Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT
Digital
- Autores:
-
Ortiz-Rivera, Karen Paola
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2024
- Institución:
- Universidad de Santander
- Repositorio:
- Repositorio Universidad de Santander
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.udes.edu.co:001/10952
- Palabra clave:
- Bacillus thuringiensis
Apoptosis
Parasporinas
Cáncer
MOLT-4
JURKAT
Cancro
Apoptose
- Rights
- embargoedAccess
- License
- http://purl.org/coar/access_right/c_f1cf
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Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| dc.title.translated.none.fl_str_mv |
Evaluation of The Effect of new Parasporins Obtained by Directed Evolution in MOLT 4 and JURKAT Leukemia cells Avaliação do efeito de novas parasporinas obtidas por evolução dirigida em células leucêmicas MOLT 4 e JURKAT |
| title |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| spellingShingle |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT Bacillus thuringiensis Apoptosis Parasporinas Cáncer MOLT-4 JURKAT Cancro Apoptose |
| title_short |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| title_full |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| title_fullStr |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| title_full_unstemmed |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| title_sort |
Evaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKAT |
| dc.creator.fl_str_mv |
Ortiz-Rivera, Karen Paola |
| dc.contributor.advisor.none.fl_str_mv |
Suarez-Barrera, Miguel Orlando |
| dc.contributor.author.none.fl_str_mv |
Ortiz-Rivera, Karen Paola |
| dc.contributor.jury.none.fl_str_mv |
Herrera-Pineda,Diego Fernando Valdivieso-Quintero, Wilfredo |
| dc.contributor.researchgroup.none.fl_str_mv |
Biología Molecular y Biotecnología |
| dc.subject.proposal.other.fl_str_mv |
Bacillus thuringiensis Apoptosis |
| topic |
Bacillus thuringiensis Apoptosis Parasporinas Cáncer MOLT-4 JURKAT Cancro Apoptose |
| dc.subject.proposal.spa.fl_str_mv |
Parasporinas Cáncer |
| dc.subject.proposal.eng.fl_str_mv |
MOLT-4 JURKAT |
| dc.subject.proposal.por.fl_str_mv |
Cancro |
| dc.subject.proposal.deu.fl_str_mv |
Apoptose |
| description |
Digital |
| publishDate |
2024 |
| dc.date.accessioned.none.fl_str_mv |
2024-10-01T14:20:02Z |
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2024-10-01T14:20:02Z 2025-03-30 |
| dc.date.issued.none.fl_str_mv |
2024-06-14 |
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Trabajo de grado - Pregrado |
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Universidad de Santander |
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T 17.24 O784e |
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Repositorio Digital Universidad de Santander |
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https://repositorio.udes.edu.co |
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https://repositorio.udes.edu.co/handle/001/10952 |
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Universidad de Santander T 17.24 O784e Repositorio Digital Universidad de Santander |
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spa |
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spa |
