Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO3 or NH4NO3 for 24 hours.<p>Nitrate was cytotoxic to both cell types at high concentrations, with EC50 values between 1557 mg/L (approximately) 5852mg/L for viability, and ~2.5 mg/L 3631 mg/L for proliferation. Several EC50 values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO3 Neutral Red assay. Here, HEK293 viability increased with increasing KNO3 concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown.<p>No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation.<p>In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC50 values in these experiments. More research is required to determine if this poses a direct threat to human health.

The cytotoxic effects of exposure to low concentrations of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) that are typically found in groundwater were investigated, in vitro. Most 2,4-D toxicology studies use high concentrations of the herbicide that are above those typically found in groundwater and measure overt biological endpoints. In contrast, this thesis examines the effects of low concentrations of 2,4-D and measures more subtle and sensitive endpoints such as gene expression and the generation of reactive oxygen species. This work derives from recent cDNA microarray analysis conducted in our laboratory that revealed significant alterations in the expression of 238 genes in cells exposed to nanomolar (nM) concentrations of a commercial formulation of 2,4-D. These findings are extended in this thesis to include the in vitro cytotoxic effects of low concentrations of both technical and commercial 2,4-D on two cell lines. Cells derived from liver (HepG2) and kidney (HEK293) respectively, were chosen, since liver and kidney are known to metabolize 2,4-D in vivo. Cell viability was measured using the Resazurin assay, reactive oxygen species (ROS) were measured with 2,7-dichlorofluorescin diacetate (2,7-DCFH-DA), and real timepolymerase chain reaction (RT-PCR) was used to assess changes in mRNA expression while protein expression was examined by Western blot.<p>Cell viability studies revealed that low environmental concentrations (0.1 to 100 nM) of 2,4-D induced small, but statistically significant decreases in cell viability. No concentration or time-dependent decreases in cell viability were observed in cells exposed to either forms of low environmental 2,4-D concentrations. HEK293 cells were more susceptible than HepG2 cells to the toxic effects of both forms of 2,4-D, having statistically significant lower viability at all exposure concentrations and durations. Both forms of 2,4-D reduced cell viability in both cell lines, suggesting that cytotoxicity was induced directly by 2,4-D, and not by the inert ingredients in the commercial formulation.<p>The ROS assays illustrated that 2,4-D induced statistically significant ROS production in HepG2 and HEK293 cell cultures at concentrations greater than 10 µM and 100 nM respectively. This was both a concentration and time-dependent effect in both cell lines. Although HEK293 cells were more susceptible to 2,4-D, they had 50 to 70% less ROS production than HepG2 cells, at all exposure concentrations and times.<p>The RT-PCR and Western blot analyses showed that exposure of HepG2 and HEK293 cells to low 2,4-D concentrations induced (< 2 fold) alterations in mRNA and protein levels of FTL, FTH1 and PCNA however these changes did not consistently vary with concentration.<p>Taken together, cell viability, ROS and gene expression studies show that low environmental 2,4-D concentrations induced subtle in vitro cytotoxic effects. However we have no evidence that these subtle changes pose a serious health threat to exposed humans.

