No description available.

The efficient fermentation of lignocellulosic biomass would enable more economically and environmentally friendly production of biofuels and biochemicals. Yet, Saccharomyces cerevisiae, a platform organism for biofuels and biochemicals production, is unable to convert all of the sugars in lignocellulosic biomass into biofuels and biochemicals mainly due to the lack of a pentose catabolic pathway. Though the advance of genetic engineering enabled S. cerevisiae to utilize pentose sugars, the efficiency of pentose sugar catabolism in S. cerevisiae is still limited. Here, the goal of this research was to confer efficient pentose sugar catabolism to S. cerevisiae by combinatorial and evolutionary engineering. To this end, pentose catabolic pathways were 1) constructed by heterologous expression of pentose catabolic genes, 2) optimized through rational engineering, and 3) further improved through evolutionary engineering. Through these efforts, we reported the highest ethanol yield (0.45 g ethanol / g xylose) and the second highest xylose consumption and ethanol production rates (0.98 g xylose g cell⁻¹ h⁻¹ and 0.44 g ethanol g cell⁻¹ h⁻¹, respectively) in xylose fermentation reported to date. The high performance in xylose fermentation was achieved based on the mutant xylose isomerase (xylA3), which showed 77% increased enzyme activity, engineered through directed evolution. In addition, we have established the first cells capable of growing on arabinose in mimimal medium and demonstrated ethanol production from xylan in minimal medium. The arabinose and xylan catabolic pathways were constructed in S. cerevisiae by expressing novel pentose catabolic genes from a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. In doing so, a complete workflow of bioprospecting to pathway engineering and evolution was detailed as an effective way to transfer a desired phenotype from a non-model organism to a model organism. This study substantially improved the prospect of biofuels and biochemicals production from lignocellulosic biomass by developing efficient pentose utilizing strains, finding new pentose catabolic genes, and suggesting alternative pentose catabolic pathway. Furthermore, the general tools for metabolic engineering demonstrated in this study would also advance microbial strain engineering. / text

No description available.

Thesis (doctoral)--University of Helsinki, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Cancer est un terme qui rassemble plusieurs ensembles hétérogène de maladies et il est caractérisé par la perte de contrôle physiologique et la transformation maligne des cellules saines. Il est essentiel de comprendre le cancer de la biologie cellulaire afin d'identifier de nouveaux biomarqueurs pour le diagnostic précoce et la conception de nouvelles stratégies thérapeutiques. Reprogrammation métabolique est une caractéristique émergente de cancer, ce qui signifie que les cellules cancéreuses passent leur métabolisme de base pour répondre aux exigences accrues de la croissance et la division cellulaire. Par conséquent, explorer reprogrammant métabolique que les cellules cancéreuses subissent est une stratégie clé pour identifier de nouvelles cibles pour le traitement du cancer. Dans cette thèse, de nouvelles possibilités pour le traitement du cancer ont été explorés en analysant la reprogrammation métabolique de la tumeur. À cet égard, nous avons étudié et proposé voie des pentoses phosphates (PPP) enzymes cibles thérapeutiques putatifs contre les cancers du sein et du côlon. En outre, nous avons exploré le métabolisme de la glutamine dans les cellules du cancer du sein et les adaptations du réseau métaboliques qu'ils subissent dans le but de contourner la privation de glutamine et la déficience mitochondriale générale. Ainsi, le ciblage PPP est l'intérêt des chercheurs d'utiliser à la fois oxydantes et non oxydantes phases de cette voie métabolique comme une cible de médicament thérapeutique. Pour tester cela, nous inhibés bœuf PPP enzymes 6PGD dans les cellules cancéreuses du sein et G6PD dans les cellules du côlon.Nous avons effectué la caractérisation de la reprogrammation métabolique