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Who. Cáncer. Who.int. 2023: https://www.who.int/es/news-room/fact-sheets/detail/cancer Campo, E., Swerdlow, S. H., Harris, N. L., Pileri, S., Stein, H., & Jaffe, E. S. The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. Blood. 2011; 117(19): 5019–5032. https://doi.org/10.1182/blood-2011-01-293050 The International Agency for Research on Cancer (IARC). (s/f). Global Cancer Observatory. Iarc.Fr. Recuperado el 22 de marzo de 2023, de https://gco.iarc.fr/ Sistema. Toxicidad de los tratamientos oncológicos. Seom.org.2019: https://seom.org/guia-actualizada-de-tratamientos/toxicidad-de-los-tratamientos-oncologicos?showall=1 Pineda-Castellanos, ML, & Núñez-Valdez, ME. Uso de bacterias y sus productos en la terapia del cáncer. Gaceta mexicana de oncología. 2011; 10 (6): 366–372. https://www.elsevier.es/es-revista-gaceta-mexicana-oncologia-305-articulo-uso-bacterias-sus-productos-terapia-X1665920111969264 Muñoz Cendales, D. R., & Cuca Suárez, L. E. Compuestos citotóxicos de origen vegetal y su relación con proteínas inhibidoras de apoptosis (IAP). Revista colombiana de cancerologia. 2020; 20(3): 124–134. https://doi.org/10.1016/j.rccan.2015.10.002 Mizuki E, Park YS, Saitoh H, Yamashita S, Akao T, Higuchi K, etal. Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis. Clin Diagn Lab Immunol [Internet]. 2000; 7(4): 625–34. Disponible en: http://dx.doi.org/10.1128/CDLI.7.4.625-634.2000 Mendoza-Almanza, G., Esparza-Ibarra, E. L., Ayala-Luján, J. L., Mercado-Reyes, M., Godina-González, S., Hernández-Barrales, M., & Olmos-Soto, J. The cytocidal spectrum of Bacillus thuringiensis toxins: From insects to human cancer cells. Toxins. 2020; 12(5): 301. https://doi.org/10.3390/toxins12050301 Borin DB, Castrejón-Arroyo K, Cruz-Nolasco A, Peña-Rico M, Sagrillo MR, Santos RCV, et al. Parasporin A13-2 of Bacillus thuringiensis isolates from the Papaloapan region (Mexico) induce a cytotoxic effect by late apoptosis against breast cancer cells. Toxins (Basel) [Internet]. 2021; 13(7): 476. http://dx.doi.org/10.3390/toxins13070476 Crickmore N, Berry C, Panneerselvam S, Mishra R, Connor TR, Bonning BC. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. J Invertebr Pathol [Internet]. 2021;186(107438):107438. Disponible en: http://dx.doi.org/10.1016/j.jip.2020.107438 Okassov, A., Nersesyan, A., Kitada, S. e Ilin, A. Las parasporinas como nuevos agentes anticancerígenos naturales: una revisión. Diario de BUON.: Diario Oficial de la Unión Balcánica de Oncología. 2015; 20 (1): 5–16. https://www.jbuon.com/archive/20-1-5.pdf Mizuki, E., Park, Y. S., Saitoh, H., Yamashita, S., Akao, T., Higuchi, K., & Ohba, M. Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis. Clinical and Diagnostic Laboratory Immunology. 2000; 7(4): 625–634. https://doi.org/10.1128/CDLI.7.4.625-634.2000 Okumura, S., Saitoh, H., Ishikawa, T., Wasano, N., Yamashita, S., Kusumoto, K.-I., Akao, T., Mizuki, E., Ohba, M., & Inouye, K. Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines. Journal of Agricultural and Food Chemistry. 2005; 53(16): 6313–6318. https://doi.org/10.1021/jf0506129 Kitada, S., Abe, Y., Shimada, H., Kusaka, Y., Matsuo, Y., Katayama, H., Okumura, S., Akao, T., Mizuki, E., Kuge, O., Sasaguri, Y., Ohba, M., & Ito, A. Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis. The Journal of Biological Chemistry. 2009; 281(36): 26350–26360. https://doi.org/10.1074/jbc.M602589200 Ohba, M., Mizuki, E., & Uemori, A. Parasporin, a new anticancer protein group from Bacillus thuringiensis. Anticancer Research. 2009; 29(1): 427–433. http://ar.iiarjournals.org/content/29/1/427 Abe, Y., Inoue, H., Ashida, H., Maeda, Y., Kinoshita, T., & Kitada, S. Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547. Journal of Invertebrate Pathology. 