Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-09-05T20:11:17Z No. of bitstreams: 1 2016 - Fernanda Marques Peixoto.pdf: 14003225 bytes, checksum: 89c95a9ad22b1e74cdf2bda273665230 (MD5) / Made available in DSpace on 2017-09-05T20:11:17Z (GMT). No. of bitstreams: 1 2016 - Fernanda Marques Peixoto.pdf: 14003225 bytes, checksum: 89c95a9ad22b1e74cdf2bda273665230 (MD5) Previous issue date: 2016-12-08 / A lot of interest in the consumption of anthocyanins increased after the association of their intake and reduced risk of chronic diseases. Despite of in vitro evidences of anthocyanins benefits to health, there is still a gap in the knowledge of the mechanisms of absorption of anthocyanins by the human body. It is known that concentration of food anthocyanins doesn't reflect the amount of these compounds which are absorbed, metabolized, distributed and biologically active in humans. Some in vitro models have been developed to evaluate the steps of cell release and transport ( uptake) of these compounds from food. The objective of this study was to evaluate the in vitro absorption of food anthocyanins using the in vitro digestion followed by uptake and transport in Caco-2 human intestinal cell line and MKN-28 human gastric cell line. Initially, anthocyanins bioaccessibility of diverse fruits was evaluated in order to select the better sources for transport assays. The bioaccessibility assays were performed using an in vitro digestion model, which mimics the human oral, gastric and intestinal stages. Quantification and characterization of anthocyanins profile were performed by high-performance liquid chromatography (HPLC) with Thermo Scientific? C1s 2.4 (4.6 x 10mm) column. After selection of the most promising fruits, the bioaccessibility tests were followed by transport assays. To assess gastric absorption, the product from gastric digestion was applied on the MKN-28 cell monolayer, which was obtained after 7 days of culture of 2.5 x 10^5 MKN-28 cells seeded in RPMI culture media in transwell? plates. The permeate was collected after 30, 60, 120 andl80 minutes oftransport. For evaluation of intestinal absorption after digestion, the digesta from the intestinal phase was applied on the Caco-2 cell monolayer, which was obtained after 21 days of culture of 2.5 x 105 Caco-2 cells seeded in DMEM culture media in TRANSWELL? plates. The permeate was collected after 30, 60 and 120 minutes of transport. All analyses were made by forming CLUE / photodiode array detector (Thermo? Scientific) at 520nm. Peel powder from jabuticaba, jambo and Jamel?o were the most promising sources. The bioaccessibility of anthocyanins after gastric digestion was 13% for jabuticaba, 45 % for jambo and 65 % for jamel?o. In addition, the intestinal bioaccessibility was 1 O % for jabuticaba, 15 % for jambo and 45 % for jamel?o. The transport assay with the MKN-28 gastric cell line, revealed 19.7%, 9.7 % and 14.1 % of transport efficiency, respectively, for jambo, jabuticaba and jamel?o digestion products. While Caco-2 intestinal cell model showed 0.8 %, 0.2 % and 0.3 % oftransport efficiency, respectively, for jambo, jabuticaba and jamel?o. These results suggest food anthocyanins are preferentially absorbed by the human gastric mucosa and to a lesser extent by the human intestinal epithelium. / O interesse pelo consumo das antocianinas aumentou ap?s o surgimento da rela??o entre o seu consumo e a redu??o do risco de doen?as cr?nicas. Apesar das evid?ncias in vitro quanto a esses beneficios ? sa?de, ainda h? uma lacuna que permanece sob investiga??o: o mecanismo de absor??o das antocianinas pelo organismo humano. Sabe-se que a quantidade desses compostos, nos alimentos, n?o reflete a quantidade absorvida, metabolizada, distribu?da e biologicamente ativa em humanos. Alguns modelos in vitro t?m sido desenvolvidos para avaliar as etapas de digest?o e transporte celular (absor??o) de compostos dos alimentos. Assim, o objetivo deste trabalho foi avaliar o transporte in vitro de