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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- Andamios porosos de zeína: prueba de concepto de su uso en el cultivo de células de mamífero.(2017-12-14) Portales Cabrera, Cynthia Guadalupe; Trujillo-de Santiago, Grissel.; Álvarez, Mario Moises; Montes Fonseca, Silvia Lorena; Mendoza Buenrostro, Christian CarlosLas espumas son materiales porosos y de baja densidad, características convenientes en andamiajes para células de mamífero. En este proyecto se estudió la aptitud de espumas elaboradas a partir de zeína (la proteína más abundante del maíz) como andamios para adherir y mantener distintos cultivos celulares de mamíferos: fibroblastos de ratón (NIH 3T3 ATCC® CRL-1658™), fibroblastos humanos FH (fuente primaria) y células de adenocarcinoma mamario MCF-7 (ATCC® HTB-22™). Los andamios porosos se obtuvieron a partir de zeína termoplastificada, la cual fue sometida a un proceso de expansión por dióxido de carbono supercrítico. Estos andamios fueron estudiados bajo condiciones de cultivo continuo en cámaras microfluídicas o lote en placas de ultrabaja adherencia. Los dispositivos microfluídicos, con un volumen efectivo de 100 µl, se fabricaron a partir de polidimetilsiloxano (PDMS) y se conectaron a una bomba de jeringa utilizando tubería flexible. Cortes rectangulares de espuma de zeína fueron colocados al interior de la cámara microfluídica, se inocularon con células de mamífero y se sometieron a condiciones de flujo continuo de medio modificado de Dulbecco (DMEM) a 3 µL/min. Resultados de microscopía confocal demuestran que todas las líneas celulares exploradas presentaron adhesión celular a los andamios de zeína. Resultados de consumo de glucosa confirman que las células mantuvieron su viabilidad en todos los experimentos, 60- 84 horas en cultivo continuo y hasta 9 días en cultivo por lote. Estos resultados sugieren que los andamios elaborados a base de zeína se pueden utilizar como un andamio adecuado para el cultivo de tejidos e ingeniería celular. Foams are low-density porous materials, a property that makes them attractive for their use as scaffolds for mammalian cells. In this project, micro foams made from thermo-plasticized zein (the main protein of maize) were used as scaffolds to attach and culture mouse fibroblasts (cell line NIH 3T3 ATCC® CRL-1658™), human fibroblasts (primary cell line FH), and adenocarcinoma cell line MCF-7 (ATCC® HTB-22™) in continuous flow chambers or ultra-low attachment multiwall plates (batch system). Zein foams were produced by supercritical-CO2 expansion. We produced polydimethylsiloxane (PDMS) devices with 100 µl of effective-volume micro-channels that were connected with tubing to a syringe pump to implement continuous culture experiments. Rectangular slices of zein foam were placed into the flow chamber, they were seeded with the corresponding cell line, and the devices were continuously perfused with Dulbecco's modified Eagle medium (DMEM) at a flow rate of 3 µl. The systems were incubated at 37°C and 5% CO2. Different behaviors were observed for each cell line, but cell adhesion was found in all experiments and it was documented using confocal microscopy. Trends of the glucose consumption curves show that the cultures maintained their viability throughout of experiment, 60-84 h in continuous cultures and up to 9 days in batch cultures. These proof-of-concept experiments suggest that zein can be used as a surface scaffold for continuous cell culture and tissue engineering.
