A Prolactina humana hPRL é um hormônio protéico com 199 aminoácidos (MM ~ 23.000 Da) com um amplo espectro de atividades biológicas, sendo mais conhecido por estimular a lactação e regular o crescimento e diferenciação da glândula mamária. Além de quebra proteolítica, a maioria dos variantes de prolactina podem ser resultantes de outros processos pós-traducionais como polimerização, fosforilação, desamidação, sulfatação e glicosilação. Essa proteína contém apenas um sítio potencial de glicosilação por ligação à asparagina, localizada no aminoácido 31, que é parcialmente ocupado (10%) quando a proteína é sintetizada em células eucariotas. Apesar da atividade biológica in vitro da prolactina glicosilada (G-hPRL) ser muito menor (~4 vezes) quando comparada à não glicosilada, sua função fisiológica ainda não é bem definida e a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e clearance plasmático do hormônio. Com o objetivo de melhor caracterizar e estudar esta variante hormonal, foi realizada sua purificação em escala laboratorial a partir de células de ovário de hamster chinês (CHO) modificadas geneticamente, utilizando meio de cultura suplementado com cicloheximida, aumentando ~4 vezes sua concentração absoluta e ~10 vezes a razão entre a isoforma glicosilada e a não-glicosilada. A purificação da G-hPRL seguiu um processo simples e efetivo de duas etapas principais baseado em uma coluna de troca catiônica e uma coluna preparativa de exclusão molecular acoplada a um sistema de cromatografia líquida de excusão molecular de alta eficiência (HPSEC). A caracterização foi feita por HPLC de fase reversa (RP-HPLC) e exclusão molecular, SDS-PAGE, Western Blotting, espectometria de massa (MALDI-TOF) e um bioensaio in vitro utilizando células Nb2 e Ba/F3-LLP. Nossos resultados demostram que a cicloheximida pode ser uma importante ferramenta para aumentar a produção de prolactina glicosilada, facilitando a purificação e caracterização dessa isoforma. / Human prolactin hPRL is a 199 aminoacid protein hormone (MM ~23.000 Da) with a wide spectrum of biological activities being, however, best known for its stimulation of lactation and development of mammary gland. Besides proteolytic cleavage, the majority of prolactin variants can be the result of other posttranslational processing of the mature molecule in the anterior pituitary gland or the plasma. These include polymerization, phosphorylation, deamidation, sulfation, and glycosylation. This protein contains only one potential asparagine-linked glycosylation site which is partially ( ~10%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found ~ 4-fold lower compared to that of hPRL, its physiological function is not well defined yet, and the carbohydrate moiety seems to play an important role in the biosynthesis, secretion, biological activity, and plasma clearance of the hormone. In order to better characterize and study this hormone variant, we carried out its laboratory scale synthesis and purification from genetically modified CHO cells medium that had been supplemented with cycloheximide, increasing thus ~4-fold its absolute concentration and ~10-fold the glycosylated versus non-glycosylated hPRL concentration ratio. G-hPRL purification was carried via a simple and effective two-step process based on a cationic exchanger and a preparative size-exclusion HPLC column (HPSEC). Characterization was carried out by reversed-phase and size-exclusion HPLC, SDS-PAGE, western blotting, MALDI-TOF MS and in vitro bioassay utilizing Nb2 and Ba/F3-LLP cells. Ours results show that cycloheximide can be an important tool to increase the production of glycosylated proteins hPRL facilitating the purification and characterization of these isoforms.