antocianinas em alimentos utilizando modelos de digest?o in vitro seguido do transporte em c?lulas intestinais Caco-2 e c?lulas g?stricas MKN-28. Na 1? etapa, oito frutos foram analisados quanto aos valores de bioacessibilidade (BCSS) fornecidos pelas antocianinas presentes, para posterior sele??o para os ensaios de transporte. Os ensaios de BCSS foram realizados com um modelo de digest?o in vitro, para simula??o das fases oral, g?strica e intestinal humana. A quantifica??o e determina??o do perfil de antocianinas foram realizadas por Cromatografia l?quida de alta efici?ncia (CLAE), com coluna Thermo? Scientific C1s 2,4 (4,6 x 100mm). Na 2? etapa, realizou-se os ensaios de BCSS, anteriormente aos ensaios de transporte, nos frutos potencialmente mais promissores. Para a avalia??o do transporte g?strico, na sequ?ncia, o digerido g?strico foi aplicado sobre a monocamada de c?lulas MKN-28, com 2,5 x 10^5 c?lulas, em meio RPMI, em placa transwell? e, ap?s 7 dias de cultivo, o permeado foi coletado nos tempos 30, 60, 120, 180 minutos. Para o transporte intestinal, sequencial, o digerido intestinal foi aplicado sobre a monocamada celular Caco-2, com 2,5 x 105 c?lulas, em meio DMEM, em placas transwell? e, ap?s 21 dias de cultivo, o permeado foi coletado nos tempos 30, 60 e 120 minutos de transporte. Todas as an?lises foram realizadas por CLUE/detector de arranjo fotodiodo (Thermo? Scientific), a 520 nm. Os p?s da casca da jabuticaba, jambo e jamel?o foram as matrizes mais promissoras. A BCSS das antocianinas, ap?s a digest?o g?strica, foi de 13 % parajabuticaba, 45 % parajambo e 65 % parajamel?o, enquanto a BCSS intestinal foi de 10% para jabuticaba, 15 % para jambo e 45 % para jamel?o. Os ensaios de transporte (ET) com os modelos de c?lula MKN-28 resultaram em 19,7; 9,7 e 14,1 % de ET, respectivamente, para os p?s do jambo, jabuticaba, e jamel?o, enquanto que o modelo Caco-2, resultaram em 0,8, 0,2 e 0,3 % de ET, respectivamente. Estes resultados sugerem que as antocianinas s?o preferencialmente absorvidas pela mucosa g?strica.

To examine the effect of buttermilk fractions on growth, iron transport, and uptake, Caco-2 cells (human colon adenocarcinoma) were grown in a bicameral chamber. The Caco-2 cell culture system is a useful model to study micronutrient utilization in the human enterocyte, because Caco-2 cells continuously differentiate and form a monolayer, which has high polarity, a well-developed brush border, and a tight junction. Iron bioavailabilty in various milks is very different depending upon milk composition. The fat fraction especially is known to be associated with iron absorption, because the fat fraction has milk fat globule membrane (MFGM), which contains bioactive molecules such as sphingolipids. Composition of buttermilk that was concentrated by 10 K molecular sieving (MS) or by bacterial fermentation (Lactococcus latis PN-l) was reduced in lactose concentration and increased in protein concentration. Percent fat in MS buttermilk was concentrated to two times higher than in the original buttermilk (P < 0.05). Growth of Caco-2 cells with molecular sieved (MS) or fermented (FM) buttermilk in the growth medium was not significantly different. Transport and uptake of 59Fe was performed with/without cold iron (1 mmol/L) by iron-depleted or iron-repleted cells. Molecular sieved or fermented buttermilk and ganglioside or sphingomyelin standards with dimethyl sulfoxide (DMSO) were added to the Hank's balance salt solution (HBSS) in the apical chamber. With cold iron, addition of MS and FM buttermilk (1, 2, or 3 percent) increased 59Fe transport across iron-repleted cells (P < 0.01). Without cold iron, ganglioside depressed 59Fe transport (P < 0.01). Uptake of 59Fe was not significantly affected by buttermilk concentrates; however, more effective uptake was shown across iron-depleted cells. It is not clear from these studies that buttermilk fractions or their components influence iron uptake or transport by Caco-2 cell cultures.

