Ciencias Exactas y Ciencias de la Salud
Permanent URI for this collectionhttps://hdl.handle.net/11285/551039
Pertenecen a esta colección Tesis y Trabajos de grado de las Maestrías correspondientes a las Escuelas de Ingeniería y Ciencias así como a Medicina y Ciencias de la Salud.
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- Immunoaffinity aqueous two-phase systems to establish novel bioprocesses for the primary recovery of CD133+ stem cells(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2018-05-25) Ornelas González, Alonso; Rito Palomares, Marco Antonio; Zavala Arcos, Judith; González González, Mirna Alejandra; Rito Palomares, Marco Antonio; Zavala Arcos, Judith; González González, Mirna AlejandraA short processing time and efficient scale-up stem cell isolation bioprocess is essential to exploit the potential of these cells for the treatment of multiple chronic diseases. Various methodologies have been used for stem cell recovery, however, most of them present economical and/or time-consuming drawbacks. In this work, the characterization and optimization of immunoaffinity aqueous two-phase systems, a liquid-liquid based separation technology enhanced with the PEGylation of the antibody, was conducted with the aim of increasing the specificity for the recovery of CD133+ stem cells from human umbilical cord blood samples. The methodology consisted in evaluating the partitioning of the different PEGylated antibodies (amine, carboxyl, thiol, succinimidyl ester, methoxy PEG and maleimide) in three previously studied aqueous two-phase systems (ATPS); PEG-dextran (DEX), Ucon-DEX and Ficoll-DEX. Subsequently, an optimization step was accomplished to manipulate the partition behavior of the CD133/2-pure antibody to the desired phase in the selected systems by varying (increasing and decreasing) two parameters closely related with the partitioning of molecules in aqueous two-phase systems; tie-line length (TLL) and volume ratio (VR). Afterwards, the partitioning behavior of the six different PEGylated antibodies in the optimized systems was tested. According to the results, the PEGylation of the CD133/2-biotin antibody induced a favorable change with respect to the non-PEGylated one when Ucon-DEX system was used, fractionating it to both phases. Likewise, the optimization of the systems showed to be effective to induce a change in the partition preference of the antibody. The best results were obtained when Ucon-DEX or PEG-DEX systems with TLL 15% w/w or 20% w/w with VR 3 were combined. Finally, PEGylated antibodies were added to the selected optimized systems. Even though a shift in the fractionation preference of the PEGylated CD133/2-biotin antibody was achieved in the optimized systems, it was not the adequate partition to justify the evaluation of this immunoaffinity ATPS with human umbilical cord samples. Both PEGylation and optimization showed to be effective to induce a change in the partition preference of the antibody, however, further studies are required to find the optimal system composition that will fractionate 100% of the antibody to the contaminants opposite phase, making this system an ideal candidate to be tested for the selectivity of CD133+ stem cells.
- Mejoramiento de la producción de Royalactina (MRJP1) en Pichia pastoris, mediante generación de una cepa sobreproductora y su cultivo en biorreactor(2017-12-04) Castañeda Aponte, Héctor Manuel; Rito-Palomares, Marco; Aguilar-Yañes, Jose Manuel; Rodriguez, Mirna AlejandraLa proteína MRJP1 (del inglés Major Royal Jelly Protein 1) mejor conocida como Royalactina (RY) tiene potencial para su explotación en la industria farmacéutica, utilizado como agente hepático-protector, factor estimulante de eritropoyesis, adyuvante para reducir los niveles de colesterol en sangre. En trabajos anteriores se han propuesto diferentes modelos para la expresión de MRJP1, sin embargo, ninguno ha sido idóneo para la producción a escala piloto debido a títulos y rendimientos bajos. El presente trabajo tiene como objetivo aumentar la expresión de la proteína RY en Pichia pastoris, incrementando el número de copias del gen ry, mejorando las condiciones del cultivo en la fase de producción y cultivo en fed-batch en biorreactor. Resultados. Se realizó la construcción de dos diferentes plásmidos para la expresión de RY, uno bajo el control del promotor pAOX1 (inducible con metanol) y un segundo con una versión mejorada del promotor pPPGAP (inducible con glucosa). Los plásmidos se transformaron de forma independiente a una comercial de P. pastoris. Se realizó el fenómeno de hiperresistencia para aumentar el número de copias de RY integradas al genoma de Pichia, al final del proceso se obtuvieron 119 candidatas productoras. Todas las candidatas fueron evaluadas mediante cinéticas realizadas en microplaca. Al final del proceso se obtuvieron 14 cepas sobreproductoras (10 bajo el promotor pAOX1 y 4 con el promotor pPPGAP. Estas cepas productoras se evaluaron en matraces en medio complejo. De las cepas evaluadas se encontró que la cepa G3-5 (portando el promotor pPPGAP) presentaba el mejor rendimiento de producción de RY, cerca de 0.5 g L-1 Posteriormente se llevaron a cabo estudios de mejoramiento de condiciones de cultivo, donde se evaluaron tres medios diferentes y dos temperaturas diferentes para la fase de expresión (25 y 30ºC). Los resultados muestran que se alcanzan rendimientos superiores en medio mineral y 25 ºC con un título final de 1.2 g L -1 . Conclusiones. Se ha generado y caracterizado una cepa de P. pastoris cuyo título y rendimiento son los más altos reportados para la producción recombinante de Royalactina. Dados los parámetros cinticos y de producción de la cepa es una candidata adecuada para futuros trabajos de escalamiento y optimización del bioproceso.