induite par l'inhibition de l'enzyme de bœuf PPP par l'ARN interferase (ARNi) silençage médiation, afin d'explorer le potentiel de cette enzyme comme une cible de médicament thérapeutique dans deux lignées de cellules de cancer du sein. Nous avons demontré que l'inhibition 6PGD a entraîné une diminution taux de prolifération, arrêt du cycle cellulaire et induction de l'apoptose médiée par l'activation de p53, en diminuant les capacités de formation mammosphere et le métabolisme altéré de carbone central par modulation de Warburg phenomenan et en améliorant le métabolisme de la glutamine. D'autre part, nous avons montré l'effet de l'inhibition de la G6PD sur la prolifération des cellules du cancer du côlon et du PPP est régulée par la disponibilité de la glutamine dans les cellules cancéreuses du côlon.De plus, nous avons caractérisé les adaptations métaboliques que les cellules cancéreuses du sein subissent la privation de glutamine ou lorsque les mitochondries sont fait défection. Nous avons effectué une analyse des flux métaboliques utilisant métabolomique et Fluxomique et nous avons utilisé la biologie des systèmes afin d'estimer une vision globale des modifications de flux dans différentes conditions de culture. Nous avons observé une augmentation du cycle de pyruvate avec privation glutamine, ce qui indique que le ciblage des enzymes de cette voie telle que l'enzyme malique pourrait être une approche prometteuse combinée à l'inhibition de l'enzyme de glutaminase. D'autre part, nous avons observé que mimant une hypoxie par des cellules de cancer du sein de traitement redirigée oligomycine pour augmenter la carboxylation réductrice. Considérant que l'hypoxie est une condition commune dans l'environnement de la tumeur, le ciblage mécanisme de carboxylation réductrice pourrait être une nouvelle stratégie de lutte contre le cancer. Collectivement, les résultats présentés dans cette thèse démontre l'importance du métabolisme de la prolifération des cellules cancéreuses et la survie. Ce travail met également en évidence l'importance de la biologie des systèmes se rapproche de comprendre les mécanismes moléculaires sous-jacents des maladies multifactorielles complexes afin de souligner de nouvelles cibles thérapeutiques potentielles. / Moreover, we characterized the metabolic adaptations that breast cancer cells undergo in the deprivation of glutamine or when mitochondria are defected. We conducted metabolic flux analysis using metabolomics and fluxomics approaches and we employed Systems Biology approaches in order to estimate a global view of flux alterations in different culture conditions. We observed an increased pyruvate cycle with glutamine deprivation, thus indicating that targeting the enzymes of this pathway such as malic enzyme could be a promising approach combined with inhibition of glutaminase enzyme. On the other hand, we observed that mimicking hypoxia by oligomycin treatment redirected breast cancer cells to increase reductive carboxylation. Considering that hypoxia is a common condition in the tumor environment, targeting reductive carboxylation mechanism could be a novel strategy to fight against cancer. Collectively, all the results provided in this thesis demosntrate the importance of metabolism in cancer cell proliferation and survival. This work also highlights the importance of Systems Biology approaches to comprehend the molecular mechanisms underlying complex multifactorial diseases in order to point out new potential therapeutic targets.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-08-30T17:21:18Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriela_maria_pereira_barbosa.pdf: 2753973 bytes, checksum: e196ae4be05114b62809f147ce9f80df (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-08-30T18:48:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriela_maria_pereira_barbosa.pdf: 2753973 bytes, checksum: e196ae4be05114b62809f147ce9f80df (MD5) / Made available in DSpace on 2017-08-30T18:48:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriela_maria_pereira_barbosa.pdf: 2753973 bytes, checksum: e196ae4be05114b62809f147ce9f80df (MD5) Previous issue date: 2017 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / As pentoses oriundas da hidr?lise da fra??o hemicelul?sica de biomassas vegetais podem ser fermentadas a etanol. Entretanto, s?o poucas as esp?cies conhecidas de micro-organismos capazes de converter pentoses a etanol e, mesmo essas, mostram-se ineficientes e suscept?veis ? presen?a de inibidores produzidos durante o pr?