Doctora en Ciencias de la Ingeniería, Mención Química / La venta de biofármacos representa una industria billonaria que ha crecido exponencialmente desde la década de los 70's debido al aumento de la demanda por estas proteínas terapéuticas altamente específicas. Estas proteínas recombinantes son sintetizadas por diferentes líneas celulares, especialmente aquella derivada de ovarios de hámster chino CHO ya que presentan altas tasas específicas de producción de proteína recombinante y son fácilmente adaptables a escalas industriales. Hoy en día, más de la mitad de los anticuerpos monoclonales que se encuentran en el mercado son producidos por células CHO. Para incrementar el rendimiento de los procesos productivos, diferentes metodologías han sido usadas por investigadores. Resultados positivos han sido obtenidos aplicando principios de ingeniería y utilizando herramientas de biología molecular para modificar reacciones bioquímicas. Proyectos de Ingeniería Metabólica han sido exitosos en reducir la producción de metabolitos indeseados, especialmente lactato, e incrementar la síntesis de proteína recombinante. En este trabajo se estudia el efecto que tienen diferentes estrategias de Ingeniería Metabólica sobre el metabolismo y cultivo de células CHO. Se ha probado que el metabolismo de carbono de células CHO es altamente ineficiente, consumiendo una cantidad de glucosa mayor de la necesaria para mantener el metabolismo energético y proliferación celular. En este trabajo se construyeron clones de células CHO productoras de IgG recombinante que sobre-expresan PYC2, MDH II y trasportador de fructosa. La expresión de estos genes permitiría aliviar cuellos de botella en el metabolismo central del carbono de las células. En este trabajo se estudiaron y contrastaron los efectos de la sobre-expresión de estos genes sobre la extensión de los cultivos, metabolismo y productividad. Los resultados indican que todos los clones estudiados presentan un metabolismo más eficiente, caracterizado por una menor producción de lactato por glucosa consumida y que no todos mejoraron su proliferación celular y/o productividad específica. Células CHO sobre expresando PYC2 mejoraron su tasa máxima de crecimiento, pero redujeron la tasa de producción de proteína recombinante; la sobre-expresión de MDH II conduce a la reducción del crecimiento celular y síntesis de proteína; finalmente, sobre-expresar el transportador de fructosa aumenta la proliferación celular y síntesis de proteína recombinante en medios con fructosa. Proponemos que las diferencias en producción de proteína se deben a alteraciones del estado RedOx de la célula que afectan en ensamblado de las cadenas peptídicas y la secreción de éstas. Reducir la expresión del gen Lactato deshidrogenasa A ha sido el objetivo de numerosos trabajos con el fin de reducir la síntesis de lactato e incrementar la producción de proteína recombinante. Utilizando el nuevo sistema para edición genómica CRISPR-Cas logramos interrumpir una de las copias del gen. Resultados del análisis de cultivos fed-batch de estas células indican que el crecimiento celular y el metabolismo fueron afectados y la síntesis específica y volumétrica de la proteína recombinante incrementó considerablemente. Se realizó un análisis a profundidad del metabolismo de las células deficientes en LDHa. Se midió la razón NAD+/NADH de éstas y el valor indicó que las células mutadas presentan niveles de NAD +/NADH menores que las del cultivo control, sugiriendo una mejora en su metabolismo energético debido a la mayor acumulación de NADH. Mediante Análisis de Flujos Metabólicos se estimó los flujos entre las reacciones más importantes del metabolismo central de las células. Los resultados confirmaron que en las células mutantes existen mayores flujos en el ciclo del TCA, debido principalmente a un mayor aporte de carbonos provenientes del catabolismo de amino ácidos. Estas células también presentan un menor flujo en la vía de la glicólisis, lo que se correlaciona con la menor proliferación que estas presentaron, y esto último puede explicar el aumento de síntesis de proteínas. En este trabajo se aplicaron exitosamente conceptos de Ingeniería Metabólica para la construcción de clones con distintos metabolismos. Este trabajo revela los efectos de varias modificaciones, lo que lo hace una fuente útil información acerca de los efectos que tienen variadas estrategias metabólicas sobre cultivos de células CHO. Finalmente, este trabajo resulta ser un aporte para la comunidad científica contribuyendo con la primera comparación de diferentes clones que sobre-expresan genes claves del metabolismo central del carbono y entregando el primer estudio en profundidad del efecto de reducir la expresión del gen de la LDHa.