- Combined effect of ultrasound treatment and phytohormones on the accumulation of bioactive compunds in broccoli florets(2017-05-27) Aguilar Camacho, Miguel; Jacobo Velázquez, Daniel Alberto; Wrlti Chanes, Jorge Santos; Benavidez Lozano, Jorge AlejandroIn the past years, postharvest abiotic stresses such as wounding, modified atmospheres, UV-radiation, and the exogenous application of ethylene (ET) and methyl jasmonate (MJ) have been studied as an effective tool to improve the accumulation of bioactive compounds in fresh produce. In this context, previous reports in literature suggest that non-thermal processing technologies such as ultrasound (US) activate the biosynthesis of nutraceuticals in crops by a similar mechanism exerted by wounding stress. However, there is still limited information in literature regarding emerging technologies and their effect on the biosynthesis of bioactive compounds. Therefore, the present thesis evaluated the immediate and late response to US treatment (20 min, frequency 24 kHz, amplitude 100 μm) on glucosinolates, isothiocyanates, phenolic compounds and ascorbic acid in broccoli florets. Moreover, the combined effects of US with exogenous MJ (250 ppm) and/or ethylene ET (1000 ppm) on the content of bioactive compounds were also evaluated during storage (72 h at 15 ºC) of broccoli florets. As an immediate response to US treatment, the extractability of glucosinolates such as glucoraphanin (795%), 4-hydroxy glucobrassicin (153%), and glucobrassicin (78.6%) increased as compared with the control (CT), and induced the detection of gluconasturtiin (0.30 mmol/kg) not identified in the control. Furthermore, US treatment increased the extractability of 1- sinapoyl-2-feruloylgentiobiose (57.23%) as compared with the control, whereas the concentration of the other phenolic compounds remained unaltered. Similarly, the content of total ascorbic acid remained unchanged immediately after US treatment. The enhanced extractability of some bioactive compounds was attributed to cell disruption by US treatment. During storage, gluconasturtiin and 4-methoxy glucobrassicin, which were not detected in CT samples before storage, showed accumulation at 24 h (0.69 mmol/kg) and 72 h (0.29 mmol/kg) of storage, respectively. On the other hand, the content of glucoraphanin and 4- hidroxy glucobrassicin, which were immediately increased by US treatment, remained stable during storage in US samples. However, the high increased content of gluconasturtiin and 4- methoxy glucobrassicin at 24 h and 72 h of storage in CT samples, was not detected in US samples. The combined application of MJ and US in broccoli florets, induced a synergistic effect on the accumulation of 4-hydroxy glucobrassicin, glucoerucin, gluconasturtiin and neoglucobrassicin at 72 h of storage, showing 187.1%, 111.92%, 755.9% and 232.8%, respectively, higher content as compared with US 72 h samples. When ET was applied on US samples, it impeded the accumulation of gluconasturtiin at 24 h of storage, whereas the accumulation of 4-methoxy glucobrassicin (0.52 mmol/kg) was slightly induced at 72 h. Interestingly, when the three stressors where applied together (US+MJ+ET) the synergistic effect of US and MJ observed on the accumulation of 4-hydroxy glucobrassicin, glucoerucin, gluconasturtiin and neoglucobrassicin at 72 h of storage was repressed. Regarding with phenolic compounds the application of US increased the accumulation of 5-O-CQA (70.6%) and 1,2,2-TSG (56.4%) at 72 h and 24 h of storage, respectively, whereas the content of CA (29.8%) decreased at 48 h, as compared with CT samples. Similar to glucosinolates, after 72 h of storage, US and MJ showed a synergistic effect on the accumulation of 3-O-CQA, 1-S-2-FG, and 1,2,2-TSG, showing 73.4%, 56.0% and 136.7%, respectively, as compared with 72h CT+MJ samples. Interestingly, the application of ET did not modify the effect exerted by US on the accumulation of individual phenolic compounds. Likewise, the application of the three stressors in broccoli florets (US+MJ+ET samples) inhibited the accumulation of 3-O-CQA, 1-S-2-FG, and 1,2,2-TSG observed in US+MJ samples stored for 72 h. In general, the ascorbic acid content decreased in most samples during storage. Samples treated with US alone did not show significant differences in the ascorbic acid content during storage when compared with CT samples. However, when MJ+ET were applied, the content of total ascorbic acid was significantly reduced in CT+MJ+ET and US+MJ+ET samples after 72 h of storage by 53.4% and 86.6%, respectively, as compared with CT 0h samples. Based on the results herein obtained, the application of US can be an effective tool to enhance the extractability of certain glucocosinolate and pheniolic compounds in broccoli. Moreover, due to the synergistic effect observed on the accumulation of bioactive compounds, the combined application of US and MJ could be an effective approach to yield higher levels of glucosinolates and phenolic compounds in broccoli during storage. Additionally, recommendations for future investigations such as different US conditions, individual isothiocyanates analysis and analysis of gene expression were proposed in order to better understand the synergistic effect excerted by US and MJ on the accumulation of bioactive compounds in broccoli florets.
