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Atividade antioxidante e antitumoral em extratos de Jatropha curcas L.Rocha, Nubia Ferreira January 2013 (has links)
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ROCHA, Núbia Ferreira.pdf: 2379770 bytes, checksum: 79e3ae0d02aa9ab0df2209233f207277 (MD5) / O pinhão manso (Jatropha curcas L.) é uma planta versátil que ultimamente tem sido utilizada como opção em diversas áreas e na medicina popular. Estudos demonstram seu potencial para o tratamento de constipação, doenças parasitárias, como antimicrobiano, antifúngico, antiinflamatório, analgésico, antipirético, antiviral, antioxidante e atividade contra algumas linhagens tumorais. Dentre os diversos fatores extrínsecos e intrínsecos apontados como responsáveis para o surgimento do câncer, um dos mais discutidos atualmente têm sido os radicais livres ou espécies reativas de oxigênio (ERO), moléculas instáveis, quimicamente reativos produzidos ao longo do metabolismo celular normal, no entanto, podem causar estresse oxidativo que tem sido descrito como um dos principais precursores de doenças como aterosclerose, catarata, doenças neurodegenerativas em especial o câncer. Antioxidantes são moléculas que têm como propriedade o bloqueio, a inibição ou o retardo da deterioração oxidativa, reduzindo ação de radicais livres. O arsenal terapêutico antineoplásico atualmente disponível não são específicos levando à morte de células cancerígenas, como de células normais desencadeando o aparecimento dos efeitos colaterais. Assim, novos compostos que apresentem seletividade são requeridos e recentemente foram introduzidos quimioterápicos de origem natural no tratamento do câncer, o que valida à busca de novos alvos farmacológicos a partir de produtos naturais, principalmente, direcionado às plantas usadas na medicina popular. Nesse contexto, este estudo objetivou avaliar o perfil fitoquímico, a atividade antioxidante e a ação antitumoral de extratos etanólicos de Jatropha curcas L., sob linhagem tumoral in vitro. Para tanto, foram preparados extratos frescos e secos de raiz, caule, folha e sementes por dois métodos de extração (maceração e uso de Soxhlet), foi então realizada a identificação dos grupos fitoquímicos e a triagem destes extratos através do efeito antiproliferativo sob a linhagem HepG2. Para avaliação da atividade antioxidante foram realizadas análises através dos métodos de sequestro do radical livre 2,2-difenil-1- picrilhidrazil (DPPH) e sequestro do radical livre 2,2′-Azino-bis (3-etilbenzotiazolina-6-ácido sulfônico) (ABTS). Os extratos de forma geral apresentaram a presença de flavonoides, taninos catéquicos e fenóis simples. Os rendimentos dos extratos obtidos por ambas as metodologias de extração mostraram diferenças significativas em termos de percentual. O rendimento dos extratos obtidos pelo método de soxhlet apresentou de forma geral menor percentual em relação ao rendimento dos extratos obtidos por maceração, variando entre 1,24 a 24, 96 %, e não tendo relação direta com a atividade antioxidante do extrato. Verificou-se que o extrato preparado com soxhlet de amostras secas de folha de J. curcas apresentou melhor atividade antioxidante atingindo o percentual superior a 80%, quando avaliado pelo método do DPPH enquanto que os extratos obtidos por maceração de sementes frescas (98,54%) e por soxhlet de sementes secas (97,69%) apresentaram melhor capacidade antioxidante usando o método do ABTS. O extrato de folhas secas obtido por soxhlet apresentou atividade antioxidante pelo método do DPPH de 83,1% / EC50 - 47,46 μg/mL,enquanto que o extrato obtido por maceração de folhas frescas foi de 68,1% / EC50 – 52,88 μg/mL, correspondendo aos menores valores de EC50 dos extratos brutos avaliados pelo método do DPPH. Os extratos etanólicos secos de raiz, caule e folhas obtidos por soxhlet, apresentaram melhor efeito antitumoral sobre as células tumorais da linhagem HepG2 e não foram citotóxicos quando avaliados em macrófagos peritoneias. Conclui-se que extratos etanólicos brutos de folhas de Jatropha curcas L. possui atividade antioxidantee extratos etanólicos de raiz e caule promovem efeito antiproliferativo em células tumorais da linhagem HepG2. Conclui-se que a atividade antintumoral sob linhagem HepG2 e antioxidante dos extratos etanólicos de Jatropha curcas L. avaliada através do método de sequestro do radical DPPH e do ensaio ABTS varia em função do método de preparo do extrato, das amostras utilizadas (peso úmido e peso seco) e das partes botânicas analisadas. Este trabalho confirma o potencial antioxidante e antitumoral de extratos etanólicos de Jatropha curcas L.