The interplay of transporters and enzymes and their transporter-enzyme was examined in Caco-2 cell monolayer and recirculating perfused rat liver preprations via both theoretical and experimental approaches. First, a Caco-2 catenary model that consisted of the apical, cellular, basolateral compartments and encompasses influx, efflux transporters and enzymes was shown to be superior to the single barrier approach for data interpretation on transporter- and enzyme- mediated processes. The kinetics of baicalein, a flavonoid that undergoes glucuronidation and sulfation, were found to be described better by the catenary model for the complex kinetics of substrate inhibition in metabolism. Second, estradiol-17beta-D-glucuronide (E217G), a protypic substrate of Oatp1a1, 1a4, and 1b2 and Mrp2 that underwent futile cycling with its 3-sulfate metabolite (E23S17G) via estrogen sulfotransferase (Sult1e1) and arylsulfatase C, was examined in the perfused rat liver preparation. Solutions of the AUC and clearances were solved to relate the intrinsic clearances of transporters and enzymes to understand how these affected the apparent clearances in the presence of futile cycling. Transporters and enzymes were perturbed experimentally by the intraportal injection of CC531 colon carcinoma cells for tumor induction in Wag/Rij rat livers. The protein expression of Oatp1a1 and Oatp1b2 were reduced to half whereas Sult1e1 was increased by 40% with tumor development versus the sham-operated control. These data were well predicted by the physiologically-based liver model, showing the impact of increased sulfation intrinsic clearance but not the decreased influx clearance. The TR- (Mrp2 mutant) rat model was used to examine how the absence of Mrp2 for biliary secretion of both E217G and E23S17G affected futile cycling. Absence of Mrp2 was found to result in a pseudo steady-state and reduction of the total, excretion, and metabolic clearances in the liver. The work shed new insight on the interplay between enzymes and transporters and how kinetic processes mediated by enzymes or efflux transporters affected futile cycling.

The objective of this study was to precipitate CaCO₃in a groundwater media to reduce dissolved arsenic concentrations. In this study a mixture of ureolytic calcite and aragonite were precipitated using groundwater as the media. Although precipitation of carbonate was successful using Ardkenneth groundwater, arsenic concentrations were not reduced. Ureolytic calcite and aragonite precipitated using broth as the media and resulted in a decrease in arsenic concentrations of up to 88% from the initial 0.7 μg L-1 concentration. Ureolytic carbonate precipitation required the inoculation of ureolytic cultures isolated from groundwater into both the groundwater and broth media. Precipitates in the inoculation experiments were identified using infrared spectroscopy techniques.<p>The decrease in arsenic concentrations in the inoculated urea treated broth samples compared to the groundwater samples was attributed to the greater amounts of precipitate formed in the broth media. The broth had a free Ca(II) concentration of 1300 mg L-1 whereas the Ardkenneth groundwater had a free Ca(II) concentration of 36 to 42 mg L-1. The higher free Ca(II) concentrations in the broth media would account for the higher yield of carbonate precipitate, making Ca(II) concentration a limiting factor in ureolytic CaCO3 remediation techniques. <p>Formation of a visible precipitate required the addition of nitrate to the broth and groundwater samples. The inoculated cultures, being denitrifiers, required a nitrate source. Ca(II) ion concentrations decreased in the different media without the addition of nitrate, but no visible precipitate formed.<p> Laboratory experiments using Ardkenneth groundwater and treatments of 0.03 M urea did not decrease the Ca(II) ion concentrations or reduce arsenic in solution. These results suggest that inoculation with selected ureolytic cultures was needed to optimize the precipitation of CaCO3 in a natural groundwater system.<p> The results of this study suggest that arsenic was reduced by the precipitation of ureolytic CaCO₃. Arsenic reduced by ureolytic CaCO₃ precipitation required adequate levels of Ca(II) ions, higher than those found in the Ardkenneth aquifer. Successful precipitation of CaCO₃ by ureolytic organisms also required an adequate cell density. Thus, inoculation with ureolytic cultures optimized the broth and groundwater media for CaCO₃ precipitation.