2017; 142: 71–81. https://doi.org/10.1016/j.jip.2016.11.008 Ekino, K., Okumura, S., Ishikawa, T., Kitada, S., Saitoh, H., Akao, T., Oka, T., Nomura, Y., Ohba, M., Shin, T., & Mizuki, E. Cloning and characterization of a unique cytotoxic protein parasporin-5 produced by Bacillus thuringiensis A1100 strain. Toxins. 2014; 6(6): 1882–1895. https://doi.org/10.3390/toxins6061882. Xu, C., Wang, B.-C., Yu, Z., & Sun, M. Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins. Toxins, 6(9). 2014: 2732–2770. https://doi.org/10.3390/toxins6092732 Abe, Y., Shimada, H., & Kitada, S. Raft-targeting and oligomerization of Parasporin-2, a bacillus thuringiensis crystal protein with anti-tumour activity. The Journal of Biochemistry. 2008;143(2): 269–275. https://doi.org/10.1093/jb/mvm220 Suárez-Barrera, M. O., Visser, L., Pinzón-Reyes, E. H., Rondón Villarreal, P., Alarcón-Aldana, J. S., & Rueda-Forero, N. J. Site-directed mutants of parasporin PS2Aa1 with enhanced cytotoxic activity in colorectal cancer cell lines. Molecules (Basel, Switzerland). 2022; 27(21): 7262. https://doi.org/10.3390/molecules27217262 Bravo, A; Gill, SS; & Soberón, M. Bacillus thurin-giensis mechanisms and use. In: Comprehensive molecular insect science. Elsevier B.V. 2005: 175-206. Crickmore, N; Baum, J; Bravo, A; Lereclus, D; Narva, K; Sampson, K; Schnepf, E; Sun, M; & Zeigler, D.R. Bacillus thuringiensis toxin nomenclature. 2015. http://www.lifesci.sussex ac.uk/Home/Neil_Crickmore/Bt/>. Espino-Vázquez, A; Gómez-Treviño, A; Galán-Wong, L; & Pereyra-Alférez, B. Isolation of Bacillus thuringiensis strains with cytotoxic activity against MOLT-4, a leukemia cell line. In Microbes in Applied Research: Current Advantages and Challenges. 2012: 147-151. Ito, A; Sasaguri, Y; Kitada, S; Kusaka, Y; Kuwano, K; Masutomi, K; & Ohba, M. A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells. Journal of Biological Chemistry. 2004; 279 (20): 21282-21286. Akiba, T; Abe, Y; Kitada, S; Kusaka, Y; Ito, A; Ichimatsu, T; Katayama, H; Akao, T; Higuchi, K; Mizuki, E; Oh-ba, M; Kanai, R; & Harata, K. Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells. journal of Molecular Biology. 2009; 386: 121-133 Ohba, M; Mizuki, E; & Uemori, A. Parasporin, a new anticancer protein group from Bacillus thurin-giensis. Anticancer Research. 2009; 29(1): 427-433. Velásquez, L; Rojas, D; Cerón, J. Proteínas de Bacillus thuringiensis con actividad citotóxica: Parasporinas. Revista Colombiana de Biotecnología. 2018; 20(2): 89-100. Yamashita, S; Katayama, H; Saitoh, H; Akao, T; Shin, Y; Park,YS; Mizuki, E.; Ohba, M; & Ito, A. Typical Three-Domain Cry Proteins of Bacillus thuringien-sis Strain A1462 Exhibit Cytocidal Activity on Limited Human Cancer Cells. J. Biochem. 2005; b3138: 663-672. |
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Bucaramanga |
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Facultad de Ciencias Médicas y de la Salud |
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Bucaramanga, Colombia |
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Bacteriología y Laboratorio Clínico |
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Suarez-Barrera, Miguel Orlando15349590-dcf9-4b0a-a9a3-95cc2ab8353c600Ortiz-Rivera, Karen Paola0491d3c4-db21-4032-85f9-6aa24971ef08-1Herrera-Pineda,Diego Fernando03ff609c-b9e7-4b55-a4b6-30eaa54cc92b-1Valdivieso-Quintero, Wilfredo869192ef-b635-464e-91ff-f62395e0944c600Biología Molecular y Biotecnología2024-10-01T14:20:02Z2025-03-302024-10-01T14:20:02Z2024-06-14DigitalIntroducción: Las parasporinas (PS) son proteínas sintetizadas por Bacillus thuringiensis que exhiben actividad citotóxica contra diversas líneas celulares humanas, mostrando