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Health-promoting phytochemicals: (1) in response to environmental factors in lettuce, spinach and tomatoes; (2) development of 3D cell culture model for potential anticancer roleXu, Jingwen January 1900 (has links)
Doctor of Philosophy / Food Science Institute / Channa B. Rajashekar / Weiqun Wang / As health-promoting agents, phytochemicals are biosynthesized in the plants that typically respond to environmental stresses. This study focused on the analysis of phytochemical contents in vegetables in response to environmental changes of high tunnel and light spectra. A potential anticancer activity was further studied by developing a novel 3D cell culture model. Three specific studies were conducted as follows.
Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific.
Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species.
Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 μM. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies.
Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application.
Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific.
Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species.
Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 M. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies.
Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application.
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Avaliação do efeito do micronutriente ferro (Fe) na viabilidade celular e estabilidade genômica de culturas celulares de fibroblasto pulmonar (MRC5) e hepatorcarcinoma (HepG2) humanosArigony, Ana Lúcia Vargas January 2013 (has links)
Micronutrientes, vitaminas e minerais, são indispensáveis para as vias de metabolismo do DNA e, além disso, são tão importantes para a manutenção da vida quanto os macronutrientes. Na ausência dos nutrientes adequados, a instabilidade genômica compromete a homeostase, ocasionando doenças crônicas e certos tipos de câncer. Meios de cultura celular tem por finalidade mimetizar o ambiente in vivo, proporcionando aos modelos in vitro condições adequadas para que se avalie a resposta celular aos diferentes estímulos. O artigo de revisão sumariza e discute os micronutrientes usados na suplementação das culturas celulares e sua influência na a viabilidade celular e a estabilidade genômica, focando nos estudos in vitro previamente realizados. Nestes estudos, os meios de cultura celular incluem certas vitaminas e minerais em concentrações distintas das fisiológicas in vivo. Em muitos meios de cultura comumente usados, a única fonte de micronutrientes é o Soro Fetal Bovino (SFB), o qual contribui com 5-10% da composição final do meio. Atenção insuficiente tem sido direcionada à composição de SFB, micronutrientes e culturas celulares como um todo, ou à influência de micronutrientes na viabilidade e genética de culturas celulares. Estudos adicionais avaliando melhor o papel de micronutrientes no nível molecular e a sua influência na estabilidade genômica de células ainda se fazem necessários. O micronutriente foco dessa tese é o Ferro (Fe), que por sua vez é um micronutriente essencial, sendo requerido para o crescimento, desenvolvimento e condições normais de funcionamento das células. Tanto seu excesso quanto a sua deficiência podem causar estresse oxidativo e dano ao DNA. Uma vez que os meios de cultura usualmente utilizados para culturas celulares têm níveis de Fe abaixo das concentrações encontradas no soro fisiológico humano, os objetivos deste estudo foram a avaliação do papel da suplementação com Fe na viabilidade celular, na produção de espécies reativas de oxigênio (ERO), na atividade da catalase, na integridade genômica, na expressão de proteínas de reparo de DNA que contém clusters Fe/S em sua estrutura (TFIIH e MutyH) e na expressão de receptores de absorção de Fe (CD71 e Nramp2). Duas linhagens celulares – MRC5 (fibroblasto pulmanar humano) e HepG2 (hepatocarcinoma) - e dois tipos de suplementação com Fe foram utilizados, holo-Transferrina (h-Tf) e FeSO4. Ambas suplementações foram capazes de aumentar os níveis intracelulares de Fe e a viabilidade genômica. A suplementação com Fe também aumentou a formação de