- Efecto del tratamiento con altas presiones hidrostáticas en la neurotoxicidad del ácido okadaico. Evaluación en cultivos de células granulares de cerebelo de rata Wistar.(2017-05-15) Hernández Ortega, César Augusto; Welti Chanes, Jorge; Fernández-Sánchez, María Teresa; Escobedo Avellaneda, Zamantha; Jorge Welti Chanes; Fernández-Sánchez, María Teresa; Escobedo Avellaneda, Zamantha; Chuck Hernández, Cristina; Novelli Ciotti, AntonelloExiste una gran diversidad de toxinas marinas que son producidas por diferentes tipos de algas. Dichas toxinas pueden acumularse dentro de moluscos y pescados cuya dieta es a base de algas. Esto representa un riesgo de salud para los consumidores de productos marinos ya que están expuestos a las toxinas que se han acumulado en la carne. El ácido okadaico (OKA) es una de las principales toxinas que se acumula en moluscos, y es responsable de ocasionar envenenamiento diarreico en humanos. El OKA es un fuerte inhibidor de proteínas fosfatasa, las cuales son responsables de controlar varios mecanismos moleculares dentro de las células. Si bien los síntomas son raramente letales, la contaminación de productos marinos con OKA ocasiona grandes pérdidas económicas. Debido a esto, es deseable buscar tratamientos que permitan detoxificar los moluscos. Lamentablemente tratamientos comunes en la industria alimentaria, tal como la cocción, no han sido capaces de eliminar el OKA de los moluscos. Las altas presiones hidrostáticas (APH) son una alternativa de tratamiento que podrían ser útiles para eliminar el OKA, con el beneficio adicionar de no alterar las propiedades sensoriales del molusco. En este trabajo se evaluó el efecto del tratamiento con APH en la toxicidad del OKA, considerando que en estudios previos se presentaron las primeras evidencias de que las APH pueden reducir la toxicidad del OKA. Se realizó el tratamiento con APH de soluciones de OKA a presión de 600 MPa durante 30 minutos, utilizando cuatro equipos de APH diferentes. La temperatura inicial del tratamiento fue de 25 ºC en todas las cosas, con excepción de un tratamiento único a temperatura inicial de 10 ºC. Se evaluó la toxicidad de las muestras de OKA (concentración 5 nM) con y sin tratamiento APH en cultivos primarios de neuronas de cerebelo de rata. Los resultados obtenidos indicaron que no existió diferencia significativa entre la neurotoxicidad del OKA sin tratamiento y la neurotoxicidad del OKA con tratamiento APH a 25 ºC. No obstante, se observó una reducción notable de la neurotoxicidad de muestras de OKA con tratamiento APH a 10 ºC. Esta disminución de la toxicidad a causa del tratamiento con APH se relacionó con una reducción de la activación de la apoptosis durante las primeras 24 horas de exposición de las células a la toxina, manteniendo el estado sano del núcleo celular y la estabilidad del ADN durante este periodo. Se concluyó que las APH a condiciones de 600 MPa y 30 minutos pueden reducir la neurotoxicidad del OKA cuando el tratamiento es realizado a temperaturas de refrigeración (10 ºC) pero no a temperaturas de nivel ambiente (25 ºC). Esta reducción de la toxicidad puede atribuirse a una disminución de la capacidad del OKA de interactuar con proteínas fosfatasas debido al tratamiento con APH, lo cual implica un posible cambio en el OKA que impida que la toxina interactúe correctamente con las fosfatasas.