una baja o nula citotoxicidad hacia células no tumorales, Sin embargo, se han observado diferencias significativas en los perfiles de citotoxicidad y niveles de actividad frente a diferentes tipos de células cancerosas. El objetivo es generar mutantes de PS2Aa1 (Mpp46Aa1) con actividad modificada para elucidar mejor su mecanismo de acción. Esto implica recopilar información crucial para guiar la mutagénesis dirigida al sitio específico, asegurando la obtención de mutantes mejorados. Se llevará a cabo una evaluación de nuevas parasporinas obtenidas mediante evolución dirigida en células de leucemia MOLT 4 y JURKAT. Materiales y Métodos: Para el desarrollo de este artículo, se realizaron varias fases: primero, se hizo cultivo de Cepa BMB171 de Bacillus thuringiensis obtenida en el laboratorio de Biología Molecular y Biotecnología de la Universidad de Santander; segundo, se aplicaron cultivos celulares a partir de la línea celular MOLT-4 Y JURKAT manteniéndose en un medio Gibco Roswell Park Memorial Institute (RPMI) 1640 mientras que la línea celular CHO-K1 (células de ovario de hámster chino), se mantuvo en un medio DMEM 10%; luego se determinó la concentración celular mediante una dilución 1:2, con 30 μL del cultivo celular (MOLT-4 O JURKAT) y 30 μL de azul de tripano de invitrogen TM, posteriormente, se cuantificó la actividad metabólica a traves de Alarmar blue, la detección de Caspasas 3, 7; finalmente se realizó un análisis estadístico. Resultados: La mutante N65, con las sustituciones K92R (dominio I), L175D (dominio II) y S218G (dominio III), y la mutante 3-35, con la sustitución G257V (dominio I), mostraron valores de IC50 de 0.16-1.33 y 0.54-0.88 μg/mL respectivamente, en comparación con la PS2Aa1, que presentó IC50 de 0.013-1.86 y 0.26-1.24 μg/mL. La mutante 0-15, con la sustitución G256A, mostró un rendimiento similar a la PS2Aa1 frente a las líneas celulares cancerosas MOLT-4 y JURKAT. Discusión: Las sustituciones en PS2Aa1 generaron modificaciones en su conformación que pueden haber aumentado o no su actividad citotóxica y su capacidad para inducir caspasas 3/7, según se ha sugerido en estudios previos. Conclusiones: la mejora genética de proteínas es una herramienta desplegada para ampliar la funcionalidad de biomoléculas naturales para su uso como bioterapéuticos y biocatalizadores; este trabajo, encuentra que las proteínas modificadas genéticamente se han convertido en la clave para dilucidar la relación estructura-función de cualquier proteína. Las alteraciones en PS2Aa1 destacan la eficacia de la mutagénesis dirigida para desarrollar variantes que intensifican la actividad citotóxica contra células de leucemia, manteniendo o incluso mejorando su selectividad hacia estas líneas celulares específicas.Introduction: Parasporins (PS) are proteins synthesized by Bacillus thuringiensis that exhibit cytotoxic activity against various human cell lines, showing low or no cytotoxicity towards non-tumor cells. However, significant differences have been observed in the cytotoxicity profiles and levels of activity against different types of cancer cells. The objective is to generate mutants of PS2Aa1 (Mpp46Aa1) with modified activity to better elucidate its mechanism of action. This involves collecting crucial information to guide specific site-directed mutagenesis, ensuring improved mutants are obtained. An evaluation of new parasporins obtained by directed evolution in MOLT 4 and JURKAT leukemia cells will be carried out. Materials and Methods: For the development of this article, several phases were carried out: first, Bacillus thuringiensis strain BMB171 was cultured, obtained in the Molecular Biology and Biotechnology laboratory of the University of Santander; Second, cell cultures were applied from the MOLT-4 and JURKAT cell lines, maintaining them in Gibco Roswell Park Memorial Institute (RPMI) 1640 medium, while the CHO-K1 cell line (Chinese hamster ovary cells) was maintained in a 10% DMEM medium; then the cell concentration was determined by means of a 1:2 dilution with 30 μL of the cell culture (MOLT-4 OR JURKAT) and 30 μL of trypan blue from invitrogen TM, subsequently, the metabolic activity was quantified through Alarm blue, the detection of Caspases 3, 7; Finally, a statistical analysis was performed. Results: The N65 mutant, with the K92R (domain I), L175D (domain II) and S218G (domain III) substitutions, and the 3-35 mutant, with the G257V substitution (domain I), showed IC50 values of 0.16- 1.33 and 0.54-0.88 μg/mL respectively, compared to PS2Aa1, which presented IC50 of 0.013-1.86 and 0.26-1.24 μg/mL. The 0-15 mutant, with the G256A substitution, showed similar performance to PS2Aa1 against the MOLT-4 and JURKAT cancer cell lines. Discussion: Substitutions in PS2Aa1 generated modifications in its conformation that may or may not have increased its cytotoxic activity and its ability to induce caspases 3/7, as has been suggested in previous studies. Conclusions: Protein genetic improvement is a tool deployed to expand the functionality of natural biomolecules for use as biotherapeutics and biocatalysts; This work finds that genetically modified proteins have become the key to elucidating the structure-function relationship of any protein. Alterations in PS2Aa1 highlight the effectiveness of targeted mutagenesis to develop variants that enhance cytotoxic activity against leukemia cells, maintaining or even improving their selectivity towards these specific cell lines.Introdução: As parasporinas (PS) são proteínas sintetizadas por Bacillus thuringiensis que apresentam atividade citotóxica contra diversas linhagens celulares humanas, apresentando baixa ou nenhuma citotoxicidade para células não tumorais. Porém, diferenças significativas foram observadas nos perfis de citotoxicidade e níveis de atividade contra diferentes. tipos de células cancerígenas. O objetivo é gerar mutantes de PS2Aa1 (Mpp46Aa1) com atividade modificada para melhor elucidar seu mecanismo de ação. Isto envolve a recolha de informações cruciais para orientar a mutagénese dirigida específica, garantindo a obtenção de mutantes melhorados. Será realizada uma avaliação de novas parasporinas obtidas por evolução dirigida em células leucêmicas MOLT 4 e JURKAT. Materiais e Métodos: Para o desenvolvimento deste artigo foram realizadas diversas fases: primeiro foi cultivada a cepa Bacillus thuringiensis BMB171, obtida no laboratório de Biologia Molecular e Biotecnologia da Universidade de Santander; Em segundo lugar, foram aplicadas culturas celulares a partir das linhas celulares MOLT-4 e JURKAT, mantendo-as em meio Gibco Roswell Park Memorial Institute (RPMI) 1640, enquanto a linha celular CHO-K1 (células de ovário de hamster chinês) foi mantida em meio DMEM a 10%. médio; em seguida a concentração celular foi determinada por meio de uma diluição 1:2 com 30 μL da cultura celular (MOLT-4 OR JURKAT) e 30 μL de azul de tripano da invitrogen TM, posteriormente a atividade metabólica foi quantificada através do Alarm blue, o detecção de Caspases 3, 7; Por fim, foi realizada uma análise estatística. Resultados: O mutante N65, com as substituições K92R (domínio