-tratamento das biomassas lignocelul?sicas. Neste contexto, o presente trabalho teve por objetivo identificar e avaliar seis linhagens de leveduras isoladas a partir de biomassas vegetais quanto a capacidade de realizar a fermenta??o alco?lica da D-xilose utilizando diferentes formula??es de meios sint?ticos. As linhagens foram submetidas a identifica??o morfol?gica e molecular. Os processos fermentativos foram conduzidos em batelada simples em frascos c?nicos sob agita??o a 150 rpm e temperatura de 30?C por at? 145 horas. Os bioprocessos foram monitorados a intervalos regulares de tempo quanto ? concentra??o de a??cares redutores, glicose, etanol e biomassa celular. Ao final do processo, foram determinados os valores das vari?veis de resposta rendimento (YP/S), produtividade (QP) e taxa de consumo de a??cares (qS). Todas as linhagens avaliadas foram capazes de produzir ?lcool em meio sint?tico contendo somente xilose como carboidrato. Os maiores valores de produ??o e rendimento alc?olico foram obtidos em meios que continham al?m da xilose, a glicose. O maior valor de YP/S observado foi de 0,38, em condi??es experimentais com produ??o de 3,33 g L-1 de etanol. A composi??o de microelementos e fontes de nitrog?nio afetou a fermenta??o alc?olica. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Biocombust?veis, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The pentoses from the hydrolysis of the hemicellulosic fraction of plant biomass can be fermented to ethanol. However, few known species of microorganisms are capable of converting pentoses to ethanol, and even those, are inefficient and susceptible to the presence of inhibitors produced during pretreatment of lignocellulosic biomasses. In this context, main objective of this work is to identify and evaluate six yeast strains isolated from plant biomasses in order to perform the alcoholic fermentation of D-xylose using different formulations of synthetic media. The strains were submitted to morphological and molecular identification at the species level. The fermentative processes were conducted in single batch in conical flasks under agitation at 150 rpm and at 30 ?C for up to 145 hours. The bioprocesses were monitored at regular intervals for the concentration of reducing sugars, glucose, ethanol and cell biomass. At the end of the process, the values of the response variables YP/S, QP e qS were determined. All the evaluated strains were able to produce alcohol in synthetic medium containing only xylose as a carbohydrate. The highest values of production and alcoholic yield were obtained in media that contained beyond xylose, glucose. The highest value of YP/S observed was 0.38, under experimental conditions with production of 3.33 g L-1 of ethanol. The composition of microelements and nitrogen sources affected the alcoholic fermentation.

Made available in DSpace on 2017-05-12T14:46:55Z (GMT). No. of bitstreams: 1 Lillian Leonel.pdf: 1330884 bytes, checksum: 72d7ed478436a2a5c72fa3f74e8d44d4 (MD5) Previous issue date: 2014-03-18 / To supply the increasing demand for bioethanol it is necessary to search for new sources of renewable raw materials and emerging technologies. Most studies have been directed to the cellulose, since this fraction is more abundant in biomass and its hydrolysis leads to glucose, a more easily metabolizable sugar for the commercially available yeasts. Considering that the hemicellulose represents 20-30% of plant biomass and is easily removable, either by acid or enzymatic hydrolysis, the use of this fraction by pentose-fermenting yeasts as Scheffersomyces (Pichia) stipitis has been promising. However, the process of hydrolysis of lignocellulosic biomass to the release of fermentable sugars, specifically the acid hydrolysis, generates toxic compounds that impair the microbial metabolism and ethanol