Research was undertaken to examine different <i>in vitro</i> characteristics of probiotic bacteria, including <i>Lactobacillus acidophilus</i> ATCC® 11975, <i>Bifidobacterium infantis</i> ATCC 15697D, <i>Bifidobacterium catenulatum</i> ATCC® 27675 and <i>Bifidobacterium adolescentis</i> ATCC® 15703 in order to identify suitable strain(s) for encapsulation. Under simulated gastric conditions (pH 2.0), <i>L. acidophilus</i> was the most acid-tolerant strain (D-value 10.2 ± 0.8 min), and was able to survive for 30 min; whereas, the other tested probiotics underwent a rapid (within the first 5 min at pH 2.0) 4-5 log colony forming units (cfu)/mL loss in viability. All probiotics tested were able to survive 5 h exposure to 0.3% Oxgall bile at pH 5.8. The relative ranking of probiotic adherence to Caco-2 cells was determined to be: <i>L. acidophilus</i> > <i>B. catenulatum</i> > <i>B. adolescentis</i> > <i>B. infantis</i>, which correlated with 4.5 104, 3.1 103, 2.6 101, and 1.5 101 cfu/mL associated with Caco-2 cell monolayers, respectively. The most hydrophobic probiotics included <i>L. acidophilus</i> (46.5 ± 6.1%) and B. catenulatum (65.5 ± 5.2%); their hydrophobicity were positively correlated with auto-aggregation ability. Addition of divalent cations, EDTA, and bile salts were found to affect hydrophobicity as well; for example, 0.5 mM MgCl2 resulted in a 20% increase in cell surface hydrophobicity of <i>L. acidophilus</i> from baseline levels; whereas, the addition of 0.1 and 0.5% bile salts decreased <i>L. acidophilus</i> hydrophobicity from control levels by 60 and 90%, respectively. Cell free culture supernatant of <i>L. acidophilus</i> effectively inhibited the growth of <i>Escherichia coli</i> O157:H7, and <i>Clostridium sordelli</i>. Bactericidal activity of <i>L. acidophilus</i> cell-free supernatant (the lethal factor was determined to be both heat and trypsin-resistant) against Escherichia coli O157:H7 and <i>Clostridium sordelli</i> ATCC 9714 over 24 h resulted in reductions of 5.5 and 3.5 log cfu/mL, respectively. Further examination of probiotics revealed varying degrees of resistance to the iv antimicrobial agents ciprofloxacin (4 ìg/mL), naladixic acid (32 ìg/mL), kanamycin (64 ìg/mL) and sulfisoxazone (256 ìg/mL). Determination of carbon source utilization patterns indicated that <i>B. catenulatum</i> utilized a number of carbohydrates including -methyl-D-glucoside, D-xylose, D-cellobiose, and -D-lactose; whereas,<i>L. acidophilus, B. infantis</i>, and <i>B. adolescentis</i> utilized D-xylose. <i>Lactobacillus acidophilus</i> was ultimately selected for encapsulation in a 3 mm diameter pea protein-alginate matrix followed by <i>in vitro</i> challenge to simulated gastric conditions (pH 2.0). Encapsulation of <i>L. acidophilus</i> demonstrated a significant (P < 0.05) protective effect during the 2 h exposure to simulated acidic stomach conditions; within capsules, there was approximately 1 log cfu/mL loss in cell viability, whereas unprotected cells experienced > 6 log/mL loss in cell viability over the same period.

The interplay of transporters and enzymes and their transporter-enzyme was examined in Caco-2 cell monolayer and recirculating perfused rat liver preprations via both theoretical and experimental approaches. First, a Caco-2 catenary model that consisted of the apical, cellular, basolateral compartments and encompasses influx, efflux transporters and enzymes was shown to be superior to the single barrier approach for data interpretation on transporter- and enzyme- mediated processes. The kinetics of baicalein, a flavonoid that undergoes glucuronidation and sulfation, were found to be described better by the catenary model for the complex kinetics of substrate inhibition in metabolism. Second, estradiol-17beta-D-glucuronide (E217G), a protypic substrate of Oatp1a1, 1a4, and 1b2 and Mrp2 that underwent futile cycling with its 3-sulfate metabolite (E23S17G) via estrogen sulfotransferase (Sult1e1) and arylsulfatase C, was examined in the perfused rat liver preparation. Solutions of the AUC and clearances were solved to relate the intrinsic clearances of transporters and enzymes to understand how these affected the apparent clearances in the presence of futile cycling. Transporters and enzymes were perturbed experimentally by the intraportal injection of CC531 colon carcinoma cells for tumor induction in Wag/Rij rat livers. The protein expression of Oatp1a1 and Oatp1b2 were reduced to half whereas Sult1e1 was increased by 40% with tumor development versus the sham-operated control. These data were well predicted by the physiologically-based liver model, showing the impact of increased sulfation intrinsic clearance but not the decreased influx clearance. The TR- (Mrp2 mutant) rat model was used to examine how the absence of Mrp2 for biliary secretion of both E217G and E23S17G affected futile cycling. Absence of Mrp2 was found to result in a pseudo steady-state and reduction of the total, excretion, and metabolic clearances in the liver. The work shed new insight on the interplay between enzymes and transporters and how kinetic processes mediated by enzymes or efflux transporters affected futile cycling.