I), L175D (domínio II) e S218G (domínio III), e o mutante 3-35, com a substituição G257V (domínio I), apresentaram valores de IC50 de 0,16 - 1,33 e 0,54-0,88 μg/mL respectivamente, comparado ao PS2Aa1, que apresentou IC50 de 0,013-1,86 e 0,26-1,24 μg/mL. O mutante 0-15, com a substituição G256A, apresentou desempenho semelhante ao PS2Aa1 contra as linhagens celulares de câncer MOLT-4 e JURKAT. Discussão: As substituições em PS2Aa1 geraram modificações em sua conformação que podem ou não ter aumentado sua atividade citotóxica e sua capacidade de induzir caspases 3/7, conforme sugerido em estudos anteriores. Conclusões: O melhoramento genético de proteínas é uma ferramenta utilizada para expandir a funcionalidade de biomoléculas naturais para uso como bioterapêuticos e biocatalisadores; Este trabalho constata que as proteínas geneticamente modificadas se tornaram a chave para elucidar a relação estrutura-função de qualquer proteína. Alterações no PS2Aa1 destacam a eficácia da mutagênese direcionada para desenvolver variantes que aumentam a atividade citotóxica contra células leucêmicas, mantendo ou mesmo melhorando sua seletividade para essas linhagens celulares específicas.PregradoBacteriólogo(a) y Laboratorista ClínicoBiología Molecular y Biotecnología20 papplication/pdfapplication/mswordUniversidad de SantanderT 17.24 O784eRepositorio Digital Universidad de Santanderhttps://repositorio.udes.edu.cohttps://repositorio.udes.edu.co/handle/001/10952spaUniversidad de SantanderBucaramangaFacultad de Ciencias Médicas y de la SaludBucaramanga, ColombiaBacteriología y Laboratorio ClínicoWho. Cáncer. Who.int. 2023: https://www.who.int/es/news-room/fact-sheets/detail/cancerCampo, E., Swerdlow, S. H., Harris, N. L., Pileri, S., Stein, H., & Jaffe, E. S. The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. Blood. 2011; 117(19): 5019–5032. https://doi.org/10.1182/blood-2011-01-293050The International Agency for Research on Cancer (IARC). (s/f). Global Cancer Observatory. Iarc.Fr. Recuperado el 22 de marzo de 2023, de https://gco.iarc.fr/Sistema. Toxicidad de los tratamientos oncológicos. Seom.org.2019: https://seom.org/guia-actualizada-de-tratamientos/toxicidad-de-los-tratamientos-oncologicos?showall=1Pineda-Castellanos, ML, & Núñez-Valdez, ME. Uso de bacterias y sus productos en la terapia del cáncer. Gaceta mexicana de oncología. 2011; 10 (6): 366–372. https://www.elsevier.es/es-revista-gaceta-mexicana-oncologia-305-articulo-uso-bacterias-sus-productos-terapia-X1665920111969264Muñoz Cendales, D. R., & Cuca Suárez, L. E. Compuestos citotóxicos de origen vegetal y su relación con proteínas inhibidoras de apoptosis (IAP). Revista colombiana de cancerologia. 2020; 20(3): 124–134. https://doi.org/10.1016/j.rccan.2015.10.002Mizuki E, Park YS, Saitoh H, Yamashita S, Akao T, Higuchi K, etal. Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis. Clin Diagn Lab Immunol [Internet]. 2000; 7(4): 625–34. Disponible en: http://dx.doi.org/10.1128/CDLI.7.4.625-634.2000Mendoza-Almanza, G., Esparza-Ibarra, E. L., Ayala-Luján, J. L., Mercado-Reyes, M., Godina-González, S., Hernández-Barrales, M., & Olmos-Soto, J. The cytocidal spectrum of Bacillus thuringiensis toxins: From insects to human cancer cells. Toxins. 2020; 12(5): 301. https://doi.org/10.3390/toxins12050301Borin DB, Castrejón-Arroyo K, Cruz-Nolasco A, Peña-Rico M, Sagrillo MR, Santos RCV, et al. Parasporin A13-2 of Bacillus thuringiensis isolates from the Papaloapan region (Mexico) induce a cytotoxic effect by late apoptosis against breast cancer cells. Toxins (Basel) [Internet]. 