ERO, sem alterar a atividade da catalase. No entanto, este aumento de ERO não foi acompanhado por genotoxicidade. No que se refere à expressão de proteínas de reparo ao DNA, os resultados sugerem que o pré-tratamento com h-Tf ou FeSO4 não exercem influência direta na expressão de TFIIH ou MutyH. Entretanto, na expressão de receptores de Fe, os resultados preliminares indicam que CD71 é uma via prioritária de absorção de Fe, estando relacionada com a homeostase de Fe, enquanto Nramp2 parece ter um papel secundário. Devido à importância fisiológica da h-Tf na homeostase do Fe e o acúmulo de ERO menos pronunciado, sugere-se que h-Tf seja uma melhor forma para a suplementação de Fe nas culturas in vitro. Estudos adicionais se fazem necessários para a melhor elucidação do papel do Fe na viabilidade celular e estabilidade genômica. / Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. The review summarizes and discusses studies of cellculture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of culture cells. Further studies better evaluating micronutrients’ roles at a molecular level and its influence on the genomic stability of cells is still required. The micronutrient focus on this thesis is Iron (Fe), which is an essential micronutrient and is required for growth, development, and normal cellular functioning. Either excess or deficiency of iron can cause oxidative stress and DNA damage Since the cell media commonly used for cell culture has a lower iron concentration than the human serum, this study aimed to evaluate the role of iron supplementation on viability, reactive oxygen species (ROS) production, catalase activity, genome integrity and the expression of iron-bearing DNA repair proteins (TFIIH and MutyH) and proteins associated with iron absorption (CD71 and Nramp2). Two human cell lines – MRC5 (normal lung fibroblast) and HepG2 (hepatocellular carcinoma) and 2 sources of iron - holo-Transferrin (h-Tf) or FeSO4 were used. Both iron supplements were able to increase intracellular iron levels and cell viability. Iron supplementation increased the formation of ROS, but did not alter catalase activity. However, this increase was not accompanied by genotoxicity. Regarding the DNA repair protein expressions, the results suggest that 24h pre-treatment with h-Tf or FeSO4 has no role in the TFIIH or MutyH expressions. Although, in iron receptor proteins expression, the preliminary data could indicate that CD71 is priority related with Fe homeostasis while Nramp2 seems to have a secondary role. Due to h-Tf physiological role in the iron homeostasis and the less pronounced ROS accumulation, h-Tf could be a better iron supplier in vitro. Additional studies are still required to better elucidate the role of Fe in cell viability and genomic stability.
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Effets d'une surexpression stable de l'apolipoprotéine E dans les lignées cellulaires humaines SW872 et HepG2Carmel, Jean-François January 2005 (has links)
No description available.
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Purifica??o, caracteriza??o e efeitos imunomodulat?rio e antiproliferativo de uma lectina do fungo Clavaria cristata (Holmsk.) PersCunha, Dayse Caroline Severiano da 08 October 2010 (has links)
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Previous issue date: 2010-10-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / A 140,0 kDa lectin was purified and characterized from the mushroom Clavaria cristata. The purification procedures from the crude extract of the mushroom comprised gel filtration chromatography on Sephacryl s200 and ion exchange on Resource Q column. The purified lectin agglutinated all types of human erythrocytes with preference for trypsinized type O erythrocytes. The haemagglutinating activity is dependent of Ca 2+ ions and was strongly inhibited by the glycoprotein bovine submaxillary mucin (BSM) up to the concentration of 0, 125 mg/mL. The C. cristata lectin (CcL) was stable in the pH
range of 2,5-11,5 and termostable up to 80 ?C. CcL molecular mass determined by gel filtration on a Superose 6 10 300 column was approximately 140,3 kDa. SDS polyacrilamide gel electrophoresis revealed a single band with a molecular mass of