- Effect of ultrasound treatment on bioactive compounds’ composition of soybean and red prickly pear used to develop a functional beverage(2017-12-05) Serrano Sandoval, Syra Nayely; Welti Chanes, Jorge Santos; Morales de la Peña, Mariana; Velázquez, Daniel Alberto JacoboSoymilk and red prickly pear have nutraceutical properties. It has been reported that aglycone isoflavones in soymilk as well betalains, polyphenols, and vitamin C from red prickly pears have health-related attributes. Ultrasound(US) has been explored to enhance the content of phytochemicals in plant-based foods. Hence, the aim of this research was, to evaluate the effects of US treatment on the extractability and/or biosynthesis of antioxidants of soybeans and red prickly pears to elaborate a mixed beverage. Firstly, soybeans were sonicated with 100µm-20min at 30°C and soymilk was obtained. Individual isoflavones were quantified in the resultant soymilk and b-glucosidase activity was evaluated. Secondly, two studies were conducted with red prickly pears. In the first one, US was applied at different amplitudes(50, 75 and 100µm) and treatment times(5, 10, and 15min); and betalains, phenolics, and vitamin C concentration was quantified immediately after processing(0h) and after 24 and 48h of storage. The optimal conditions were employed in the second study(50µm-15min and 100µm-10min) and in addition to the quantification of antioxidants, the respiration rate and ethylene production were measured(0-48h, every 6h). Furthermore, tyrosinase activity was measured. Finally, a mixed beverage was prepared with soymilk extracted form sonicated soybeans and US-treated prickly pears(70:30 v/v) and the physicochemical and phytochemical properties were evaluated. Obtained results indicated that the activation of bglucosidase during US-processing of soybeans induced the interconversion of isoflavone glucosides to aglycone structures in the obtained soymilk. Furthermore, US-cavitation phenomena increased the extraction of isoflavones. On the other hand, immediately after US at 100µm-10min and during storage, the concentration of betalains on the peel and pulp of prickly pears augmented significantly, which was supported by the increase of tyrosinase activity. Furthermore, changes in the concentration of phenolics and vitamin C were also observed in the US-treated fruits. The data obtained from the evaluation of the respiration rate and ethylene production support that US affects both the extractability and the stimulation of biosynthetic pathways. The mixed beverage presented a high content of aglycone isoflavones, betalains, phenolics, and vitamin C which could have functional properties
- Characterizing the role of mitochondrial Ca2+ overload and permeability transition during the BCR-dependent activation of murine B lymphocytes(2017-12-05) Torres Quintanilla, Alejandro Daniel; González Castillo, Elena Cristina; García Rivas, Gerardo; Maravillas Montero, José Luis; Torre Amione, GuillermoMitochondria have proved their role as major players for immunometabolism, regulating cell function by metabolic signals. Indeed, mitochondrial function during lymphocyte activation goes beyond ATP supply, as recent evidence shows that mitochondrial Ca2+ uptake and mitochondrial ROS (mROS) production regulate lymphocyte activation. Furthermore, the adaptive immune response depends on the differentiation of lymphocytes into various subsets. Whether metabolic signals determine lymphocyte differentiation has just began to be explored. Interestingly, recent evidence shows that mitochondrial signals determine B lymphocyte differentiation into memory cells. During lymphocyte activation, Ca2+ uptake into mitochondria has proven to be an important mechanism of regulation. However, if mitochondrial Ca2+ uptake capacity impacts B cell differentiation and activation is not yet determined. Here, we have generated an in vitro model of B lymphocyte mitochondrial Ca2+ overload to explore whether this mechanism is involved in B cell activation. Primary mice B lymphocytes were subjected to activation or thapsigargin treatment to induce Ca2+ overload. The effect on the expression of activation surface marker CD69 was explored using flow cytometry. Mitochondrial contribution was explored by Ca2+ uptake inhibition using Ru360 or treatment with the mitochondrial antioxidant MitoTEMPO. Mitochondrial membrane potential, Ca2+ uptake, and content were determined. Results show that our in vitro model effectively induces mitochondrial Ca2+ overload, as seen by higher Ca2+ content and mitochondrial membrane depolarization. Moreover, mitochondrial Ca2+ overload contributes to the expression of CD69 via mROS production. Lastly, results suggest that during BCR-dependent activation, mitochondria take up Ca2+ to increase mROS production that can alter the signaling cascade. The in vitro model generated herein, should allow for further exploration of whether mitochondrial Ca2+ overload impacts B cell differentiation.
- 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.
- Changes in the activity of CYP450 3A4 induced by kaempferol 3-O-glucoside, major flavonol found in black bean (Phaseolus vulgaris) seed coat extract.(2017-12-01) Serna Guerrero, Delia Ayled; Gutiérrez Uribe, Janet Alejandra; Antunes Ricardo, Marilena; Hernández Hernández, José Ascención; Antunes Ricardo, MarilenaSupplements have increase the popularity of herbal medicine as complementary and/or alternative therapies in the treatment of many disorders, because of this there is a potential possibility for pharmacokinetic and pharmacodynamics interaction between xenobiotics. The health promoting effects of natural foods and dietary supplements containing herbal constituents is believed to be caused by the antioxidant activity of flavonoids. Black bean flavonoids have been shown to have biological effects as antimicrobials, anti-inflammatory, anticholesterolemic, and have anticancerous properties. These interactions can attenuate or enhance drug effects and toxicity by interfering with permeability, phase I metabolism and the antioxidant activity of flavonoids. Keywords: pharmacokinetics, pharmacodynamics, drug interactions, CyP450, hepatocytes, HepG2, Caco2, permeability, cellular antioxidant activity, flavonoids, black bean, quercetin, kaempferol, CPC
- 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.