production. Thus, it is important to know the different effects of the major inhibitors present in hemicellulosic hydrolysates in the metabolism of S. stipitis ATCC 58376. For this purpose the culture was performed in synthetic medium supplemented with different concentrations of the inhibitors furfural, HMF, acetic acid, syringic acid and vanillin, following the Plackett and Burman factorial design with 12 assays (7 assays more compared to the number of independent variables) and 4 assays at the central point, totalizing 16 assays. The assays were conducted under agitation of 200 rpm at 30 °C and pH 5.25 for 96 hours. The analysis of the kinetic profiles obtained showed that the consumption of sugars, cell growth and ethanol production by S. stipitis were adversely affected by the addition of inhibitor compounds in synthetic medium promoting the inhibition of cell growth and ethanol production, as well as the delay in the consumption of sugars (xylose and glucose) according to the concentrations of the inhibitors tested. However, the acetic acid concentration of 2.0 g / L favored S. stipitis metabolism in the assays 09 (furfural 0.025 g.L-1; HMF: 0.01 g.L-1; acetic acid: 2.0 g.L-1, vanillin: 0.09 g.L-1 and syringic acid: 0.75 g.L-1), 10 (furfural: 0.75 g.L-1 HMF: 0,01 g.L-1; vanillin: 0.09 g.L-1 and syringic acid: 0.75 g.L-1) and 02 (furfural: 0.75 g.L-1; HMF: 0.03 g.L-1; acetic acid 2.0 g.L-1; vanillin: 0.09 g.L-1 and syringic acid: 0.025 g L-1), with peak concentrations of ethanol obtained in 72 hours of 14.3 g.L-1, 12.43 g.L-1 and 10.75 g.L-1, respectively, higher than the control assay (10.05 g.L-1), that lacked inhibitors. In assays with 6.0 g.L-1 of acetic acid, the metabolism of the yeast was completely inhibited. Statistical analysis for cell growth response showed that only acetic acid was significant (p < 0.10), being the most potent inhibitor. The others inhibitors also showed negative effect, except for syringic acid, which had a positive effect at 72 and 96 hours, but were not statistically significant. For the response ethanol production, among the factors evaluated, only the effect of furfural was not significant (p> 0.10). The most potent inhibitors were acetic acid and HMF, while vanillin and syringic acid promoted a positive effect on ethanol production. These results contribute to the choice of an appropriate method of detoxification for hemicellulosic hydrolyzate, which aims for a greater removal of the compounds that actually affect the metabolism of S. stipitis ATCC 58376 / Para suprir a demanda crescente de bioetanol há necessidade de buscar novas fontes de matérias-primas renováveis e tecnologias emergentes. A maior parte dos estudos tem sido direcionada à celulose, visto que esta fração é mais abundante na biomassa e sua hidrólise resulta em glicose, açúcar mais facilmente metabolizável por leveduras comercialmente disponíveis. Considerando que a hemicelulose representa 20-30% da biomassa vegetal e é facilmente extraível, seja por hidrólise ácida ou enzimática, a utilização desta fração por leveduras fermentadoras de pentoses, como Scheffersomyces (Pichia) stipitis, tem se mostrado promissora. No entanto, o processo de hidrólise da biomassa lignocelulósica para liberação dos açúcares fermentescíveis, em específico a hidrólise ácida, gera compostos tóxicos que prejudicam o metabolismo microbiano e a produção de etanol. Assim, é importante o conhecimento dos efeitos distintos dos principais inibidores presentes nos hidrolisados hemicelulósicos no metabolismo de S. stipitis ATCC 58376. Para isso foram realizados cultivos em meio sintético, adicionado de diferentes concentrações dos inibidores furfural, HMF, ácido acético, ácido siríngico e vanilina, seguindo o planejamento fatorial Plackett e Burman com 12 ensaios (7 ensaios a mais em relação ao número de variáveis independentes) e 4 ensaios no ponto central, totalizando 16 ensaios. Os ensaios foram conduzidos sob agitação de 200 rpm, temperatura de 30 °C e pH inicial 5,25 por 96 horas. A análise dos perfis cinéticos obtidos mostrou que o consumo de açúcares, o crescimento celular e a produção de etanol por S. stipitis foram afetados negativamente pela