approximately 14,5 kDa, when the lectin was heated at 100 ?C in the presence or absence of ?-mercaptoethanol. CcL induced activation of murine peritoneal macrophages in vitro resulting in the release of nitric oxide (NO), reaching the maximum production at 24 h. In experimental paw oedema model in mice, CcL showed proinflammatory activity being able to induce oedema formation. Cell viability of HepG2, MDA 435 e 3T3 cell lines was examined after 72 h of incubation with CcL in different
concentrations (0,5-50 ?g/mL). CcL inhibited HepG2 cells growth with an IC50 value of 50 ?g/mL. In the present work, the observed immunomodulatory and antiproliferative
effects indicate CcL as a possible immunomodulator compound, interfering in the macrophages immune response, taking possible anti-parasitic, anti-tumoral effects or diagnostic and/or therapeutic / Uma lectina de 140,0 kDa foi purificada e caracterizada a partir do extrato prot?ico do fungo Clavaria cristata. O processo de purifica??o a partir do extrato bruto do fungo compreendeu uma cromatografia de gel filtra??o SEPHACRYL S200 e uma cromatografia de troca i?nica Resource Q em sistema FPLC-AKTA (Fast Protein Liquid Chromatography). A lectina de C. cristata (CcL) aglutinou todos os tipos de eritr?citos
humanos com prefer?ncia pelos do tipo O tratados com tripsina. A atividade hemaglutinante de CcL se mostrou dependente do ?on c?lcio e foi fortemente inibida pela glicoprote?na mucina bovina (BSM) at? a concentra??o m?nima de 0,125 mg/mL. CcL foi est?vel numa ampla faixa de pH, que variou entre 2,5-11,5 e termoest?vel at? 80?C por uma hora. A massa molecular da CcL, determ inada por cromatografia de gel
filtra??o Superose 6 10 300 GL em sistema de FPLC-AKTA foi de aproximadamente 140,3 kDa e uma eletroforese SDS-PAGE revelou uma ?nica banda com massa molecular de aproximadamente 14,5 kDa quando a lectina foi aquecida ? temperatura de 100 ?C. CcL induziu a ativa??o de macr?fagos murinos in vitro com conseq?ente libera??o de ?xido n?trico atingindo a m?xima produ??o de ?xido n?trico no tempo de 24
h. Em modelo experimental de edema de pata em camundongos, a lectina do fungo apresentou atividade pr?-inflamat?ria sendo capaz de induzir a forma??o do edema. A viabilidade celular das linhagens celulares HepG2, MDA 435 e 3T3 foi analisada ap?s
incuba??o por 72 h com concentra??es de CcL (0,5-50 ?g/mL). O valor do IC50 foi obtido com a concentra??o de CcL de 50 ?g/mL para linhagem de c?lulas HepG2. No presente trabalho, os efeitos imunomodulat?rios e antiproliferativos foram observados
apontando a CcL como um poss?vel imunomodulador, interferindo na resposta imune de macr?fagos levando a poss?veis efeitos anti-parasit?rios, anti-tumorais ou agente diagn?stico e/ou terap?utico
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Avaliação do efeito do micronutriente ferro (Fe) na viabilidade celular e estabilidade genômica de culturas celulares de fibroblasto pulmonar (MRC5) e hepatorcarcinoma (HepG2) humanosArigony, Ana Lúcia Vargas January 2013 (has links)
Micronutrientes, vitaminas e minerais, são indispensáveis para as vias de metabolismo do DNA e, além disso, são tão importantes para a manutenção da vida quanto os macronutrientes. Na ausência dos nutrientes adequados, a instabilidade genômica compromete a homeostase, ocasionando doenças crônicas e certos tipos de câncer. Meios de cultura celular tem por finalidade mimetizar o ambiente in vivo, proporcionando aos modelos in vitro condições adequadas para que se avalie a resposta celular aos diferentes estímulos. O artigo de revisão sumariza e discute os micronutrientes usados na suplementação das culturas celulares e sua influência na a viabilidade celular e a estabilidade genômica, focando nos estudos in vitro previamente realizados. Nestes estudos, os meios de cultura celular incluem certas vitaminas e minerais em concentrações distintas das fisiológicas in vivo. Em muitos meios de cultura comumente usados, a única fonte de micronutrientes é o Soro Fetal Bovino (SFB), o qual contribui com 5-10% da composição final do meio. Atenção insuficiente tem sido direcionada à composição de SFB, micronutrientes e culturas