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Eyre Sánchez Elena - One of the best experts on this subject based on the ideXlab platform.
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Aportacions a l’estudi de la relació inflamació-tumorigènesi en el còlon.
'Universitat Autonoma de Barcelona', 2015Co-Authors: Eyre Sánchez ElenaAbstract:La colitis ulcerosa (UC) s’associa a un alt risc de desenvolupar càncer colorectal (CRC). La resposta inflamatòria i, en particular, alguns receptors de la immunitat innata com els TLR, condicionen la resposta immune, intervenen en el manteniment de la funció barrera intestinal i en la modulació de mecanismes reparadors, processos relacionats amb la resolució o cronificació de la inflamació. En aquesta tesi hem explorat la relació entre mecanismes inflamatoris i tumorigènics en models animals, que permeten estudiar la transició inflamació-tumorigènesi sense les influències degudes a la farmacoteràpia, i en mostres humanes. En models murins de colitis, càncer colorectal i càncer colorectal associat a colitis hem analitzat les relacions entre els canvis histopatològics i l’expressió de gens, i hem valorat quines modificacions de l’expressió de microRNA les acompanyen, buscant la seva possible repercussió en processos d’inflamació, proliferació, apoptosi i distorsió de l’arquitectura tissular. També hem estudiat com es modifiquen els perfils fenotípics de cèl·lules mononuclears de cara a establir-ne el valor com a biomarcadors putatius de la transició colitis-càncer. En material biològic humà, els nostres esforços s’han adreçat a valorar l’expressió gènica en biòpsies de còlon obtingudes d’individus sans, de pacients amb UC i d’adenomes, de cara a seleccionar els gens que presenten canvis d’expressió associats a processos tumorals i inflamatoris. També hem valorat in vitro fins quin punt la funció barrera intestinal és afectada per les secrecions dels monòcits, per a establir la possible relació causal entre un comportament anòmal dels monòcits i la permeabilitat intestinal incrementada que es dóna en la UC. Els resultats obtinguts en els models murins ens han permès validar la correlació entre la inflamació intestinal i la promoció dels processos tumorigènics i constatar que la simple cronificació de la inflamació desorganitza la resposta reparadora provocant una marcada hiperplàsia i l’aparició de focus de criptes aberrants, fins i tot sense induir mutagènesi per administració d’Agents alquilants. En els animals que reben un Agent mutagen i pateixen episodis recurrents de colitis, els tumors són més nombrosos, més grans i presenten major grau de displàsia en relació als que s’observen sense el concurs d’episodis d’inflamació. Els tumors que apareixen en un context inflamatori presenten alteracions significatives en l’expressió dels gens tlr4, tlr9, cnnd1, cox2 i bcl2. Això suggereix una implicació dels gens esmentats en el desenvolupament de tumors associats a colitis. En aquests tumors s’evidencia també una disminució significativa de l'expressió de miR-135a, miR-21, miR-145, i miR-34a, fet que suposaria un increment d'expressió de les seves dianes. L’anàlisi predictiva amb eines bioinformàtiques ha permès seleccionar dianes d’interès en funció de la seva relació amb els processos de proliferació, inflamació, apoptosi, angiogènesi i organització tissular. Entre elles destaquem tiam1, vcl, stat3, ptk2, wnt1, pak4, p120, birc5, btg2, klf4, rtkn, i peli1. L’anàlisi de l’expressió gènica sobre micromatrius partint de còlon humà sà, de pacients d’UC i d’adenomes, ha permès verificar quins gens es troben significativament alterats. De l'anàlisi GO, la comparació entre els diferents grups, i la literatura, hem seleccionat un llistat de gens biomarcadors putatius del CRC i associats també a UC: brca1, brca2, nbn, tp53, smad3, fcer1g, a2m, dock2, atf4, cxcl12, itga8, eps8, rhou, nrp1 i ptk2b. Pel que fa als monòcits de sang perifèrica, l’estimulació amb lligands de TLR4 determina una resposta secretora diferent en els monòcits de pacients d’UC en comparació amb els d’individus sans. Aquest perfil secretor diferencial pot ser parcialment responsable d’un deteriorament més acusat la funció barrera intestinal en pacients d’UC, afavorint la cronificació de la inflamació i l’aparició de displàsies.Ulcerative colitis (UC) is associated to an increased risk to develop colorectal cancer (CRC). In the gut, inflammation and activation of receptors of innate immunity such as TLRs, accomplish several functions, including tailoring adaptive immune responses, deploying repair mechanisms and maintaining the intestinal barrier function. The tuning of such functions may result in resolution or chronification of the inflammation. Here we have explored the relationship between inflammatory and tumourigenic mechanisms in animal models - which allow the study of the inflammation-tumourigenesis transition without the biases due to pharmacotherapy -, and in human samples. We have analyzed the relationship between histopathological findings and gene expression variations in murine models of colitis, colorectal cancer and colitis associated colorectal cancer. The expression of microRNAs in these conditions has also been quantified and their potential repercussions in the expression of genes related to inflammation, proliferation, apoptosis and tissue organization has been explored. Changes in mononuclear cell phenotypic profiles have also been studied in order to assess their putative value as biomarkers of inflammation-tumourigenesis transition. In human samples, we intended to study gene expression in colon biopsies obtained from healthy individuals, UC patients and adenomas, in an attempt to find expression changes associated both to UC and CRC. Furthermore, we have evaluated in vitro to what extent the intestinal barrier function is affected by monocyte secretions, to establish whether an anomalous monocyte responsiveness may contribute to the enhanced intestinal permeability usually found in UC patients. The results obtained in the murine model show a strong correlation between intestinal inflammation and enhanced tumour development, probably resulting from a disregulation of repair mechanisms, as shown by the finding of a significant hyperplasia and increased occurrence of aberrant crypt foci. In animals receiving a Mutagenic Agent and undergoing recurrent colitic episodes, the tumours are larger, more abundant and exhibit a higher degree of dysplasia compared to tumours observed in mice receiving only the Mutagenic Agent. Tumours appearing in an inflammatory context display significant gene expression alterations in tlr4, tlr9, cnnd1, cox2, and bcl2. This suggests an involvement of these genes in colitis associated tumour development. Inflammation associated tumours also showed a significant decrease in miR-135a, miR-21, miR-145 and miR-34a expression, which may lead to an increased expression of their target mRNAs. Among them we have highlighted those involved in proliferation, inflammation, apoptosis, angiogenesis and tissue organization processes. We propose tiam1, vcl, stat3, ptk2, wnt1, pak4, p120, birc5, btg2, klf4, rtkn and peli1 as candidate genes for the follow up of the bias inflammation-tumourigenesis. Gene expression microarray analysis has allowed us to identify genes whose expression is significantly altered in biopsies taken from UC patients and colon adenomas compared to healthy tissue. On the basis of this comparison, literature and gene ontology analysis, we have selected a list of genes altered in CRC and also associated to UC, which may be useful as putative biomarker genes: bcra1, bcra2, ndn, tp53, smad3, fcer1g, a2m, dock2, atf4, cxcl12, itga8, eps8, rhou, nrp1 and ptk2b. Our results show too that TLR4 ligands elicit an enhanced secretory response in monocytes of UC patients compared to those of healthy individuals. This enhanced response induces increased disruptive effects on the intestinal barrier function in vitro, suggesting that an anomalous monocyte secretory profile may be partly responsible for the increased intestinal permeability found in UC patients, which may contribute to the chronification of colitis and promotion of dysplasia
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aportacions a l'estudi de la relació inflamació-tumorigènesi en el còlon.