The objective of this study was to precipitate CaCO₃in a groundwater media to reduce dissolved arsenic concentrations. In this study a mixture of ureolytic calcite and aragonite were precipitated using groundwater as the media. Although precipitation of carbonate was successful using Ardkenneth groundwater, arsenic concentrations were not reduced. Ureolytic calcite and aragonite precipitated using broth as the media and resulted in a decrease in arsenic concentrations of up to 88% from the initial 0.7 μg L-1 concentration. Ureolytic carbonate precipitation required the inoculation of ureolytic cultures isolated from groundwater into both the groundwater and broth media. Precipitates in the inoculation experiments were identified using infrared spectroscopy techniques.<p>The decrease in arsenic concentrations in the inoculated urea treated broth samples compared to the groundwater samples was attributed to the greater amounts of precipitate formed in the broth media. The broth had a free Ca(II) concentration of 1300 mg L-1 whereas the Ardkenneth groundwater had a free Ca(II) concentration of 36 to 42 mg L-1. The higher free Ca(II) concentrations in the broth media would account for the higher yield of carbonate precipitate, making Ca(II) concentration a limiting factor in ureolytic CaCO3 remediation techniques. <p>Formation of a visible precipitate required the addition of nitrate to the broth and groundwater samples. The inoculated cultures, being denitrifiers, required a nitrate source. Ca(II) ion concentrations decreased in the different media without the addition of nitrate, but no visible precipitate formed.<p> Laboratory experiments using Ardkenneth groundwater and treatments of 0.03 M urea did not decrease the Ca(II) ion concentrations or reduce arsenic in solution. These results suggest that inoculation with selected ureolytic cultures was needed to optimize the precipitation of CaCO3 in a natural groundwater system.<p> The results of this study suggest that arsenic was reduced by the precipitation of ureolytic CaCO₃. Arsenic reduced by ureolytic CaCO₃ precipitation required adequate levels of Ca(II) ions, higher than those found in the Ardkenneth aquifer. Successful precipitation of CaCO₃ by ureolytic organisms also required an adequate cell density. Thus, inoculation with ureolytic cultures optimized the broth and groundwater media for CaCO₃ precipitation.

Research was undertaken to examine different <i>in vitro</i> characteristics of probiotic bacteria, including <i>Lactobacillus acidophilus</i> ATCC® 11975, <i>Bifidobacterium infantis</i> ATCC 15697D, <i>Bifidobacterium catenulatum</i> ATCC® 27675 and <i>Bifidobacterium adolescentis</i> ATCC® 15703 in order to identify suitable strain(s) for encapsulation. Under simulated gastric conditions (pH 2.0), <i>L. acidophilus</i> was the most acid-tolerant strain (D-value 10.2 ± 0.8 min), and was able to survive for 30 min; whereas, the other tested probiotics underwent a rapid (within the first 5 min at pH 2.0) 4-5 log colony forming units (cfu)/mL loss in viability. All probiotics tested were able to survive 5 h exposure to 0.3% Oxgall bile at pH 5.8. The relative ranking of probiotic adherence to Caco-2 cells was determined to be: <i>L. acidophilus</i> > <i>B. catenulatum</i> > <i>B. adolescentis</i> > <i>B. infantis</i>, which correlated with 4.5 104, 3.1 103, 2.6 101, and 1.5 101 cfu/mL associated with Caco-2 cell monolayers, respectively. The most hydrophobic probiotics included <i>L. acidophilus</i> (46.5 ± 6.1%) and B. catenulatum (65.5 ± 5.2%); their hydrophobicity were positively correlated with auto-aggregation ability. Addition of divalent cations, EDTA, and bile salts were found to affect hydrophobicity as well; for example, 0.5 mM MgCl2 resulted in a 20% increase in cell surface hydrophobicity of <i>L. acidophilus</i> from baseline levels; whereas, the