2021; 13(7): 476. http://dx.doi.org/10.3390/toxins13070476Crickmore N, Berry C, Panneerselvam S, Mishra R, Connor TR, Bonning BC. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. J Invertebr Pathol [Internet]. 2021;186(107438):107438. Disponible en: http://dx.doi.org/10.1016/j.jip.2020.107438Okassov, A., Nersesyan, A., Kitada, S. e Ilin, A. Las parasporinas como nuevos agentes anticancerígenos naturales: una revisión. Diario de BUON.: Diario Oficial de la Unión Balcánica de Oncología. 2015; 20 (1): 5–16. https://www.jbuon.com/archive/20-1-5.pdfMizuki, E., Park, Y. S., Saitoh, H., Yamashita, S., Akao, T., Higuchi, K., & Ohba, M. Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis. Clinical and Diagnostic Laboratory Immunology. 2000; 7(4): 625–634. https://doi.org/10.1128/CDLI.7.4.625-634.2000Okumura, S., Saitoh, H., Ishikawa, T., Wasano, N., Yamashita, S., Kusumoto, K.-I., Akao, T., Mizuki, E., Ohba, M., & Inouye, K. Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines. Journal of Agricultural and Food Chemistry. 2005; 53(16): 6313–6318. https://doi.org/10.1021/jf0506129Kitada, S., Abe, Y., Shimada, H., Kusaka, Y., Matsuo, Y., Katayama, H., Okumura, S., Akao, T., Mizuki, E., Kuge, O., Sasaguri, Y., Ohba, M., & Ito, A. Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis. The Journal of Biological Chemistry. 2009; 281(36): 26350–26360. https://doi.org/10.1074/jbc.M602589200Ohba, M., Mizuki, E., & Uemori, A. Parasporin, a new anticancer protein group from Bacillus thuringiensis. Anticancer Research. 2009; 29(1): 427–433. http://ar.iiarjournals.org/content/29/1/427Abe, Y., Inoue, H., Ashida, H., Maeda, Y., Kinoshita, T., & Kitada, S. Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547. Journal of Invertebrate Pathology. 2017; 142: 71–81. https://doi.org/10.1016/j.jip.2016.11.008Ekino, K., Okumura, S., Ishikawa, T., Kitada, S., Saitoh, H., Akao, T., Oka, T., Nomura, Y., Ohba, M., Shin, T., & Mizuki, E. Cloning and characterization of a unique cytotoxic protein parasporin-5 produced by Bacillus thuringiensis A1100 strain. Toxins. 2014; 6(6): 1882–1895. https://doi.org/10.3390/toxins6061882.Xu, C., Wang, B.-C., Yu, Z., & Sun, M. Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins. Toxins, 6(9). 2014: 2732–2770. https://doi.org/10.3390/toxins6092732Abe, Y., Shimada, H., & Kitada, S. Raft-targeting and oligomerization of Parasporin-2, a bacillus thuringiensis crystal protein with anti-tumour activity. The Journal of Biochemistry. 2008;143(2): 269–275. https://doi.org/10.1093/jb/mvm220Suárez-Barrera, M. O., Visser, L., Pinzón-Reyes, E. H., Rondón Villarreal, P., Alarcón-Aldana, J. S., & Rueda-Forero, N. J. Site-directed mutants of parasporin PS2Aa1 with enhanced cytotoxic activity in colorectal cancer cell lines. Molecules (Basel, Switzerland). 2022; 27(21): 7262. https://doi.org/10.3390/molecules27217262Bravo, A; Gill, SS; & Soberón, M. Bacillus thurin-giensis mechanisms and use. In: Comprehensive molecular insect science. Elsevier B.V. 2005: 175-206.Crickmore, N; Baum, J; Bravo, A; Lereclus, D; Narva, K; Sampson, K; Schnepf, E; Sun, M; & Zeigler, D.R. Bacillus thuringiensis toxin nomenclature. 2015. http://www.lifesci.sussex ac.uk/Home/Neil_Crickmore/Bt/>.Espino-Vázquez, A; Gómez-Treviño, A; Galán-Wong, L; & Pereyra-Alférez, B. Isolation of Bacillus thuringiensis strains with cytotoxic activity against MOLT-4, a leukemia cell line. In Microbes in Applied Research: Current Advantages and Challenges. 2012: 147-151.Ito, A; Sasaguri, Y; Kitada, S; Kusaka, Y; Kuwano, K; Masutomi, K; & Ohba, M. A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells. Journal of Biological Chemistry. 