<p>Cadmium is a human carcinogen and a persistent environmental pollutant of increasing concern. Yet, the exact molecular targets of cadmium toxicity and the molecular mechanisms by which cadmium influences gene expression have not been fully elucidated. Therefore, the characterization of cadmium-inducible genes will provide a better understanding of the underlying mechanism involved in sensing cadmium-stress and the subsequent signaling pathways important for cellular defense against cadmium toxicity. To this end, we characterized two cadmium-responsive genes of no known biological function from the nematode Caenorhabditis elegans (C. elegans), numr-1 and numr-2.</p><p>Expression analysis of numr-1 and numr-2 revealed the same temporal and spatial expression patterns of both genes in the absence and presence of metal treatment. In the absence of metal, constitutive expression of numr-1/-2 was developmentally regulated. When adult animals were exposed to metal, numr-1/-2 expression dramatically increased. We show that worms overexpressing numr-1/-2 were more resistant to metal stress and longer lived than control animals; whereas reducing numr-1/-2 activity resulted in increased sensitivity to metal exposure. Furthermore, in the absence of metal, the two numr-1 mutant alleles, tm2775 and ok2239, exhibited decreased muscular functions. The molecular characterization of numr-1 and numr-2 also revealed that the expression of these two genes, at least in part, was regulated by changes in intracellular calcium concentrations ([Ca2+]i). This finding lead us to reevaluate the role of calcium mobilization in cadmium-induced transcription. </p><p>While several studies have indicated that exposure to cadmium resulted in increased [Ca2+]i, the mechanism by which cadmium can effect [Ca2+]i and concurrent effects on gene expression remain poorly understood. Therefore, we investigated the effects of low-level cadmium exposure, sufficient to induce transcription of cadmium-responsive genes, on the regulation of [Ca2+]i. In these studies, we utilized the protein-based calcium sensor YC 3.60 stably expressed in a HEK293 cell line. YC 3.60 is insensitive to cadmium ions, and thus is useful to monitor changes in [Ca2+]i following cadmium treatment. Exposing HEK293 cells to 1-30 µM cadmium was sufficient to induce transcription of cadmium-responsive genes such as metallothionein. Cadmium exposure from 1-10 µM had no effect on cell viability, [Ca2+]i mobilization, or increased transcriptional activity of calcium-responsive genes. In contrast, exposure to 30 µM cadmium significantly decreased cell viability, reduced intracellular calcium stores, and significantly altered the transcriptional activity of calcium-responsive genes. Taken together, these data indicate that low-level cadmium exposures (1-10 µM) can induce transcription of cadmium-responsive genes such as metallothionein independent of [Ca2+]i mobilization. </p><p>To gain further insight into the mechanistic relationship between cadmium and calcium we investigated the effects of cadmium exposure on the defecation cycle of C. elegans. Defecation is a highly rhythmic behavior that is regulated by calcium oscillations. We found that low-level cadmium exposures, sufficient to induce expression of cadmium-responsive genes such as numr-1/-2, significantly shortened the defecation cycle but did not alter the rhythm of the cycle or the magnitude of the intestinal calcium oscillations. Modulation of lipid metabolism in C. elegans results in a similar shortened defecation cycle, whereas modulation of [Ca2+]i results in lengthened and arrhythmic defection cycles, suggesting that the mechanism by which cadmium alters defecation is independent of [Ca2+]i mobilization.</p><p>In summary, the data in this work demonstrates that low-level cadmium exposure induces expression of cadmium-responsive genes independent of calcium mobilization. Thus, modulation of intracellular calcium is unlikely the primary mechanism by which cadmium regulates transcription at low-levels of exposure.</p> / Dissertation

Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.