adição de compostos inibidores em meio sintético, promovendo a inibição do crescimento celular e produção de etanol, bem como o retardo no consumo de açúcares (glicose e xilose) de acordo com as concentrações dos inibidores testadas. No entanto, o ácido acético em concentração de 2,0 g/L favoreceu o metabolismo de S. stipitis, nos ensaios 09 (furfural: 0,025 g.L-1; HMF: 0,01 g.L-1; ácido acético 2,0 g.L-1; vanilina: 0,09 g.L-1 e ácido siríngico: 0,75 g.L-1), 10 (furfural: 0,75 g.L-1; HMF: 0,01 g.L-1; ácido acético 2,0 g.L-1; vanilina: 0,09 g.L-1 e ácido siríngico: 0,75 g.L-1) e 02 (furfural: 0,75 g.L-1; HMF: 0,03 g.L-1; ácido acético 2,0 g.L-1; vanilina: 0,09 g.L-1 e ácido siríngico: 0,025 g.L-1), sendo as máximas concentrações de etanol obtidas em 72 horas de 14,3 g.L-1, 12,43 g.L-1 e 10,75 g.L-1,respectivamente, sendo valores superiores ao ensaio controle (10,05 g.L-1), que não possuía inibidores. Nos testes com 6,0 g.L-1 de ácido acético, o metabolismo da levedura foi completamente inibido. A análise estatística para a resposta crescimento celular mostrou que somente o ácido acético foi significativo (p<0,10), mostrando-se o mais potente inibidor. Os demais inibidores também apresentaram efeito negativo, exceto o ácido siríngico, que apresentou efeito positivo em 72 e 96 horas, porém não foram estatisticamente significativos. Para a resposta produção de etanol, entre os fatores avaliados, somente o efeito do furfural não foi significativo (p>0,10). Os mais potentes inibidores foram o ácido acético e o HMF, enquanto vanilina e ácido siríngico promoveram um efeito positivo na produção de etanol. Esses resultados contribuem para a escolha de um método de destoxificação adequado para o hidrolisado hemicelulósico, que vise uma maior remoção dos compostos que realmente afetam o metabolismo de S. stipitis ATCC 58376

Made available in DSpace on 2017-07-10T19:23:42Z (GMT). No. of bitstreams: 1 Lillian Leonel.pdf: 1330884 bytes, checksum: 72d7ed478436a2a5c72fa3f74e8d44d4 (MD5) Previous issue date: 2014-03-18 / To supply the increasing demand for bioethanol it is necessary to search for new sources of renewable raw materials and emerging technologies. Most studies have been directed to the cellulose, since this fraction is more abundant in biomass and its hydrolysis leads to glucose, a more easily metabolizable sugar for the commercially available yeasts. Considering that the hemicellulose represents 20-30% of plant biomass and is easily removable, either by acid or enzymatic hydrolysis, the use of this fraction by pentose-fermenting yeasts as Scheffersomyces (Pichia) stipitis has been promising. However, the process of hydrolysis of lignocellulosic biomass to the release of fermentable sugars, specifically the acid hydrolysis, generates toxic compounds that impair the microbial metabolism and ethanol production. Thus, it is important to know the different effects of the major inhibitors present in hemicellulosic hydrolysates in the metabolism of S. stipitis ATCC 58376. For this purpose the culture was performed in synthetic medium supplemented with different concentrations of the inhibitors furfural, HMF, acetic acid, syringic acid and vanillin, following the Plackett and Burman factorial design with 12 assays (7 assays more compared to the number of independent variables) and 4 assays at the central point, totalizing 16 assays. The assays were conducted under agitation of 200 rpm at 30 °C and pH 5.25 for 96 hours. The analysis of the kinetic profiles obtained showed that the consumption of sugars, cell growth and ethanol production by S. stipitis were adversely affected by the addition of inhibitor compounds in synthetic medium promoting the inhibition of cell growth and ethanol production, as well as the delay in the consumption of sugars (xylose and glucose) according to the concentrations of the inhibitors tested. However, the acetic acid concentration of 2.0 g / L favored S. stipitis metabolism in the assays 09 (furfural 0.025 g.L-1; HMF: 0.01 g.L-1; acetic acid: 2.0 g.L-1, vanillin: 0.09 g.L-1 and syringic acid: 0.75 g.L-1), 10 (furfural: 0.75 g.L-1 HMF: 0,01 g.L-1; vanillin: 0.09 g.L-1 and syringic acid: 0.75 g.L-1) and 02 (furfural: 0.75 g.L-1; HMF: 0.03 g.L-1; acetic acid 2.0 g.L-1; vanillin: 0.09 g.L-1 and syringic acid: 0.025 g L-1), with peak concentrations of ethanol obtained in 72 hours of 14.3 g.L-1, 12.43 g.L-1 and 10.75 g.L-1, respectively, higher than the control assay (10.05 g.L-1), that lacked inhibitors. In assays with 6.0 g.L-1 of acetic acid, the metabolism of the yeast was completely inhibited. Statistical analysis for cell growth response showed that only acetic acid was significant (p < 0.10), being the most potent inhibitor. The others inhibitors also showed negative effect, except for syringic acid, which had a positive effect at 72 and 96 hours, but were not statistically significant. For the response ethanol production, among the factors evaluated, only the effect of furfural was not significant (p> 0.10). The most potent inhibitors were acetic acid and HMF, while vanillin and syringic acid promoted a positive effect on ethanol production. These results contribute to the choice of an appropriate method of detoxification for hemicellulosic hydrolyzate, which aims for a greater removal of the compounds that actually affect the metabolism of S. stipitis ATCC 58376 / Para suprir a demanda crescente de bioetanol há necessidade de buscar novas fontes de matérias-primas renováveis e tecnologias emergentes. A maior parte dos estudos tem sido direcionada à celulose, visto que esta fração é mais abundante na biomassa e sua hidrólise resulta em glicose, açúcar mais facilmente metabolizável por leveduras comercialmente disponíveis. Considerando que a hemicelulose representa 20-30% da biomassa vegetal e é facilmente extraível, seja por hidrólise ácida ou enzimática, a utilização desta fração por leveduras fermentadoras de pentoses, como Scheffersomyces (Pichia) stipitis, tem se mostrado promissora. No entanto, o processo de hidrólise da biomassa lignocelulósica para liberação dos açúcares fermentescíveis, em específico a hidrólise ácida, gera compostos tóxicos que prejudicam o metabolismo microbiano e a produção de etanol. Assim, é importante o conhecimento dos efeitos distintos dos principais inibidores presentes nos hidrolisados hemicelulósicos no metabolismo de S. stipitis ATCC 58376. Para isso foram realizados cultivos em meio sintético, adicionado de diferentes concentrações dos inibidores furfural, HMF, ácido acético, ácido siríngico e vanilina, seguindo o planejamento fatorial Plackett e Burman com 12 ensaios (7 ensaios a mais em relação ao número de variáveis independentes) e 4 ensaios no ponto central, totalizando 16 ensaios. Os ensaios foram conduzidos sob agitação de 200 rpm, temperatura de 30 °C e pH inicial 5,25 por 96 horas. A análise dos perfis cinéticos obtidos mostrou que o consumo de açúcares, o crescimento celular e a produção de etanol por S. stipitis foram afetados negativamente pela adição de compostos inibidores em meio sintético, promovendo a inibição do crescimento celular e produção de etanol, bem como o retardo no consumo de açúcares (glicose e xilose) de acordo com as concentrações dos inibidores testadas. No entanto, o ácido acético em concentração de 2,0 g/L favoreceu o metabolismo de S. stipitis, nos ensaios 09 (furfural: 0,025 g.L-1; HMF: 0,01 g.L-1; ácido acético 2,0 g.L-1; vanilina: 0,09 g.L-1 e ácido siríngico: 0,75 g.L-1), 10 (furfural: 0,75 g.L-1; HMF: 0,01 g.L-1; ácido acético 2,0 g.L-1; vanilina: 0,09 g.L-1 e ácido siríngico: 0,75 g.L-1) e 02 (furfural: 0,75 g.L-1; HMF: 0,03 g.L-1; ácido acético 2,0 g.L-1; vanilina: 0,09 g.L-1 e ácido siríngico: 0,025 g.L-1), sendo as máximas concentrações de etanol obtidas em 72 horas de 14,3 g.L-1, 12,43 g.L-1 e 10,75 g.L-1,respectivamente, sendo valores superiores ao ensaio controle (10,05 g.L-1), que não possuía inibidores. Nos testes com 6,0 g.L-1 de ácido acético, o metabolismo da levedura foi completamente inibido. A análise estatística para a resposta crescimento celular mostrou que somente o ácido acético foi significativo (p<0,10), mostrando-se o mais potente inibidor. Os demais inibidores também apresentaram efeito negativo, exceto o ácido siríngico, que apresentou efeito positivo em 72 e 96 horas, porém não foram estatisticamente significativos. Para a resposta produção de etanol, entre os fatores avaliados, somente o efeito do furfural não foi significativo (p>0,10). Os mais potentes inibidores foram o ácido acético e o HMF, enquanto vanilina e ácido siríngico promoveram um efeito positivo na produção de etanol. Esses resultados contribuem para a escolha de um método de destoxificação adequado para o hidrolisado hemicelulósico, que vise uma maior remoção dos compostos que realmente afetam o metabolismo de S. stipitis ATCC 58376