[Barcelona] : Universitat Autònoma de Barcelona, 2015Co-Authors: Eyre Sánchez Elena, Universitat Autònoma De Barcelona. Departament De Biologia Cel·lular, DAbstract:La colitis ulcerosa (UC) s'associa a un alt risc de desenvolupar càncer colorectal (CRC). La resposta inflamatòria i, en particular, alguns receptors de la immunitat innata com els TLR, condicionen la resposta immune, intervenen en el manteniment de la funció barrera intestinal i en la modulació de mecanismes reparadors, processos relacionats amb la resolució o cronificació de la inflamació. En aquesta tesi hem explorat la relació entre mecanismes inflamatoris i tumorigènics en models animals, que permeten estudiar la transició inflamació-tumorigènesi sense les influències degudes a la farmacoteràpia, i en mostres humanes. En models murins de colitis, càncer colorectal i càncer colorectal associat a colitis hem analitzat les relacions entre els canvis histopatològics i l'expressió de gens, i hem valorat quines modificacions de l'expressió de microRNA les acompanyen, buscant la seva possible repercussió en processos d'inflamació, proliferació, apoptosi i distorsió de l'arquitectura tissular. També hem estudiat com es modifiquen els perfils fenotípics de cèl·lules mononuclears de cara a establir-ne el valor com a biomarcadors putatius de la transició colitis-càncer. En material biològic humà, els nostres esforços s'han adreçat a valorar l'expressió gènica en biòpsies de còlon obtingudes d'individus sans, de pacients amb UC i d'adenomes, de cara a seleccionar els gens que presenten canvis d'expressió associats a processos tumorals i inflamatoris. També hem valorat in vitro fins quin punt la funció barrera intestinal és afectada per les secrecions dels monòcits, per a establir la possible relació causal entre un comportament anòmal dels monòcits i la permeabilitat intestinal incrementada que es dóna en la UC. Els resultats obtinguts en els models murins ens han permès validar la correlació entre la inflamació intestinal i la promoció dels processos tumorigènics i constatar que la simple cronificació de la inflamació desorganitza la resposta reparadora provocant una marcada hiperplàsia i l'aparició de focus de criptes aberrants, fins i tot sense induir mutagènesi per administració d'Agents alquilants. En els animals que reben un Agent mutagen i pateixen episodis recurrents de colitis, els tumors són més nombrosos, més grans i presenten major grau de displàsia en relació als que s'observen sense el concurs d'episodis d'inflamació. Els tumors que apareixen en un context inflamatori presenten alteracions significatives en l'expressió dels gens tlr4, tlr9, cnnd1, cox2 i bcl2. Això suggereix una implicació dels gens esmentats en el desenvolupament de tumors associats a colitis. En aquests tumors s'evidencia també una disminució significativa de l'expressió de miR-135a, miR-21, miR-145, i miR-34a, fet que suposaria un increment d'expressió de les seves dianes. L'anàlisi predictiva amb eines bioinformàtiques ha permès seleccionar dianes d'interès en funció de la seva relació amb els processos de proliferació, inflamació, apoptosi, angiogènesi i organització tissular. Entre elles destaquem tiam1, vcl, stat3, ptk2, wnt1, pak4, p120, birc5, btg2, klf4, rtkn, i peli1. L'anàlisi de l'expressió gènica sobre micromatrius partint de còlon humà sà, de pacients d'UC i d'adenomes, ha permès verificar quins gens es troben significativament alterats. De l'anàlisi GO, la comparació entre els diferents grups, i la literatura, hem seleccionat un llistat de gens biomarcadors putatius del CRC i associats també a UC: brca1, brca2, nbn, tp53, smad3, fcer1g, a2m, dock2, atf4, cxcl12, itga8, eps8, rhou, nrp1 i ptk2b. Pel que fa als monòcits de sang perifèrica, l'estimulació amb lligands de TLR4 determina una resposta secretora diferent en els monòcits de pacients d'UC en comparació amb els d'individus sans. Aquest perfil secretor diferencial pot ser parcialment responsable d'un deteriorament més acusat la funció barrera intestinal en pacients d'UC, afavorint la cronificació de la inflamació i l'aparició de displàsies.Ulcerative colitis (UC) is associated to an increased risk to develop colorectal cancer (CRC). In the gut, inflammation and activation of receptors of innate immunity such as TLRs, accomplish several functions, including tailoring adaptive immune responses, deploying repair mechanisms and maintaining the intestinal barrier function. The tuning of such functions may result in resolution or chronification of the inflammation. Here we have explored the relationship between inflammatory and tumourigenic mechanisms in animal models - which allow the study of the inflammation-tumourigenesis transition without the biases due to pharmacotherapy -, and in human samples. We have analyzed the relationship between histopathological findings and gene expression variations in murine models of colitis, colorectal cancer and colitis associated colorectal cancer. The expression of microRNAs in these conditions has also been quantified and their potential repercussions in the expression of genes related to inflammation, proliferation, apoptosis and tissue organization has been explored. Changes in mononuclear cell phenotypic profiles have also been studied in order to assess their putative value as biomarkers of inflammation-tumourigenesis transition. In human samples, we intended to study gene expression in colon biopsies obtained from healthy individuals, UC patients and adenomas, in an attempt to find expression changes associated both to UC and CRC. Furthermore, we have evaluated in vitro to what extent the intestinal barrier function is affected by monocyte secretions, to establish whether an anomalous monocyte responsiveness may contribute to the enhanced intestinal permeability usually found in UC patients. The results obtained in the murine model show a strong correlation between intestinal inflammation and enhanced tumour development, probably resulting from a disregulation of repair mechanisms, as shown by the