addition of 0.1 and 0.5% bile salts decreased <i>L. acidophilus</i> hydrophobicity from control levels by 60 and 90%, respectively. Cell free culture supernatant of <i>L. acidophilus</i> effectively inhibited the growth of <i>Escherichia coli</i> O157:H7, and <i>Clostridium sordelli</i>. Bactericidal activity of <i>L. acidophilus</i> cell-free supernatant (the lethal factor was determined to be both heat and trypsin-resistant) against Escherichia coli O157:H7 and <i>Clostridium sordelli</i> ATCC 9714 over 24 h resulted in reductions of 5.5 and 3.5 log cfu/mL, respectively. Further examination of probiotics revealed varying degrees of resistance to the iv antimicrobial agents ciprofloxacin (4 ìg/mL), naladixic acid (32 ìg/mL), kanamycin (64 ìg/mL) and sulfisoxazone (256 ìg/mL). Determination of carbon source utilization patterns indicated that <i>B. catenulatum</i> utilized a number of carbohydrates including -methyl-D-glucoside, D-xylose, D-cellobiose, and -D-lactose; whereas,<i>L. acidophilus, B. infantis</i>, and <i>B. adolescentis</i> utilized D-xylose. <i>Lactobacillus acidophilus</i> was ultimately selected for encapsulation in a 3 mm diameter pea protein-alginate matrix followed by <i>in vitro</i> challenge to simulated gastric conditions (pH 2.0). Encapsulation of <i>L. acidophilus</i> demonstrated a significant (P < 0.05) protective effect during the 2 h exposure to simulated acidic stomach conditions; within capsules, there was approximately 1 log cfu/mL loss in cell viability, whereas unprotected cells experienced > 6 log/mL loss in cell viability over the same period.

Ph.D.

Le récepteur ionotropique P2X7 est impliqué dans diverses fonctions physiologiques telles que la prolifération, l’apoptose, la réponse inflammatoire et le trafic membranaire dans plusieurs types cellulaires. Cependant, peu est connu quant aux rôles physiologiques de P2X7 dans les cellules épithéliales intestinales (CEIs). Dernièrement, un collègue a démontré une nouvelle fonction pour P2X7 qui est de réguler l’internalisation du transporteur à glucose GLUT2. Comme l’un des rôles physiologiques majeurs des CEIs est l’absorption du glucose, la régulation de ce transporteur est d’une importance capitale. Par contre, nous ne savons pas ce qui permet de réguler l’expression du récepteur P2X7 dont la disponibilité pourrait être un des facteurs déterminants dans la régulation de l’absorption de glucose par le transporteur GLUT2. Ceci nous a menés à l’hypothèse suivante qui dit que le glucose lui-même pourrait stimuler certaines voies de signalisation menant à l’activation de facteurs de transcription, qui pourraient réguler l’expression du récepteur P2X7. Les objectifs de mes travaux de maîtrise étaient de déterminer si des variations dans la concentration de glucose affectaient l’expression de P2X7 dans les CEIs et d’identifier les facteurs de transcription impliqués dans ce processus. Les résultats obtenus démontrent que l’expression de P2X7 semble être affectée par la concentration de glucose. Cette régulation de l’expression de P2X7 en réponse au glucose semble impliquer les facteurs de transcription C/EBP? et ß. En fait, les essais d’immunoprécipitation de la chromatine (ChIP) effectués confirment que ces facteurs pouvaient lier le promoteur de P2X7 et que la liaison de C/EBPß au promoteur varie en fonction de la concentration de glucose. La capacité de liaison de C/EBPß au promoteur est comparable au niveau d’expression du gène du récepteur P2X7. In vivo, nous avons observé une diminution de l’expression du récepteur P2X7 dans les extraits de jéjunum de souris invalidées pour C/EBPß. Finalement, dans le modèle de souris diabétique NOD, il semble y avoir un dérèglement dans l’expression du récepteur P2X7, ce qui pourrait indiquer que P2X7 a bel et bien un rôle dans la régulation de l’homéostasie du glucose. [symboles non conformes]