29sPgogICAgICA8L2xpPgogICAgICA8bGk+R2VzdGnDs24gZGUgRGVyZWNob3MgZGUgQXV0b3Igc29icmUgSW50ZXJwcmV0YWNpb25lcyB5IEVqZWN1Y2lvbmVzIERpZ2l0YWxlcyAoV2ViQ2FzdGluZykuIFBhcmEgZXZpdGFyIHRvZGEgY29uZnVzacOzbiwgZWwgTGljZW5jaWFudGUgYWNsYXJhIHF1ZSwgY3VhbmRvIGxhIG9icmEgc2VhIHVuIGZvbm9ncmFtYSwgZWwgTGljZW5jaWFudGUgc2UgcmVzZXJ2YSBlbCBkZXJlY2hvIGV4Y2x1c2l2byBkZSBhdXRvcml6YXIgbGEgZWplY3VjacOzbiBww7pibGljYSBkaWdpdGFsIGRlIGxhIG9icmEgKHBvciBlamVtcGxvLCB3ZWJjYXN0KSB5IGRlIHJlY29sZWN0YXIsIGluZGl2aWR1YWxtZW50ZSBvIGEgdHJhdsOpcyBkZSB1bmEgc29jaWVkYWQgZGUgZ2VzdGnDs24gY29sZWN0aXZhIGRlIGRlcmVjaG9zIGRlIGF1dG9yIHkgZGVyZWNob3MgY29uZXhvcyAocG9yIGVqZW1wbG8sIEFDSU5QUk8pLCBsYXMgcmVnYWzDrWFzIHBvciBsYSBlamVjdWNpw7NuIHDDumJsaWNhIGRpZ2l0YWwgZGUgbGEgb2JyYSAocG9yIGVqZW1wbG8sIHdlYmNhc3QpLCBzdWpldGEgYSBsYXMgZGlzcG9zaWNpb25lcyBhcGxpY2FibGVzIGRlbCByw6lnaW1lbiBkZSBEZXJlY2hvIGRlIEF1dG9yLCBzaSBlc3RhIGVqZWN1Y2nDs24gcMO6YmxpY2EgZGlnaXRhbCBlc3TDoSBwcmltb3JkaWFsbWVudGUgZGlyaWdpZGEgYSBvYnRlbmVyIHVuYSB2ZW50YWphIGNvbWVyY2lhbCBvIHVuYSBjb21wZW5zYWNpw7NuIG1vbmV0YXJpYSBwcml2YWRhLjwvbGk+CiAgICA8L29sPgogIDwvbGk+CiAgPGJyLz4KICA8bGk+CiAgICBSZXByZXNlbnRhY2lvbmVzLCBHYXJhbnTDrWFzIHkgTGltaXRhY2lvbmVzIGRlIFJlc3BvbnNhYmlsaWRhZC4KICAgIDxwPkEgTUVOT1MgUVVFIExBUyBQQVJURVMgTE8gQUNPUkRBUkFOIERFIE9UUkEgRk9STUEgUE9SIEVTQ1JJVE8sIEVMIExJQ0VOQ0lBTlRFIE9GUkVDRSBMQSBPQlJBIChFTiBFTCBFU1RBRE8gRU4gRUwgUVVFIFNFIEVOQ1VFTlRSQSkg4oCcVEFMIENVQUzigJ0sIFNJTiBCUklOREFSIEdBUkFOVMONQVMgREUgQ0xBU0UgQUxHVU5BIFJFU1BFQ1RPIERFIExBIE9CUkEsIFlBIFNFQSBFWFBSRVNBLCBJTVBMw41DSVRBLCBMRUdBTCBPIENVQUxRVUlFUkEgT1RSQSwgSU5DTFVZRU5ETywgU0lOIExJTUlUQVJTRSBBIEVMTEFTLCBHQVJBTlTDjUFTIERFIFRJVFVMQVJJREFELCBDT01FUkNJQUJJTElEQUQsIEFEQVBUQUJJTElEQUQgTyBBREVDVUFDScOTTiBBIFBST1DDk1NJVE8gREVURVJNSU5BRE8sIEFVU0VOQ0lBIERFIElORlJBQ0NJw5NOLCBERSBBVVNFTkNJQSBERSBERUZFQ1RPUyBMQVRFTlRFUyBPIERFIE9UUk8gVElQTywgTyBMQSBQUkVTRU5DSUEgTyBBVVNFTkNJQSBERSBFUlJPUkVTLCBTRUFOIE8gTk8gREVTQ1VCUklCTEVTIChQVUVEQU4gTyBOTyBTRVIgRVNUT1MgREVTQ1VCSUVSVE9TKS4gQUxHVU5BUyBKVVJJU0RJQ0NJT05FUyBOTyBQRVJNSVRFTiBMQSBFWENMVVNJw5NOIERFIEdBUkFOVMONQVMgSU1QTMONQ0lUQVMsIEVOIENVWU8gQ0FTTyBFU1RBIEVYQ0xVU0nDk04gUFVFREUgTk8gQVBMSUNBUlNFIEEgVVNURUQuPC9wPgogIDwvbGk+CiAgPGJyLz4KICA8bGk+CiAgICBMaW1pdGFjacOzbiBkZSByZXNwb25zYWJpbGlkYWQuCiAgICA8cD5BIE1FTk9TIFFVRSBMTyBFWElKQSBFWFBSRVNBTUVOVEUgTEEgTEVZIEFQTElDQUJMRSwgRUwgTElDRU5DSUFOVEUgTk8gU0VSw4EgUkVTUE9OU0FCTEUgQU5URSBVU1RFRCBQT1IgREHDkU8gQUxHVU5PLCBTRUEgUE9SIFJFU1BPTlNBQklMSURBRCBFWFRSQUNPTlRSQUNUVUFMLCBQUkVDT05UUkFDVFVBTCBPIENPTlRSQUNUVUFMLCBPQkpFVElWQSBPIFNVQkpFVElWQSwgU0UgVFJBVEUgREUgREHDkU9TIE1PUkFMRVMgTyBQQVRSSU1PTklBTEVTLCBESVJFQ1RPUyBPIElORElSRUNUT1MsIFBSRVZJU1RPUyBPIElNUFJFVklTVE9TIFBST0RVQ0lET1MgUE9SIEVMIFVTTyBERSBFU1RBIExJQ0VOQ0lBIE8gREUgTEEgT0JSQSwgQVVOIENVQU5ETyBFTCBMSUNFTkNJQU5URSBIQVlBIFNJRE8gQURWRVJUSURPIERFIExBIFBPU0lCSUxJREFEIERFIERJQ0hPUyBEQcORT1MuIEFMR1VOQVMgTEVZRVMgTk8gUEVSTUlURU4gTEEgRVhDTFVTScOTTiBERSBDSUVSVEEgUkVTUE9OU0FCSUxJREFELCBFTiBDVVlPIENBU08gRVNUQSBFWENMVVNJw5NOIFBVRURFIE5PIEFQTElDQVJTRSBBIFVTVEVELjwvcD4KICA8L2xpPgogIDxici8+CiAgPGxpPgogICAgVMOpcm1pbm8uCiAgICA8b2wgdHlwZT0iYSI+CiAgICAgIDxsaT5Fc3RhIExpY2VuY2lhIHkgbG9zIGRlcmVjaG9zIG90b3JnYWRvcyBlbiB2aXJ0dWQgZGUgZWxsYSB0ZXJtaW5hcsOhbiBhdXRvbcOhdGljYW1lbnRlIHNpIFVzdGVkIGluZnJpbmdlIGFsZ3VuYSBjb25kaWNpw7NuIGVzdGFibGVjaWRhIGVuIGVsbGEuIFNpbiBlbWJhcmdvLCBsb3MgaW5kaXZpZHVvcyBvIGVudGlkYWRlcyBxdWUgaGFuIHJlY2liaWRvIE9icmFzIERlcml2YWRhcyBvIENvbGVjdGl2YXMgZGUgVXN0ZWQgZGUgY29uZm9ybWlkYWQgY29uIGVzdGEgTGljZW5jaWEsIG5vIHZlcsOhbiB0ZXJtaW5hZGFzIHN1cyBsaWNlbmNpYXMsIHNpZW1wcmUgcXVlIGVzdG9zIGluZGl2aWR1b3MgbyBlbnRpZGFkZXMgc2lnYW4gY3VtcGxpZW5kbyDDrW50ZWdyYW1lbnRlIGxhcyBjb25kaWNpb25lcyBkZSBlc3RhcyBsaWNlbmNpYXMuIExhcyBTZWNjaW9uZXMgMSwgMiwgNSwgNiwgNywgeSA4IHN1YnNpc3RpcsOhbiBhIGN1YWxxdWllciB0ZXJtaW5hY2nDs24gZGUgZXN0YSBMaWNlbmNpYS48L2xpPgogICAgICA8bGk+U3VqZXRhIGEgbGFzIGNvbmRpY2lvbmVzIHkgdMOpcm1pbm9zIGFudGVyaW9yZXMsIGxhIGxpY2VuY2lhIG90b3JnYWRhIGFxdcOtIGVzIHBlcnBldHVhIChkdXJhbnRlIGVsIHBlcsOtb2RvIGRlIHZpZ2VuY2lhIGRlIGxvcyBkZXJlY2hvcyBkZSBhdXRvciBkZSBsYSBvYnJhKS4gTm8gb2JzdGFudGUgbG8gYW50ZXJpb3IsIGVsIExpY2VuY2lhbnRlIHNlIHJlc2VydmEgZWwgZGVyZWNobyBhIHB1YmxpY2FyIHkvbyBlc3RyZW5hciBsYSBPYnJhIGJham8gY29uZGljaW9uZXMgZGUgbGljZW5jaWEgZGlmZXJlbnRlcyBvIGEgZGVqYXIgZGUgZGlzdHJpYnVpcmxhIGVuIGxvcyB0w6lybWlub3MgZGUgZXN0YSBMaWNlbmNpYSBlbiBjdWFscXVpZXIgbW9tZW50bzsgZW4gZWwgZW50ZW5kaWRvLCBzaW4gZW1iYXJnbywgcXVlIGVzYSBlbGVjY2nDs24gbm8gc2Vydmlyw6EgcGFyYSByZXZvY2FyIGVzdGEgbGljZW5jaWEgbyBxdWUgZGViYSBzZXIgb3RvcmdhZGEgLCBiYWpvIGxvcyB0w6lybWlub3MgZGUgZXN0YSBsaWNlbmNpYSksIHkgZXN0YSBsaWNlbmNpYSBjb250aW51YXLDoSBlbiBwbGVubyB2aWdvciB5IGVmZWN0byBhIG1lbm9zIHF1ZSBzZWEgdGVybWluYWRhIGNvbW8gc2UgZXhwcmVzYSBhdHLDoXMuIExhIExpY2VuY2lhIHJldm9jYWRhIGNvbnRpbnVhcsOhIHNpZW5kbyBwbGVuYW1lbnRlIHZpZ2VudGUgeSBlZmVjdGl2YSBzaSBubyBzZSBsZSBkYSB0w6lybWlubyBlbiBsYXMgY29uZGljaW9uZXMgaW5kaWNhZGFzIGFudGVyaW9ybWVudGUuPC9saT4KICAgIDwvb2w+CiAgPC9saT4KICA8YnIvPgogIDxsaT4KICAgIFZhcmlvcy4KICAgIDxvbCB0eXBlPSJhIj4KICAgICAgPGxpPkNhZGEgdmV6IHF1ZSBVc3RlZCBkaXN0cmlidXlhIG8gcG9uZ2EgYSBkaXNwb3NpY2nDs24gcMO6YmxpY2EgbGEgT2JyYSBvIHVuYSBPYnJhIENvbGVjdGl2YSwgZWwgTGljZW5jaWFudGUgb2ZyZWNlcsOhIGFsIGRlc3RpbmF0YXJpbyB1bmEgbGljZW5jaWEgZW4gbG9zIG1pc21vcyB0w6lybWlub3MgeSBjb25kaWNpb25lcyBxdWUgbGEgbGljZW5jaWEgb3RvcmdhZGEgYSBVc3RlZCBiYWpvIGVzdGEgTGljZW5jaWEuPC9saT4KICAgICAgPGxpPlNpIGFsZ3VuYSBkaXNwb3NpY2nDs24gZGUgZXN0YSBMaWNlbmNpYSByZXN1bHRhIGludmFsaWRhZGEgbyBubyBleGlnaWJsZSwgc2Vnw7puIGxhIGxlZ2lzbGFjacOzbiB2aWdlbnRlLCBlc3RvIG5vIGFmZWN0YXLDoSBuaSBsYSB2YWxpZGV6IG5pIGxhIGFwbGljYWJpbGlkYWQgZGVsIHJlc3RvIGRlIGNvbmRpY2lvbmVzIGRlIGVzdGEgTGljZW5jaWEgeSwgc2luIGFjY2nDs24gYWRpY2lvbmFsIHBvciBwYXJ0ZSBkZSBsb3Mgc3VqZXRvcyBkZSBlc3RlIGFjdWVyZG8sIGFxdcOpbGxhIHNlIGVudGVuZGVyw6EgcmVmb3JtYWRhIGxvIG3DrW5pbW8gbmVjZXNhcmlvIHBhcmEgaGFjZXIgcXVlIGRpY2hhIGRpc3Bvc2ljacOzbiBzZWEgdsOhbGlkYSB5IGV4aWdpYmxlLjwvbGk+CiAgICAgIDxsaT5OaW5nw7puIHTDqXJtaW5vIG8gZGlzcG9zaWNpw7NuIGRlIGVzdGEgTGljZW5jaWEgc2UgZXN0aW1hcsOhIHJlbnVuY2lhZGEgeSBuaW5ndW5hIHZpb2xhY2nDs24gZGUgZWxsYSBzZXLDoSBjb25zZW50aWRhIGEgbWVub3MgcXVlIGVzYSByZW51bmNpYSBvIGNvbnNlbnRpbWllbnRvIHNlYSBvdG9yZ2FkbyBwb3IgZXNjcml0byB5IGZpcm1hZG8gcG9yIGxhIHBhcnRlIHF1ZSByZW51bmNpZSBvIGNvbnNpZW50YS48L2xpPgogICAgICA8bGk+RXN0YSBMaWNlbmNpYSByZWZsZWphIGVsIGFjdWVyZG8gcGxlbm8gZW50cmUgbGFzIHBhcnRlcyByZXNwZWN0byBhIGxhIE9icmEgYXF1w60gbGljZW5jaWFkYS4gTm8gaGF5IGFycmVnbG9zLCBhY3VlcmRvcyBvIGRlY2xhcmFjaW9uZXMgcmVzcGVjdG8gYSBsYSBPYnJhIHF1ZSBubyBlc3TDqW4gZXNwZWNpZmljYWRvcyBlbiBlc3RlIGRvY3VtZW50by4gRWwgTGljZW5jaWFudGUgbm8gc2UgdmVyw6EgbGltaXRhZG8gcG9yIG5pbmd1bmEgZGlzcG9zaWNpw7NuIGFkaWNpb25hbCBxdWUgcHVlZGEgc3VyZ2lyIGVuIGFsZ3VuYSBjb211bmljYWNpw7NuIGVtYW5hZGEgZGUgVXN0ZWQuIEVzdGEgTGljZW5jaWEgbm8gcHVlZGUgc2VyIG1vZGlmaWNhZGEgc2luIGVsIGNvbnNlbnRpbWllbnRvIG11dHVvIHBvciBlc2NyaXRvIGRlbCBMaWNlbmNpYW50ZSB5IFVzdGVkLjwvbGk+CiAgICA8L29sPgogIDwvbGk+CiAgPGJyLz4KPC9vbD4K