finding of a significant hyperplasia and increased occurrence of aberrant crypt foci. In animals receiving a Mutagenic Agent and undergoing recurrent colitic episodes, the tumours are larger, more abundant and exhibit a higher degree of dysplasia compared to tumours observed in mice receiving only the Mutagenic Agent. Tumours appearing in an inflammatory context display significant gene expression alterations in tlr4, tlr9, cnnd1, cox2, and bcl2. This suggests an involvement of these genes in colitis associated tumour development. Inflammation associated tumours also showed a significant decrease in miR-135a, miR-21, miR-145 and miR-34a expression, which may lead to an increased expression of their target mRNAs. Among them we have highlighted those involved in proliferation, inflammation, apoptosis, angiogenesis and tissue organization processes. We propose tiam1, vcl, stat3, ptk2, wnt1, pak4, p120, birc5, btg2, klf4, rtkn and peli1 as candidate genes for the follow up of the bias inflammation-tumourigenesis. Gene expression microarray analysis has allowed us to identify genes whose expression is significantly altered in biopsies taken from UC patients and colon adenomas compared to healthy tissue. On the basis of this comparison, literature and gene ontology analysis, we have selected a list of genes altered in CRC and also associated to UC, which may be useful as putative biomarker genes: bcra1, bcra2, ndn, tp53, smad3, fcer1g, a2m, dock2, atf4, cxcl12, itga8, eps8, rhou, nrp1 and ptk2b. Our results show too that TLR4 ligands elicit an enhanced secretory response in monocytes of UC patients compared to those of healthy individuals. This enhanced response induces increased disruptive effects on the intestinal barrier function in vitro, suggesting that an anomalous monocyte secretory profile may be partly responsible for the increased intestinal permeability found in UC patients, which may contribute to the chronification of colitis and promotion of dysplasia
Vera Vega, Miguel Angel - One of the best experts on this subject based on the ideXlab platform.
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Determinación de la dosimetría aplicada a la semilla de espárrago (Asparagus officinalis L.)
'Universidad Nacional Agraria la Molina', 2019Co-Authors: Vera Vega, Miguel AngelAbstract:Universidad Nacional Agraria La Molina. Facultad de Agronomía. Departamento Académico de FitotecniaEl espárrago (Asparagus officinalis L.) es una hortaliza muy cotizada tanto en nuestro país como a nivel mundial. En el Perú la producción de los cultivares espárrago se expande por el litoral peruano, en los departamentos de La Libertad, Lima, Ica, Ancash y Lambayeque. En la actualidad los cultivares liberados están perdiendo su capacidad de resistencia y tolerancia a las plagas y enfermedades, lo que obliga a los fitomejoradores a generar nuevos cultivares que respondan a estos nuevos desafíos. La primera acción del fitomejorador es generar variabilidad genética dentro de la población y una alternativa para lograrlo es la inducción de mutaciones. Esta técnica ayuda a generar variabilidad genética, permitiendo obtener y seleccionar los genotipos con tolerancia y/o resistencia a estreses bióticos y abióticos. El experimento se llevó a cabo en la PIPS en Cereales y Granos Nativos de la Universidad Nacional Agraria La Molina. Se trabajó con tres genotipos: UC157F1, UC115 y la 2H-38H cuyas semillas fueron tratadas. Se tuvieron dos controles (agua, buffer fosfato a pH 3) y siete concentraciones de azida de sodio (1mM, 3mM, 5mM, 7mM y 9mM). Las semillas fueron sumergidas por una hora y sembradas en placas y macetas (100 por repetición) y se utilizó un diseño DBCA con arreglo factorial con 4 repeticiones. Las variables estudiadas fueron las siguientes: germinación, supervivencia, altura de planta, longitud de radícula, relación de la longitud plúmula-radícula, peso del germinado, la velocidad de elongación de la plántula y radícula y ganancia de peso del germinado, DL50 y el efecto promedio del Agente mutagénico. Durante el análisis de las variables se observó que hubo diferencias significativas. Los tres genotipos mostraron respuestas diferentes al efecto de la azida de sodio, siendo el cultivar UC157F1 es más quimio-sensible y la dosis de 9mM de NaN3 causó el mayor efectoAsparagus (Asparagus officinalis L.) is a vegetable that is highly valued both in our country and worldwide. In Peru, the production of asparagus crops expands along the Peruvian coast, in the departments of La Libertad, Lima, Ica, Ancash and Lambayeque. Currently, the released cultivars are losing their resistance or tolerance to pests and diseases, forcing to plant breeders to generate new cultivars that respond to these new challenges. The first action of the breeder is to generate genetic variation in the population. This technique helps to increace genetic variability, it is achieved and selected genotypes with tolerance and / or resistance to biotic and abiotic stresses. The cape was taken to the PIPS in Cereals and Native Crops of La Molina National Agrarian University. Three genotypes were evaluated: UC157F1, UC115 and 2H-38H. The seed were submerged in the following treatments: water and phosphate buffer at pH 3 as control and 1mM, 3mM, 5mM, 7mM and 9mM sodium azides for one hour and sowed in plates and pots (100 per replication) under RBD design with factorial arrangement and 4 replications were used. The studied variables were: germination, survival, plant length, root length, plumule/radicle length ratio, germination weight, seedling and root elongation speed and germination weight gain, LD50 and the average effect of the Mutagenic Agent. During the analysis of the variables there were significant differences. The genotypes had different response to the sodium azide, being the UC157F1 cultivar tha most chemo-sensitive and 9mM showed the highy effectTesi
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Determinación de la dosimetría aplicada a la semilla de espárrago (Asparagus officinalis L.)