Submitted by Michele Mologni (mologni@unoeste.br) on 2017-08-30T13:15:11Z No. of bitstreams: 1 Fláive Loyze Baldassarini Silva.pdf: 2001999 bytes, checksum: e6f3c3f0169423517f9b8ffac6f5f788 (MD5) / Made available in DSpace on 2017-08-30T13:15:11Z (GMT). No. of bitstreams: 1 Fláive Loyze Baldassarini Silva.pdf: 2001999 bytes, checksum: e6f3c3f0169423517f9b8ffac6f5f788 (MD5) Previous issue date: 2017-06-27 / The association between proline metabolism in the plant and the pentose phosphate pathway has been proposed as a model to stimulate one of the biosynthetic routes of secondary metabolism related to the production of the different phenolic compounds in plants, known as the shikimic acid pathway. In transgenic plants in which overexpression of the P5CS gene Δ1-pyrroline-5-carboxylate synthetase encoding the key enzyme of proline biosynthesis occurs, in addition to increased tolerance to abiotic stresses, there is the possibility of promoting the synthesis of phenolic compounds as a pleiotropic effect. The objective of this work was to evaluate the role of proline "per se" in relation to the production of phenolic compounds in transgenic tobacco plants accumulating this amino acid (Nicotiana tabacum cv. Petit Havana SR1) and to verify if water stress would modify this answer. The experiment was carried out under greenhouse conditions, at Campus II of Universidade do Oeste Paulista, Presidente Prudente - SP. A completely randomized design was used in the factorial scheme 3x2 consisting of 3 genotypes (two transgenic events with constitutive expression 35S::P5CSF129A and untransformed control plants) and two levels of water regime, with daily water replenishment of 100% and 30% of field capacity (water stress). The role of proline "per se" was evaluated by means of biometric growth analyzes (plant height, shoot and root dry mass and leaf number) in addition to proline analyzes, glucose-6-phosphate dehydrogenase activity (G6PDH), phenylalanine ammonia lyase (PAL), total phenolic compounds in leaves and lignin. In a natural response to water stress, a reduction in biometric parameters was observed for all genotypes. The accumulation of proline occurred in a greater proportion in the transgenic plants as expected by the constitutive expression of the P5CS gene, but the activity of the G6PDH enzyme was lower in the transgenic plants. The link between increased proline endogenous content and increased phenol synthesis occurred both under normal hydration conditions (for E2 event) and in the presence of water stress (for both transgenic events), as well as PAL activity. Lignin contents increased in all genotypes in response to stress. Thus, the results of this research lead us to affirm the existence of distinct responses between exogenous application of proline reported in the literature and endogenous cellular metabolism. It was not possible to confirm the hypothesis that the proline metabolism linked to the pentose phosphate pathway induces the synthesis of phenolic compounds, since the activity of the G6PDH enzyme was lower in the transgenic plants in the two water conditions. It is suggested that the precursors to the pathway of phenolic compounds linked to proline may be provided by other metabolic pathways such as glycolysis and the Calvin cycle. Further study will be needed to clarify this issue. / A associação entre o metabolismo de prolina na planta e a via pentose fosfato tem sido proposta como um modelo para se estimular uma das rotas do metabolismo secundário relacionada à produção dos diferentes compostos fenólicos em plantas, denominada via do ácido chiquímico. Em plantas transgênicas nas quais ocorrem a superexpressão do gene P5CS