celulares como um todo, ou à influência de micronutrientes na viabilidade e genética de culturas celulares. Estudos adicionais avaliando melhor o papel de micronutrientes no nível molecular e a sua influência na estabilidade genômica de células ainda se fazem necessários. O micronutriente foco dessa tese é o Ferro (Fe), que por sua vez é um micronutriente essencial, sendo requerido para o crescimento, desenvolvimento e condições normais de funcionamento das células. Tanto seu excesso quanto a sua deficiência podem causar estresse oxidativo e dano ao DNA. Uma vez que os meios de cultura usualmente utilizados para culturas celulares têm níveis de Fe abaixo das concentrações encontradas no soro fisiológico humano, os objetivos deste estudo foram a avaliação do papel da suplementação com Fe na viabilidade celular, na produção de espécies reativas de oxigênio (ERO), na atividade da catalase, na integridade genômica, na expressão de proteínas de reparo de DNA que contém clusters Fe/S em sua estrutura (TFIIH e MutyH) e na expressão de receptores de absorção de Fe (CD71 e Nramp2). Duas linhagens celulares – MRC5 (fibroblasto pulmanar humano) e HepG2 (hepatocarcinoma) - e dois tipos de suplementação com Fe foram utilizados, holo-Transferrina (h-Tf) e FeSO4. Ambas suplementações foram capazes de aumentar os níveis intracelulares de Fe e a viabilidade genômica. A suplementação com Fe também aumentou a formação de ERO, sem alterar a atividade da catalase. No entanto, este aumento de ERO não foi acompanhado por genotoxicidade. No que se refere à expressão de proteínas de reparo ao DNA, os resultados sugerem que o pré-tratamento com h-Tf ou FeSO4 não exercem influência direta na expressão de TFIIH ou MutyH. Entretanto, na expressão de receptores de Fe, os resultados preliminares indicam que CD71 é uma via prioritária de absorção de Fe, estando relacionada com a homeostase de Fe, enquanto Nramp2 parece ter um papel secundário. Devido à importância fisiológica da h-Tf na homeostase do Fe e o acúmulo de ERO menos pronunciado, sugere-se que h-Tf seja uma melhor forma para a suplementação de Fe nas culturas in vitro. Estudos adicionais se fazem necessários para a melhor elucidação do papel do Fe na viabilidade celular e estabilidade genômica. / Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. The review summarizes and discusses studies of cellculture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of culture cells. Further studies better evaluating micronutrients’ roles at a molecular level and its influence on the genomic stability of cells is still required. The micronutrient focus on this thesis is Iron (Fe), which is an essential micronutrient and is required for growth, development, and normal cellular functioning. Either excess or deficiency of iron can cause oxidative stress and DNA damage Since the cell media commonly used for cell culture has a lower iron concentration than the human serum, this study aimed to evaluate the role of iron supplementation on viability, reactive oxygen species (ROS) production, catalase activity, genome integrity and the expression of iron-bearing DNA repair proteins (TFIIH and MutyH) and proteins associated with iron absorption (CD71 and Nramp2). Two human cell lines – MRC5 (normal lung fibroblast) and HepG2 (hepatocellular carcinoma) and 2 sources of iron - holo-Transferrin (h-Tf) or FeSO4 were used. Both iron supplements were able to increase intracellular iron levels and cell viability. Iron supplementation increased the formation of ROS, but did not alter catalase activity. However, this increase was not accompanied by genotoxicity. Regarding the DNA repair protein expressions, the results suggest that 24h pre-treatment with h-Tf or FeSO4 has no role in the TFIIH or MutyH expressions. Although, in iron receptor proteins expression, the preliminary data could indicate that CD71 is priority related with Fe homeostasis while Nramp2 seems to have a secondary role. Due to h-Tf physiological role in the iron homeostasis and the less pronounced ROS accumulation, h-Tf could be a better iron supplier in vitro. Additional studies are still required to better elucidate the role of Fe in cell viability and genomic stability.