'Baishideng Publishing Group Inc.', 2019Co-Authors: Vera Vega, Miguel AngelAbstract:Universidad Nacional Agraria La Molina. Facultad de Agronomía. Departamento Académico de FitotecniaEl espárrago (Asparagus officinalis L.) es una hortaliza muy cotizada tanto en nuestro país como a nivel mundial. En el Perú la producción de los cultivares espárrago se expande por el litoral peruano, en los departamentos de La Libertad, Lima, Ica, Ancash y Lambayeque. En la actualidad los cultivares liberados están perdiendo su capacidad de resistencia y tolerancia a las plagas y enfermedades, lo que obliga a los fitomejoradores a generar nuevos cultivares que respondan a estos nuevos desafíos. La primera acción del fitomejorador es generar variabilidad genética dentro de la población y una alternativa para lograrlo es la inducción de mutaciones. Esta técnica ayuda a generar variabilidad genética, permitiendo obtener y seleccionar los genotipos con tolerancia y/o resistencia a estreses bióticos y abióticos. El experimento se llevó a cabo en la PIPS en Cereales y Granos Nativos de la Universidad Nacional Agraria La Molina. Se trabajó con tres genotipos: UC157F1, UC115 y la 2H-38H cuyas semillas fueron tratadas. Se tuvieron dos controles (agua, buffer fosfato a pH 3) y siete concentraciones de azida de sodio (1mM, 3mM, 5mM, 7mM y 9mM). Las semillas fueron sumergidas por una hora y sembradas en placas y macetas (100 por repetición) y se utilizó un diseño DBCA con arreglo factorial con 4 repeticiones. Las variables estudiadas fueron las siguientes: germinación, supervivencia, altura de planta, longitud de radícula, relación de la longitud plúmula-radícula, peso del germinado, la velocidad de elongación de la plántula y radícula y ganancia de peso del germinado, DL50 y el efecto promedio del Agente mutagénico. Durante el análisis de las variables se observó que hubo diferencias significativas. Los tres genotipos mostraron respuestas diferentes al efecto de la azida de sodio, siendo el cultivar UC157F1 es más quimio-sensible y la dosis de 9mM de NaN3 causó el mayor efectoAsparagus (Asparagus officinalis L.) is a vegetable that is highly valued both in our country and worldwide. In Peru, the production of asparagus crops expands along the Peruvian coast, in the departments of La Libertad, Lima, Ica, Ancash and Lambayeque. Currently, the released cultivars are losing their resistance or tolerance to pests and diseases, forcing to plant breeders to generate new cultivars that respond to these new challenges. The first action of the breeder is to generate genetic variation in the population. This technique helps to increace genetic variability, it is achieved and selected genotypes with tolerance and / or resistance to biotic and abiotic stresses. The cape was taken to the PIPS in Cereals and Native Crops of La Molina National Agrarian University. Three genotypes were evaluated: UC157F1, UC115 and 2H-38H. The seed were submerged in the following treatments: water and phosphate buffer at pH 3 as control and 1mM, 3mM, 5mM, 7mM and 9mM sodium azides for one hour and sowed in plates and pots (100 per replication) under RBD design with factorial arrangement and 4 replications were used. The studied variables were: germination, survival, plant length, root length, plumule/radicle length ratio, germination weight, seedling and root elongation speed and germination weight gain, LD50 and the average effect of the Mutagenic Agent. During the analysis of the variables there were significant differences. The genotypes had different response to the sodium azide, being the UC157F1 cultivar tha most chemo-sensitive and 9mM showed the highy effec
Luzia D. Paccola-meirelles - One of the best experts on this subject based on the ideXlab platform.