2004; 279 (20): 21282-21286.Akiba, T; Abe, Y; Kitada, S; Kusaka, Y; Ito, A; Ichimatsu, T; Katayama, H; Akao, T; Higuchi, K; Mizuki, E; Oh-ba, M; Kanai, R; & Harata, K. Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells. journal of Molecular Biology. 2009; 386: 121-133Ohba, M; Mizuki, E; & Uemori, A. Parasporin, a new anticancer protein group from Bacillus thurin-giensis. Anticancer Research. 2009; 29(1): 427-433.Velásquez, L; Rojas, D; Cerón, J. Proteínas de Bacillus thuringiensis con actividad citotóxica: Parasporinas. Revista Colombiana de Biotecnología. 2018; 20(2): 89-100.Yamashita, S; Katayama, H; Saitoh, H; Akao, T; Shin, Y; Park,YS; Mizuki, E.; Ohba, M; & Ito, A. Typical Three-Domain Cry Proteins of Bacillus thuringien-sis Strain A1462 Exhibit Cytocidal Activity on Limited Human Cancer Cells. J. Biochem. 2005; b3138: 663-672.Derechos Reservados - Universidad de Santander, 2024. Al consultar y hacer uso de este recurso, está aceptando las condiciones de uso establecidas por los autores.info:eu-repo/semantics/embargoedAccesshttp://purl.org/coar/access_right/c_f1cfAtribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)https://creativecommons.org/licenses/by-nc-nd/4.0/Bacillus thuringiensisApoptosisParasporinasCáncerMOLT-4JURKATCancroApoptoseEvaluación del Efecto de Nuevas Parasporinas Obtenidas Mediante Evolución Dirigida en Células de Leucemia MOLT 4 y JURKATEvaluation of The Effect of new Parasporins Obtained by Directed Evolution in MOLT 4 and JURKAT Leukemia cellsAvaliação do efeito de novas parasporinas obtidas por evolução dirigida em células leucêmicas MOLT 4 e JURKATTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_71e4c1898caa6e32Textinfo:eu-repo/semantics/bachelorThesisinfo:eu-repo/semantics/submittedVersionTodas las AudienciasPublicationORIGINALActa_Sustentacion.pdfActa_Sustentacion.pdfapplication/pdf597015https://repositorio.udes.edu.co/bitstreams/48dfc5d9-2a37-4bda-875f-3d2a0df78193/downloadb8c786a2837a940eae5dbf9030aa896cMD56Label.jpgLabel.jpgimage/jpeg405426https://repositorio.udes.edu.co/bitstreams/12c79145-0600-40ca-93be-a18a721fd8ab/download7cee1eebae1e3e11c85961d4a9abc90cMD58Evaluación_del_efecto_de_nuevas_Parasporinas_obtenidas_mediante_evolución_dirigida_en_células_de_leucemia_MOLT 4_y_JURKAT.docxEvaluación_del_efecto_de_nuevas_Parasporinas_obtenidas_mediante_evolución_dirigida_en_células_de_leucemia_MOLT 4_y_JURKAT.docxapplication/vnd.openxmlformats-officedocument.wordprocessingml.document340144https://repositorio.udes.edu.co/bitstreams/25cde551-53eb-4ba1-a3a2-e1618e603d08/download8feb55a9561f3284d8a4cef0930d07e2MD51Evaluación_del_efecto_de_nuevas_Parasporinas_obtenidas_mediante_evolución_dirigida_en_células_de_leucemia_MOLT 4_y_JURKAT.pdfEvaluación_del_efecto_de_nuevas_Parasporinas_obtenidas_mediante_evolución_dirigida_en_células_de_leucemia_MOLT 4_y_JURKAT.pdfapplication/pdf422471https://repositorio.udes.edu.co/bitstreams/b31b4a71-69c2-4c14-98ce-620f34d48fc7/downloadfff30d43978e02649778d7de88da020aMD52Certificado_de_Similitud_de_Texto.pdfCertificado_de_Similitud_de_Texto.pdfapplication/pdf498373https://repositorio.udes.edu.co/bitstreams/72f49c1b-f2f7-462d-8918-5047b24a4530/download58444695565a2b9d67a05a9859380c3bMD54Cesion_de_Derechos .pdfCesion_de_Derechos .pdfapplication/pdf318217https://repositorio.udes.edu.co/bitstreams/3547aea6-9dc7-4502-b972-9b5a533a038a/downloadb6f3277eea4492bdfd4d9897f212388fMD55LICENSElicense.txtlicense.txttext/plain; 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