A positive correlation between the flux of TCA cycle and productivity of Chinese Hamster Ovary (CHO) cells has been reported. Earlier work in this laboratory revealed that supplementation with nutrients that enter the TCA cycle (combination of glucose (Glc), pyruvate (Py), aspartate (Asp), asparagine (Asn) and glutamate (Glu)) significantly increased maximum viable cell density and antibody production of recombinant CHO cells. Increased amounts of extracellular citrate was associated with feeding conditions. It was hypothesized that increased flux through the TCA cycle and related metabolism was linked to enhanced growth and/or productivity of CHO cells. Therefore, the aim of this thesis is to clarify these relationships to provide routes to improve the efficiency of CHO cells by nutrient supplementation and metabolic engineering. The relationship between growth, antibody production and metabolite profiles of CHO-LB01 cells was examined in response to individual supplementation with Asn, Asp, Glu, Py and β-hydroxybutyrate (HB). Feeding HB significantly increased antibody titre while Asn feeding increased maximum cell density but led to earlier cell death. Both nutrients increased the amounts of TCA cycle intermediates and decreased the amounts of lactate, glycerol, sorbitol and amino acids. Moreover, oxygen consumption rate was increased in the presence of Asn or HB. This finding inferred that increased production of the TCA cycle intermediates in cells fed Asn or HB correlated with enhanced flux of the TCA cycle leading to enhanced oxidative metabolism. Combination of Asn or HB with Glc further improved cell growth, increased antibody titre and enhanced metabolic responses to feeds (TCA cycle intermediates). Based on these results, inhibition of sorbitol production using chemical reagent (EPBC) and siRNA designed against Akr1b1 and overexpression of malate dehydrogenase II (MDH II) were undertaken in order to increased flow of carbon atoms to TCA cycle and/or increased flux in the TCA cycle, respectively. Inhibition of sorbitol production was achieved in the presence of EBPC but there was no improvement of cell culture performance and accumulation of TCA cycle intermediates remained the same. CHO cells transfected with exogenous Mdh2 did not show improved cell culture performance. Whilst stable clones exhibited variable MDH II expression at protein level (and antibody titre), overexpression of exogenous MDH II could not be confirmed by Western blot. One CHO-MDH II clone showed greater antibody titre and exhibited similar metabolite profiling with cells fed Asn or HB. This contrasted to the majority of clones that were low producers. Comparison by RNA-Seq transcriptomic profiling of high- and low-producing CHO-MDH II clones showed that the majority of differentially expressed genes were genes related to cytoskeleton-related element and cell signaling pathways. Overall, these results confirmed the relationship between increased the amount of TCA cycle intermediates and increased antibody production. Increased amount of TCA cycle intermediates could result in increased the flow of TCA cycle lead to enhance energy and antibody production. In addition, this work represents the first study on addition of HB offers a simple effective strategy to increase antibody production.

The irradiation of cells in culture is an essential part of many radiation biology experiments. Since these experiments necessarily involve the irradiation of cell culture vessels and nutrient medium, the possibility of effects due to the interactions of irradiated material with growing cells needed to be investigated. In the present study the micronucleus frequency in Chinese hamster ovary (CHO) cells as a function of such parameters as type of radiation, type of cell substrate, changes in cell environment, and time course of the effect were characterized. Observations of the persistence of micronucleus formation in irradiated CHO cells reveal that the number of cells containing micronuclei reaches its maximum within nine hours after irradiation and remain elevated for at least five days. The influence of the cell environment on micronucleus formation in CHO cells was examined by plating cells in preirradiated nutrient medium or on preirradiated cell culture vessels. In all experiments, pre-irradiation of the cell substrate (the culture dish or culture dish filled with medium) led to a significantly higher micronucleus frequency than when cells were plated on un-irradiated substrate. The difference is most pronounced at the lowest doses examined. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be confounding factors, particularly in the experiments which are intended to examine the response of cells exposed to low doses of ionizing radiation.

Thesis (Th. M.)--Dallas Theological Seminary, 1989. / Includes bibliographical references (leaves : (60)-62).

Thesis (Th. M.)--Dallas Theological Seminary, 1989. / Includes bibliographical references (leaves : (60)-62).

Thesis (Th. M.)--Dallas Theological Seminary, 1989. / Includes bibliographical references (leaves : (60)-62).

Thesis (Ph. D.)--Bob Jones University, 1992. / Abstract. Includes bibliographical references (leaves 318-336).

Thesis (Ph. D.)--Bob Jones University, 1992. / Abstract. Includes bibliographical references (leaves 318-336).

Thesis (Ph. D.)--Bob Jones University, 1992. / Abstract. Includes bibliographical references (leaves 318-336).