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ANTIMutagenic ACTION OF LENTINULA EDODES AND AGARICUS BLAZEI ON ASPERGILLUS NIDULANS CONIDIA
2015Co-Authors: Edneia A. Souza-paccola, Cleide A. Bomfeti, Léia C. L, Fávaro Inês, C. B. Fonseca, Luzia D. Paccola-meirellesAbstract:The antiMutagenic effect of the mushrooms Lentinula edodes and Agaricus blazei was studied on conidia of Aspergillus nidulans when exposed to short wave ultraviolet light. Two strains of A. nidulans were used. For the preparation of the extracts, the fresh mushrooms were left in aqueous infusion for 12 hours and heated in a water bath for 15 min at 100ºC, and then the material was filtered. The dehydrated mushrooms were left in aqueous infusion for 12 hours and to filtrated. Both filtrates were used as extracts. A. nidulans conidia were incubated for three hours in water and in mushroom extracts and only after were exposed to UV light (pre-treatment). A. nidulans conidia were suspended in water and in mushroom extracts and immediately submitted to UV light (post-treatment). Conidial suspension in water and in mushroom extracts but without exposure to the Mutagenic Agent were used as controls. After Mutagenic treatment, it was observed an increase in the survival rate of the A. nidulans and a decrease in the percentage of morphologic mutants on conidia treated with mushroom extracts. Our results demonstrated the radioprotective and antiMutagenic effect of L. edodes and A. blazei mushrooms on eukaryotic cells when exposed to UV radiation
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AntiMutagenic action of Lentinula edodes and Agaricus blazei on Aspergillus nidulans conidia Ação antimutagênica de Lentinula edodes e Agaricus blazei em conídios de Aspergillus nidulans
Sociedade Brasileira de Microbiologia, 2004Co-Authors: Edneia A. Souza-paccola, Cleide A. Bomfeti, Léia C.l. Fávaro, Inês C.b. Fonseca, Luzia D. Paccola-meirellesAbstract:The antiMutagenic effect of the mushrooms Lentinula edodes and Agaricus blazei was studied on conidia of Aspergillus nidulans when exposed to short wave ultraviolet light. Two strains of A. nidulans were used. For the preparation of the extracts, the fresh mushrooms were left in aqueous infusion for 12 hours and heated in a water bath for 15 min at 100ºC, and then the material was filtered. The dehydrated mushrooms were left in aqueous infusion for 12 hours and to filtrated. Both filtrates were used as extracts. A. nidulans conidia were incubated for three hours in water and in mushroom extracts and only after were exposed to UV light (pre-treatment). A. nidulans conidia were suspended in water and in mushroom extracts and immediately submitted to UV light (post-treatment). Conidial suspension in water and in mushroom extracts but without exposure to the Mutagenic Agent were used as controls. After Mutagenic treatment, it was observed an increase in the survival rate of the A. nidulans and a decrease in the percentage of morphologic mutants on conidia treated with mushroom extracts. Our results demonstrated the radioprotective and antiMutagenic effect of L. edodes and A. blazei mushrooms on eukaryotic cells when exposed to UV radiation.O efeito antimutagênico dos cogumelos Lentinula edodes e Agaricus blazei foram estudados sobre conídios de Aspergillus nidulans quando expostos à luz ultravioleta de comprimento de onda curto. Duas linhagens de A. nidulans foram usadas. Para o preparo dos extratos, os cogumelos frescos permaneceram em infusão aquosa por 12 horas e em seguida foram aquecidos em banho-maria por 15 min à 100ºC e a seguir o material foi filtrado. Os cogumelos desidratados foram deixados em infusão aquosa por 12 horas e a seguir filtrados. Ambos os filtrados foram usados como extratos. Os conídios de A. nidulans foram incubados por três horas em água e em extrato de cogumelo e somente após foram expostos a luz ultravioleta (pré-tratamento). Conídios de A. nidulans foram incubados em água e em extrato de cogumelo e imediatamente submetidos à luz ultravioleta (pós-tratamento). Conídios incubados em água e em extrato de cogumelo, mas sem exposição ao Agente mutagênico, foram usados como controle. Após tratamento mutagênico, observou-se um aumento na taxa de sobrevivência de A. nidulans e uma diminuição na porcentagem de mutantes morfológicos em conídios tratados com extrato de cogumelos. Nossos resultados demonstram o efeito radioprotetor e antimutagênico dos cogumelos L. edodes e A. blazei sobre células eucarióticas submetidas à radiação UV
Universitat Autònoma De Barcelona. Departament De Biologia Cel·lular, D - One of the best experts on this subject based on the ideXlab platform.
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aportacions a l'estudi de la relació inflamació-tumorigènesi en el còlon.
[Barcelona] : Universitat Autònoma de Barcelona, 2015Co-Authors: Eyre Sánchez Elena, Universitat Autònoma De Barcelona. Departament De Biologia Cel·lular, DAbstract:La colitis ulcerosa (UC) s'associa a un alt risc de desenvolupar càncer colorectal (CRC). La resposta inflamatòria i, en particular, alguns receptors de la immunitat innata com els TLR, condicionen la resposta immune, intervenen en el manteniment de la funció barrera intestinal i en la modulació de mecanismes reparadors, processos relacionats amb la resolució o cronificació de la inflamació. En aquesta tesi hem explorat la relació entre mecanismes inflamatoris i tumorigènics en models animals, que permeten estudiar la transició inflamació-tumorigènesi sense les influències degudes a la farmacoteràpia, i en mostres humanes. En models murins de colitis, càncer colorectal i càncer colorectal associat a colitis hem analitzat les relacions entre els canvis histopatològics i l'expressió de gens, i hem valorat quines modificacions de l'expressió de microRNA les acompanyen, buscant la seva possible repercussió en processos d'inflamació, proliferació, apoptosi i distorsió de l'arquitectura tissular. També hem estudiat com es modifiquen els perfils fenotípics de cèl·lules mononuclears de cara a establir-ne el valor com a biomarcadors putatius de la transició colitis-càncer. En material biològic humà, els nostres esforços s'han adreçat a valorar l'expressió gènica en biòpsies de còlon obtingudes d'individus sans, de pacients amb UC i d'adenomes, de cara a seleccionar els gens que presenten canvis d'expressió associats a processos tumorals i inflamatoris. També hem valorat in vitro fins quin punt la funció barrera intestinal és afectada per les secrecions dels monòcits, per a establir la possible relació causal entre un comportament anòmal dels monòcits i la