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Avaliação do efeito do micronutriente ferro (Fe) na viabilidade celular e estabilidade genômica de culturas celulares de fibroblasto pulmonar (MRC5) e hepatorcarcinoma (HepG2) humanosArigony, Ana Lúcia Vargas January 2013 (has links)
Micronutrientes, vitaminas e minerais, são indispensáveis para as vias de metabolismo do DNA e, além disso, são tão importantes para a manutenção da vida quanto os macronutrientes. Na ausência dos nutrientes adequados, a instabilidade genômica compromete a homeostase, ocasionando doenças crônicas e certos tipos de câncer. Meios de cultura celular tem por finalidade mimetizar o ambiente in vivo, proporcionando aos modelos in vitro condições adequadas para que se avalie a resposta celular aos diferentes estímulos. O artigo de revisão sumariza e discute os micronutrientes usados na suplementação das culturas celulares e sua influência na a viabilidade celular e a estabilidade genômica, focando nos estudos in vitro previamente realizados. Nestes estudos, os meios de cultura celular incluem certas vitaminas e minerais em concentrações distintas das fisiológicas in vivo. Em muitos meios de cultura comumente usados, a única fonte de micronutrientes é o Soro Fetal Bovino (SFB), o qual contribui com 5-10% da composição final do meio. Atenção insuficiente tem sido direcionada à composição de SFB, micronutrientes e culturas celulares como um todo, ou à influência de micronutrientes na viabilidade e genética de culturas celulares. Estudos adicionais avaliando melhor o papel de micronutrientes no nível molecular e a sua influência na estabilidade genômica de células ainda se fazem necessários. O micronutriente foco dessa tese é o Ferro (Fe), que por sua vez é um micronutriente essencial, sendo requerido para o crescimento, desenvolvimento e condições normais de funcionamento das células. Tanto seu excesso quanto a sua deficiência podem causar estresse oxidativo e dano ao DNA. Uma vez que os meios de cultura usualmente utilizados para culturas celulares têm níveis de Fe abaixo das concentrações encontradas no soro fisiológico humano, os objetivos deste estudo foram a avaliação do papel da suplementação com Fe na viabilidade celular, na produção de espécies reativas de oxigênio (ERO), na atividade da catalase, na integridade genômica, na expressão de proteínas de reparo de DNA que contém clusters Fe/S em sua estrutura (TFIIH e MutyH) e na expressão de receptores de absorção de Fe (CD71 e Nramp2). Duas linhagens celulares – MRC5 (fibroblasto pulmanar humano) e HepG2 (hepatocarcinoma) - e dois tipos de suplementação com Fe foram utilizados, holo-Transferrina (h-Tf) e FeSO4. Ambas suplementações foram capazes de aumentar os níveis intracelulares de Fe e a viabilidade genômica. A suplementação com Fe também aumentou a formação de ERO, sem alterar a atividade da catalase. No entanto, este aumento de ERO não foi acompanhado por genotoxicidade. No que se refere à expressão de proteínas de reparo ao DNA, os resultados sugerem que o pré-tratamento com h-Tf ou FeSO4 não exercem influência direta na expressão de TFIIH ou MutyH. Entretanto, na expressão de receptores de Fe, os resultados preliminares indicam que CD71 é uma via prioritária de absorção de Fe, estando relacionada com a homeostase de Fe, enquanto Nramp2 parece ter um papel secundário. Devido à importância fisiológica da h-Tf na homeostase do Fe e o acúmulo de ERO menos pronunciado, sugere-se que h-Tf seja uma melhor forma para a suplementação de Fe nas culturas in vitro. Estudos adicionais se fazem necessários para a melhor elucidação do papel do Fe na viabilidade celular e estabilidade genômica. / Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells’ responses to different stimuli. The review summarizes and discusses studies of cellculture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of culture cells. Further studies better evaluating micronutrients’ roles at a molecular level and its influence on the genomic stability of cells is still required. The micronutrient focus on this thesis is Iron (Fe), which is an essential micronutrient and is required for growth, development, and normal cellular functioning. Either excess or deficiency of iron can cause oxidative stress and DNA damage Since the cell media commonly used for cell culture has a