El avance en la investigación en terapia génica durante los últimos años ha generado interés en la construcción y producción de vectores virales, siendo actualmente los adenovirus los más utilizados en ensayos clínicos de terapia génica. Para satisfacer la demanda de estos vectores, es necesaria su producción a gran escala. Debido a esto, la caracterización del crecimiento de las células de riñón de embrión humano (HEK293) es de gran importancia para la identificación de factores que permitan la optimización de las condiciones de producción y una mejora en el rendimiento en la producción de adenovirus. En este trabajo se realizó una comparación del estado metabólico y del nivel de proteínas para cultivos de células HEK293 durante el crecimiento y producción de adenovirus. Se determinaron los parámetros de crecimiento para células creciendo adheridas en medio suplementado con 10% de suero fetal bovino (SFB) y para células adaptadas a crecer en un medio bajo en suero, suplementado con 0,5% de SFB, 40 [mg/L] de albúmina de suero bovino (BSA) e intralípidos al 0,01 [%v/v]. Las células adaptadas a crecer en el medio bajo en suero alcanzaron una concentración celular de 2x106 [cel/mL], comparable al caso base, logrando un crecimiento uniforme usando placas cebadas. En el caso de células creciendo adheridas, se observó que al consumirse completamente la glucosa éstas comenzaban a consumir lactato como fuente alternativa de carbono. Esto significó una extensión en la mantención del cultivo tanto en medio bajo en suero como en medio suplementado con 10% de SFB. Se adaptaron células a crecer en suspensión en spinners con medio bajo en suero suplementado con Pluronic F-68 y antiaglomerante, alcanzando una concentración de 1x106 [cel/mL]. Esta concentración es un 48% inferior a la obtenida creciendo adheridas, presentando además tasas de consumo de glucosa y producción de lactato un 71% y 76% menores, respectivamente. Durante la producción de adenovirus se registraron mayores tasas de consumo de glucosa y producción de lactato que las alcanzadas en el crecimiento, sin presentar un aumento de biomasa. Esto indica un cambio en el metabolismo celular durante la producción de adenovirus. Al mismo tiempo se observó una disminución de la viabilidad celular producto de la acción lítica del adenovirus. La mayor producción de virus se alcanzó en células creciendo adheridas en medio con 10% de SFB con un título de 1,6x1013 [pfu/mL] a las 48 [hpi]. A través de un ensayo de ELISA intracelular se cuantificaron enzimas claves del metabolismo del carbono, siendo las enzimas estudiadas: Fosfofructoquinasa (PFK), Piruvato deshidrogenasa (PDH), Lactato deshidrogenasa (LDH), y α-Ketoglutarato deshidrogenasa (α-KGD). Se comparó la cantidad de enzimas entre células creciendo en medio suplementado con 10% y 0,5% de SFB, tanto para el estado de crecimiento como para la producción de adenovirus. Se obtuvo diferencias significativas sólo en caso de la LDH, siendo el nivel de esta enzima mayor durante el crecimiento en el cultivo suplementado con 10% de SFB, siendo también el nivel de LDH mayor en la producción de adenovirus en comparación al crecimiento con 0,5% de SFB. Estos resultados fueron contrastados con los obtenidos a través del Análisis de Flujo Metabólico (MFA), para las vías en que participan dichas enzimas, encontrando una concordancia en el comportamiento en las condiciones comparadas, ya que el aumento en la producción de lactato en el crecimiento en medio con 10% de SFB responde a una mayor cantidad de LDH. En la producción de adenovirus el aumento del lactato sería producto de la lisis celular y no de un aumento de la LDH. Finalmente los resultados obtenidos permitirán un mejor entendimiento del comportamiento de células HEK293, y de sus requerimientos nutricionales en cultivo y producción de adenovirus.

The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.

Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...

Farmworkers in Apopka, FL, have been subjected to overhead pesticide exposure since the 1940s. Pesticides including Paraquat (PQ), Metribuzin and Aldicarb were sprayed onto the field while farmworkers worked. In "Fed Up: The High Cost of Cheap Food," farmworkers recalled the physical toll these conditions took on their bodies, blaming pesticides for their diseases, such as chronic kidney disease (CKD). While established that pesticides, specifically PQ, may be involved in some forms of Parkinson's disease, no explicit connection has been identified for SLE, CKD, and other diseases experienced by farm workers. This study evaluated whether pesticides could contribute to kidney disease. We quantified the fluorescence of reactive oxygen species (ROS) following varying PQ exposure in human embryonic kidney 293 (HEK293) cells using a microplate reader. Dosages of 75 and 150 µM were chosen based on previous literature. We also measured expression of KD biomarkers KIM-1 and NGAL upon PQ exposure with RT-qPCR. Glutathione-S-transferase pi 1 (GSTP1) served as an indicator of ROS. We predicted that ROS would increase with increasing PQ concentration, as would the fold change in the expression of the mRNA biomarker levels. The results showed a trend of increased expression of NGAL, KIM-1 and GSTP1 as PQ concentration increased. This study suggests metabolic panels may be an option when assessing patient health, especially patients susceptible to kidney disease. Future in vitro and in vivo examinations of these biomarkers are needed to clinically correlate physiological concentrations of these pesticides, and progression of kidney disease.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 &plusmn; 8,3 &mu;g/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser &cong; 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 &plusmn; 8.3 &mu;g/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be &cong; 16-fold lower than G-hPRL produced in CHO cells.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 &plusmn; 8,3 &mu;g/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser &cong; 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 &plusmn; 8.3 &mu;g/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be &cong; 16-fold lower than G-hPRL produced in CHO cells.