permeabilitat intestinal incrementada que es dóna en la UC. Els resultats obtinguts en els models murins ens han permès validar la correlació entre la inflamació intestinal i la promoció dels processos tumorigènics i constatar que la simple cronificació de la inflamació desorganitza la resposta reparadora provocant una marcada hiperplàsia i l'aparició de focus de criptes aberrants, fins i tot sense induir mutagènesi per administració d'Agents alquilants. En els animals que reben un Agent mutagen i pateixen episodis recurrents de colitis, els tumors són més nombrosos, més grans i presenten major grau de displàsia en relació als que s'observen sense el concurs d'episodis d'inflamació. Els tumors que apareixen en un context inflamatori presenten alteracions significatives en l'expressió dels gens tlr4, tlr9, cnnd1, cox2 i bcl2. Això suggereix una implicació dels gens esmentats en el desenvolupament de tumors associats a colitis. En aquests tumors s'evidencia també una disminució significativa de l'expressió de miR-135a, miR-21, miR-145, i miR-34a, fet que suposaria un increment d'expressió de les seves dianes. L'anàlisi predictiva amb eines bioinformàtiques ha permès seleccionar dianes d'interès en funció de la seva relació amb els processos de proliferació, inflamació, apoptosi, angiogènesi i organització tissular. Entre elles destaquem tiam1, vcl, stat3, ptk2, wnt1, pak4, p120, birc5, btg2, klf4, rtkn, i peli1. L'anàlisi de l'expressió gènica sobre micromatrius partint de còlon humà sà, de pacients d'UC i d'adenomes, ha permès verificar quins gens es troben significativament alterats. De l'anàlisi GO, la comparació entre els diferents grups, i la literatura, hem seleccionat un llistat de gens biomarcadors putatius del CRC i associats també a UC: brca1, brca2, nbn, tp53, smad3, fcer1g, a2m, dock2, atf4, cxcl12, itga8, eps8, rhou, nrp1 i ptk2b. Pel que fa als monòcits de sang perifèrica, l'estimulació amb lligands de TLR4 determina una resposta secretora diferent en els monòcits de pacients d'UC en comparació amb els d'individus sans. Aquest perfil secretor diferencial pot ser parcialment responsable d'un deteriorament més acusat la funció barrera intestinal en pacients d'UC, afavorint la cronificació de la inflamació i l'aparició de displàsies.Ulcerative colitis (UC) is associated to an increased risk to develop colorectal cancer (CRC). In the gut, inflammation and activation of receptors of innate immunity such as TLRs, accomplish several functions, including tailoring adaptive immune responses, deploying repair mechanisms and maintaining the intestinal barrier function. The tuning of such functions may result in resolution or chronification of the inflammation. Here we have explored the relationship between inflammatory and tumourigenic mechanisms in animal models - which allow the study of the inflammation-tumourigenesis transition without the biases due to pharmacotherapy -, and in human samples. We have analyzed the relationship between histopathological findings and gene expression variations in murine models of colitis, colorectal cancer and colitis associated colorectal cancer. The expression of microRNAs in these conditions has also been quantified and their potential repercussions in the expression of genes related to inflammation, proliferation, apoptosis and tissue organization has been explored. Changes in mononuclear cell phenotypic profiles have also been studied in order to assess their putative value as biomarkers of inflammation-tumourigenesis transition. In human samples, we intended to study gene expression in colon biopsies obtained from healthy individuals, UC patients and adenomas, in an attempt to find expression changes associated both to UC and CRC. Furthermore, we have evaluated in vitro to what extent the intestinal barrier function is affected by monocyte secretions, to establish whether an anomalous monocyte responsiveness may contribute to the enhanced intestinal permeability usually found in UC patients. The results obtained in the murine model show a strong correlation between intestinal inflammation and enhanced tumour development, probably resulting from a disregulation of repair mechanisms, as shown by the finding of a significant hyperplasia and increased occurrence of aberrant crypt foci. In animals receiving a Mutagenic Agent and undergoing recurrent colitic episodes, the tumours are larger, more abundant and exhibit a higher degree of dysplasia compared to tumours observed in mice receiving only the Mutagenic Agent. Tumours appearing in an inflammatory context display significant gene expression alterations in tlr4, tlr9, cnnd1, cox2, and bcl2. This suggests an involvement of these genes in colitis associated tumour development. Inflammation associated tumours also showed a significant decrease in miR-135a, miR-21, miR-145 and miR-34a expression, which may lead to an increased expression of their target mRNAs. Among them we have highlighted those involved in proliferation, inflammation, apoptosis, angiogenesis and tissue organization processes. We propose tiam1, vcl, stat3, ptk2, wnt1, pak4, p120, birc5, btg2, klf4, rtkn and peli1 as candidate genes for the follow up of the bias inflammation-tumourigenesis. Gene expression microarray analysis has allowed us to identify genes whose expression is significantly altered in biopsies taken from UC patients and colon adenomas compared to healthy tissue. On the basis of this comparison, literature and gene ontology analysis, we have selected a list of genes altered in CRC and also associated to UC, which may be useful as putative biomarker genes: bcra1, bcra2, ndn, tp53, smad3, fcer1g, a2m, dock2, atf4, cxcl12, itga8, eps8, rhou, nrp1 and ptk2b. Our results show too that TLR4 ligands elicit an enhanced secretory response in monocytes of UC patients compared to those of healthy individuals. This enhanced response induces increased disruptive effects on the intestinal barrier function in vitro, suggesting that an anomalous monocyte secretory profile may be partly responsible for the increased intestinal permeability found in UC patients, which may contribute to the chronification of colitis and promotion of dysplasia
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Study of production of iturin by Bacillus subtilis in solid state fermentation using as substrate soybean meal, rice meal, wheat bran and husk rice