lower iron concentration than the human serum, this study aimed to evaluate the role of iron supplementation on viability, reactive oxygen species (ROS) production, catalase activity, genome integrity and the expression of iron-bearing DNA repair proteins (TFIIH and MutyH) and proteins associated with iron absorption (CD71 and Nramp2). Two human cell lines – MRC5 (normal lung fibroblast) and HepG2 (hepatocellular carcinoma) and 2 sources of iron - holo-Transferrin (h-Tf) or FeSO4 were used. Both iron supplements were able to increase intracellular iron levels and cell viability. Iron supplementation increased the formation of ROS, but did not alter catalase activity. However, this increase was not accompanied by genotoxicity. Regarding the DNA repair protein expressions, the results suggest that 24h pre-treatment with h-Tf or FeSO4 has no role in the TFIIH or MutyH expressions. Although, in iron receptor proteins expression, the preliminary data could indicate that CD71 is priority related with Fe homeostasis while Nramp2 seems to have a secondary role. Due to h-Tf physiological role in the iron homeostasis and the less pronounced ROS accumulation, h-Tf could be a better iron supplier in vitro. Additional studies are still required to better elucidate the role of Fe in cell viability and genomic stability.
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Génotoxicité et potentiel perturbateur endocrinien de contaminants de l'aliment : modèle cellulaire Hep-G2 - mécanismes moléculaires / Genotoxicity and endocrine disruptor effects of food contaminants : hepG2 cell model - molecular mechanismsDumont, Coralie 15 October 2010 (has links)
L’alimentation peut être à l’origine d’une exposition aux xénobiotiques. Les différentes étapes entre la production et la consommation peuvent être sources de contaminations de cet aliment. L’objectif de ce travail de thèse est de vérifier deux toxicités s’exprimant à faible dose : la génotoxicité et la perturbation endocrinienne. Les xénobiotiques étudiés sont une dioxine (la TCDD, polluant de l’environnement), le glyphosate et ses formulations de Roundup (pesticides), le 5-hydroxyméthylfurfural (molécule néoformée) et le bisphénol F (contaminant d’emballage). Le modèle cellulaire choisi est la lignée cellulaire HepG2, issue d’un hépatocarcinome d’origine humaine. Ces cellules sont un modèle pertinent car elles possèdent des capacités métaboliques bien caractérisées et que les contaminants étudiés sont tous hépatotoxiques. Les résultats ont permis de montrer, avec un test d’activation transcriptionnelle, que les quatre formulations de Roundup étaient antioestrogèniques et antiandrogèniques. Le glyphosate ne présentait que des effets antiandrogèniques. De plus, les formulations modifient l’activité de l’aromatase dans les cellules HepG2. Après quatre heures de contact, le Roundup à 400 g/L présente des effets génotoxiques mais non liés à un stress oxydatif, dans le test des comètes. Il présente également des effets apoptotiques. Les différences observées entre les formulations et les différents éléments de la formulation (le glyphosate et l’adjuvant POEA) suggèrent un « effet mélange ». Le modèle HepG2 a également permis de montrer que le 5-HMF est une molécule progénotoxique à des concentrations non cytotoxiques dans le test des comètes. De plus, il a permis de mettre en évidence le fait que le BPF est la molécule la plus active en matière de génotoxicité et de perturbation endocrinienne, par comparaison avec ses métabolites. Parallèlement, la mise en place d’un modèle stablement transfecté issu des cellules HepG2 a été initiée pour vérifier les potentiels effets (anti)oestrogèniques. Parallèlement, les mécanismes cellulaires et moléculaires à l’origine des effets toxiques observés ont été étudiés. Ainsi, il a été montré que les cellules HepG2 métabolisent le BPF (30% à 25 μM) en sulfoconjugué alors que les hépatocytes humains le métabolisent en sulfoconjugué et/ou glucuroconjugué (100% à 25 μM) avec une différence interindividuelle. Enfin, il a été vérifié, in vitro et dans les conditions expérimentales de l’étude le rôle de ERalpha dans les effets toxiques de la TCDD. Ainsi, un effet anti-oestrogènique de la TCDD sur ERE et un effet potentialisateur de E2 sur l’effet de la TCDD sur XRE ont été mis en évidence. Les tests in vitro ont permis la mise en évidence d’effets toxiques de contaminants de l’alimentation et ont ainsi leur place dans l’évaluation du risque. / Food can expose Human to xenobiotics. Indeed, the various steps between production and consumption can be at the origin of food contaminations, either with natural or chemical substances. The objective of this work was to test two toxicities exhibited at low doses: genotoxicity and endocrine disruption. Xenobiotics studied are a dioxin (TCDD, environmental pollutant), glyphosate and different Roundup formulations (pesticides), 5-hydroxymethylfurfural (neoformed compound) and bisphenol F (food packaging contaminant). The model is the HepG2 cell line derived from a human hepatocarcinoma. These cells are chose because they have well-characterized metabolic capacities and all contaminants studied are hepatotoxic. Using transcriptional activation assay, we have shown that the four formulations of Roundup were anti-estrogenic and anti-androgenic. Glyphosate was only anti-androgenic. Furthermore, formulations were able to modify the aromatase activity in HepG2 cells. After 4 hours of contact, the formulation at 400g/L induced genotoxic effect (comet assay) which was not correlated to an oxidative stress or an apoptotic effect (caspase 3,7 activation). The differences observed between the formulation and its components (glyphosate and POEA) suggest a “mixture effect”. We have shown that 5-HMF is a progenotoxic molecule at noncytotoxic concentrations in the comet assay. Also, we have demonstrated that BPF is the most active molecule in genotoxicity and endocrine disruption assays compared to its metabolites. In parallel, the establishment of a stably transfected HepG2 cell line in order to assess the potential (anti)estrogenic effects was initiated.Cellular and molecular mechanisms involved in the toxic effects was also studied. Thus, HepG2 cells metabolized BPF to sulfate metabolite (30% at 25μM), whereas human hepatocytes produced the sulfate and / or glucuronide conjugates (100% at 25μM) with aninterindividual difference. Finally, in vitro and in our experimental conditions, the role of ERin the toxic effects of TCDD was investigated. Using transcriptional activity, TCDD was shown anti-estrogenic on ER. Furthermore, a potention of E2 on transcriptional activity of TCDD induced via AhR was demonstrated. Finally, in vitro assays was used to assess xenobiotic toxicity. They are relevant to in risk assessment.
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Understanding Liver Toxicity Induced by Polybrominated Diphenyl Ethers in Human HepatocytesRamoju, Siva P. January 2012 (has links)
Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.
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Metabolismus nových polysacharidických nanomateriálů pro biomedicinální aplikace / Metabolism of new polysacharidic nanomaterials for biomedicinal applicationsJirátová, Markéta January 2014 (has links)
Cancer is one of the leading cause of death in modern world, so there is an emerging demand for better diagnostic tools and more specific less toxique therapeutics. Nanoparticles offers characteristics that could fullfill such perspectives. They can easily target tumor by ehanced permeation and retention effect (EPR). Nanoparticles can combine more than one imaging properties, so we can say that they are multimodal, some of them could combine diagnostic and therapeutic molecules in one nanoparticle, which is now highly popular topic of nanoparticles for theranostics . The aim of this thesis was to characterize new multimodal glycogen-based nanoparticle. Glycogen is an ideal structure for nanoparticle design. Glycogen is part of natural dendrimers group which are easily to modify. Glycogen's size is suitable for EPR effect. We have evaluated biological characteristics of five different types of modified glycogen. The in vitro experiments were carried on HepG2 cells. We have set time curve of cellular uptake of this glycogen probes, evaluated cytoplasmatic localization and for the first time we have carried MTT assay. Biodistribution studies on CD1-Nude mice were performed by using non-invasive method for measuring in vivo fluorescence. In conlusion we've provided some of the biological characteristics of new...
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