2017Co-Authors: Cesar Augusto Piedrahita AguirreAbstract:Resumo: Este trabalho se propôs a estudar a produção da iturina A por Bacillus subtilis em fermentação semi-sólida em biorreatores de leito empacotado. O trabalho foi desenvolvido em quatro partes. Em uma primeira parte foi feito um screening com cepas silvestres e seus mutantes obtidos a partir da exposição de luz UV e acridina laranja. A cepa Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi a que apresentou maior atividade antagônica contra os fungos Aspergillus fumigatus Fresenius NRRL 164, Aspergillus fumigatus Fresenius NRRL 166 e Aspergillus flavus var. oryzae NRRL 484. O extrato metanólico obtido da fermentação semi-sólida do Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi analisado através da espectrometria de massas encontrando-se lipopeptídeos com massa molecular entre m/z 1021,43 e m/z 1087,48, mas sem a presença da iturina A. Em uma segunda etapa a cepa Bacillus Iso 1 foi isolada a partir das raízes de soja, e ante a dificuldade de identificar a iturina A através da cromatografia liquida de alta eficiência (HPLC), foi desenvolvida a metodologia de purificação da iturina A utilizando a cromatografia em coluna de vidro preenchida com sílica gel 60. A iturina A foi eluída com três sistemas de solventes compostos por 20 mL de clorofórmio-metanol-água (65:25:4,v/v/v), fração 1, seguido de 20 mL de clorofórmio-metanol-água (30:50:10, v/v/v), fração 2, e a fração final composta por 10 mL de clorofórmio-metanol-água (20:60:15, v/v/v). As frações obtidas foram analisadas através da HPLC e da espectrometria de massa, identificando 5 isômeros da iturina A (C13-C16). Na terceira etapa, foi feito um delineamento composto central rotacional (DCCR) para avaliar o efeito da casca de arroz como suporte inerte e da vazão volumétrica de ar na produção de iturina A; como substratos foram utilizados o farelo de soja desengordurado e o farelo de trigo. Nenhuma variável do DCCR foi estatisticamente significativa, mas operacionalmente foram importantes, devido à redução da oxigenação do Bacillus Iso 1 pela baixa vazão de ar e menor concentração de casca de arroz, favorecendo a produção de iturina; nestas condições obteve-se 6,88 g/kg de substrato seco de iturina A.Esta é a maior quantidade de iturina A produzida em biorreatores de leito empacotado (coluna) com aeração forçada até hoje. Na quarta etapa, a partir dos resultados obtidos no DCCR foram estudados os parâmetros do processo: queda de pressão, consumo de oxigênio e perfis de temperatura, visando entender o comportamento da fermentação a 0,4 L/min e 0,8 L/min. A máxima produção de iturina obtida foi 5,58 g/kg de substrato seco com a vazão de 0,4 L/min. O incremento na queda de pressão é ocasionado não unicamente pelo incremento da vazão volumétrica, mas também pela produção do biopolímero ?-PGA o qual ocupa os espaços livres entre as partículas, dificultando o fluxo normal de ar através do leito, reduzindo o consumo de oxigênio. A baixa oxigenação favoreceu a alta produção da iturina A e gerou baixo calor metabólico (5,75 W/kg-dry substrato·min). Os resultados obtidos podem ser úteis na elaboração de estratégias para ampliação de escala do processo em fermentadores aerados de leito empacotadoAbstract:This work covers a study of the production of iturin A by Bacillus by solid-state fermentation in packed bed bioreactors. The study was conducted in four parts. At first a screening was conducted with wild strains and their mutants obtained from exposure to UV light and Mutagenic Agent acridine orange. The strain Bacillus subtilis subsp subtilis NRRL NRS 1270 showed the highest antagonistic activity against Aspergillus fumigatus NRRL 164, Aspergillus fumigatus NRRL 166 and Aspergillus flavus var . oryzae NRRL 484. A methanolic extract obtained by solid state fermentation of Bacillus subtilis subsp subtilis NRRL NRS 1270 was analyzed with mass spectrometry showing lipopeptides with molecular mass between m/z 1021.43 and m/z 1087.48, but without the presence of iturin A. In the second stage, the strain Bacillus Iso 1 was isolated from soybean roots. Given the difficulty of identifying iturin A by high performance liquid chromatography (HPLC), a iturin A purification methodology was developed using glass column chromatography packed with activated Silica gel 60 and alumina. This methodology involved three solvent systems for elution of the iturin A from the column. A first fraction consisted of 20 ml of chloroform-methanol-water (65:25:4 , v/v/v) and was followed by 20 ml of chloroform - methanol- water (30:50 : 10, v/v/v), that was then followed by a final fraction consisting of 10 ml of chloroform-methanol-water (20:60:15, v/v/v). The fractions obtained of fermentation were analyzed by both HPLC and mass spectrometry, identifying five iturin A isomers (C13-C16). In the third stage of the study, an experimental design was constructed in the form of a central composite rotational design (CCRD) to evaluate the effect of rice husk as an inert support and air flow rates to the iturin A production, using defatted soybean meal and wheat bran as substrate. Although none of the studied variables showed statistical significance, the operational importance of reduction of oxygenation of the Bacillus Iso 1 fermentation due to the low concentration of rice husk and air flow rate was observed to favor the production of iturin; in these conditions high productivity was obtained reaching 6.88 g/kg-dry substrate of iturin A. Concluding from available literature, this is the highest concentration of iturin A ever produced in packed bed bioreactor (column) with forced aeration to date. In the fourth stage, in order to understand the behavior of the fermentation under aeration conditions between 0.4 L/min and 0.8 L/min, the following process parameters were studied, based on the results obtained from the CCRD: pressure drop, oxygen consumption and temperature profiles. The maximum production of iturin obtained was 5.58 g/kg-dry substrate with the air flow rate at 0.4 L/. The increase of the pressure gradients is caused not only by increasing the volumetric air flow rate but also by the production of biopolymer ?-PGA by Bacillus iso 1, which occupies the free interparticle space, hindering or preventing the normal flow of air through the bed and thus leading to reduced oxygen consumption. The low oxygenation favored the high iturin A production and resulted in low metabolic heat generation (5.75 W/kg-dry substrate.min). The results of this work are expected to be conducive for designing strategies to scale up the process in aerated packed bed bioreactor
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Study of production of iturin by Bacillus subtilis in solid state fermentation using as substrate soybean meal, rice meal, wheat bran and husk rice
Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos, 2013Co-Authors: Cesar Augusto Piedrahita AguirreAbstract:Este trabalho se propôs a estudar a produção da iturina A por Bacillus subtilis em fermentação semi-sólida em biorreatores de leito empacotado. O trabalho foi desenvolvido em quatro partes. Em uma primeira parte foi feito um screening com cepas silvestres e seus mutantes obtidos a partir da exposição de luz UV e acridina laranja. A cepa Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi a que apresentou maior atividade antagônica contra os fungos Aspergillus fumigatus Fresenius NRRL 164, Aspergillus fumigatus Fresenius NRRL 166 e Aspergillus flavus var. oryzae NRRL 484. O extrato metanólico obtido da fermentação semi-sólida do Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi analisado através da espectrometria de massas encontrando-se lipopeptídeos com massa molecular entre m/z 1021,43 e m/z 1087,48, mas sem a presença da iturina A. Em uma segunda etapa a cepa Bacillus Iso 1 foi isolada a partir das raízes de soja, e ante a dificuldade de identificar a iturina A através da cromatografia liquida de alta eficiência (HPLC), foi desenvolvida a metodologia de purificação da iturina A utilizando a cromatografia em coluna de vidro preenchida com sílica gel 60. A iturina A foi eluída com três sistemas de solventes compostos por 20 mL de clorofórmio-metanol-água (65:25:4,v/v/v), fração 1, seguido de 20 mL de clorofórmio-metanol-água (30:50:10, v/v/v), fração 2, e a fração final composta por 10 mL de clorofórmio-metanol-água (20:60:15, v/v/v). As frações obtidas foram analisadas através da HPLC e da espectrometria de massa, identificando 5 isômeros da iturina A (C13-C16). Na terceira etapa, foi feito um delineamento composto central rotacional (DCCR) para avaliar o efeito da casca de arroz como suporte inerte e da vazão volumétrica de ar na produção de iturina A; como substratos foram utilizados o farelo de soja desengordurado e o farelo de trigo. Nenhuma variável do DCCR foi estatisticamente significativa, mas operacionalmente foram importantes, devido à redução da oxigenação do Bacillus Iso 1 pela baixa vazão de ar e menor concentração de casca de arroz, favorecendo a produção de iturina; nestas condições obteve-se 6,88 g/kg de substrato seco de iturina A.Esta é a maior quantidade de iturina A produzida em biorreatores de leito empacotado (coluna) com aeração forçada até hoje. Na quarta etapa, a partir dos resultados obtidos no DCCR foram estudados os parâmetros do processo: queda de pressão, consumo de oxigênio e perfis de temperatura, visando entender o comportamento da fermentação a 0,4 L/min e 0,8 L/min. A máxima produção de iturina obtida foi 5,58 g/kg de substrato seco com a vazão de 0,4 L/min. O incremento na queda de pressão é ocasionado não unicamente pelo incremento da vazão volumétrica, mas também pela produção do biopolímero ?-PGA o qual ocupa os espaços livres entre as partículas, dificultando o fluxo normal de ar através do leito, reduzindo o consumo de oxigênio. A baixa oxigenação favoreceu a alta produção da iturina A e gerou baixo calor metabólico (5,75 W/kg-dry substrato·min). Os resultados obtidos podem ser úteis na elaboração de estratégias para ampliação de escala do processo em fermentadores aerados de leito empacotadoThis work covers a study of the production of iturin A by Bacillus by solid-state fermentation in packed bed bioreactors. The study was conducted in four parts. At first a screening was conducted with wild strains and their mutants obtained from exposure to UV light and Mutagenic Agent acridine orange. The strain Bacillus subtilis subsp subtilis NRRL NRS 1270 showed the highest antagonistic activity against Aspergillus fumigatus NRRL 164, Aspergillus fumigatus NRRL 166 and Aspergillus flavus var . oryzae NRRL 484. A methanolic extract obtained by solid state fermentation of Bacillus subtilis subsp subtilis NRRL NRS 1270 was analyzed with mass spectrometry showing lipopeptides with molecular mass between m/z 1021.43 and m/z 1087.48, but without the presence of iturin A. In the second stage, the strain Bacillus Iso 1 was isolated from soybean roots. Given the difficulty of identifying iturin A by high performance liquid chromatography (HPLC), a iturin A purification methodology was developed using glass column chromatography packed with activated Silica gel 60 and alumina. This methodology involved three solvent systems for elution of the iturin A from the column. A first fraction consisted of 20 ml of chloroform-methanol-water (65:25:4 , v/v/v) and was followed by 20 ml of chloroform - methanol- water (30:50 : 10, v/v/v), that was then followed by a final fraction consisting of 10 ml of chloroform-methanol-water (20:60:15, v/v/v). The fractions obtained of fermentation were analyzed by both HPLC and mass spectrometry, identifying five iturin A isomers (C13-C16). In the third stage of the study, an experimental design was constructed in the form of a central composite rotational design (CCRD) to evaluate the effect of rice husk as an inert support and air flow rates to the iturin A production, using defatted soybean meal and wheat bran as substrate. Although none of the studied variables showed statistical significance, the operational importance of reduction of oxygenation of the Bacillus Iso 1 fermentation due to the low concentration of rice husk and air flow rate was observed to favor the production of iturin; in these conditions high productivity was obtained reaching 6.88 g/kg-dry substrate of iturin A. Concluding from available literature, this is the highest concentration of iturin A ever produced in packed bed bioreactor (column) with forced aeration to date. In the fourth stage, in order to understand the behavior of the fermentation under aeration conditions between 0.4 L/min and 0.8 L/min, the following process parameters were studied, based on the results obtained from the CCRD: pressure drop, oxygen consumption and temperature profiles. The maximum production of iturin obtained was 5.58 g/kg-dry substrate with the air flow rate at 0.4 L/. The increase of the pressure gradients is caused not only by increasing the volumetric air flow rate but also by the production of biopolymer ?-PGA by Bacillus iso 1, which occupies the free interparticle space, hindering or preventing the normal flow of air through the bed and thus leading to reduced oxygen consumption. The low oxygenation favored the high iturin A production and resulted in low metabolic heat generation (5.75 W/kg-dry substrate.min). The results of this work are expected to be conducive for designing strategies to scale up the process in aerated packed bed bioreactor