Δ1-pirrolina-5-carboxilato sintetase, que codifica a enzima-chave da biossíntese de prolina, além do aumento de tolerância a estresses abióticos, existe a possibilidade de promoção da síntese de compostos fenólicos como um efeito pleiotrópico. O objetivo deste trabalho foi avaliar o papel da prolina “per se” em relação à produção de compostos fenólicos em plantas transgênicas de tabaco acumuladoras deste aminoácido (Nicotiana tabacum cv. Petit Havana SR1) e verificar se o estresse hídrico modificaria essa resposta. O experimento foi realizado em condições de casa de vegetação, no Campus II da Universidade do Oeste Paulista, Presidente Prudente - SP. Utilizou-se o delineamento experimental em arranjo inteiramente casualizado, no esquema fatorial 3x2, formado por 3 genótipos (dois eventos transgênicos com expressão constitutiva 35S::P5CSF129A e plantas controle não transformadas) e dois níveis de regime hídrico, com reposições diárias de água de 100% e de 30% da capacidade de campo (estresse hídrico). O papel da prolina “per se” foi avaliado por meio de análises biométricas de crescimento (altura de plantas, massas seca de parte aérea e raiz e número de folhas) além das análises de prolina, atividade das enzimas glicose-6-fosfato-desidrogenase (G6PDH), fenilalanina amônia liase (FAL), compostos fenólicos totais nas folhas e lignina. Em resposta natural ao estresse hídrico, foi observado uma redução nos parâmetros biométricos para todos os genótipos. O acúmulo de prolina ocorreu em maior proporção nas plantas transgênicas como esperado pela expressão constitutiva do gene P5CS, porém a atividade da enzima G6PDH foi menor nas plantas transgênicas. A vinculação entre um maior conteúdo endógeno de prolina com a síntese aumentada de fenóis ocorreu tanto em condições normais de hidratação (para o evento E2) como na presença de estresse hídrico (para ambos os eventos transgênicos), assim como a atividade da FAL. Os teores de lignina aumentou em todos os genótipos em resposta ao estresse. Desta forma, os resultados dessa pesquisa levam-nos a afirmar a existência de respostas distintas entre aplicação exógena de prolina relatada na literatura e o metabolismo endógeno celular. Não foi possível confirmar a hipótese de que o metabolismo de prolina vinculado à via pentose fosfato induz a síntese de compostos fenólicos, pois a atividade da enzima G6PDH foi menor nas plantas transgênicas nas duas condições hídricas. Sugere-se que os precursores para a via dos compostos fenólicos ligados a prolina podem ser fornecidos por outras vias metabólicas tais como a glicólise e o ciclo de Calvin. Estudos mais aprofundados serão necessários para esclarecer esta questão.

Le furfural est produit actuellement par des procédés industriels"batch" utilisant des catalyseurs acides très actifs, comme l'acide sulfurique ou l'acide chlorhydrique. Ces méthodes offrent des rendements molaires moyens en furfural de 40 à 50 % du furfural potentiel contenu dans la source de pentosans, principale composante de la fraction hémicellulosique de la biomasse. Ces rendements sont accompagnés d'une sélectivité de l'ordre de 50 %. La forte activité des acides minéraux rend la réaction de déshydratation peu sélective. En effet, sous hautes acidité et température, la formation de polymères dérivés du furfural et de sous-produits de condensation devient inévitable. Considérant que la fraction hémicellulosique, selon l'essence d'arbre, peut représenter entre 20 et 40 % en masse de la biomasse végétale, cette matière première renouvelable demeure, encore à ce jour, sous valorisée. Ce mémoire présente une nouvelle unité réactionnelle où la technologie du réacteur"plug flow" biphasique est utilisée de façon à ce que le furfural produit dans la phase aqueuse soit transféré instantanément dans un solvant organique. Ce mode d'opération solubilise le furfural dès sa formation, limitant ainsi sa dégradation/polymérisation. De plus, la méthode proposée se tourne vers des catalyseurs moins actifs et plus sélectifs que les acides minéraux (HCl, H[indice inférieur 2]SO[indice inférieur 4], etc.) : les zéolithes. La déshydratation des pentoses nécessitant une catalyse acide, l'imprégnation des zéolithes étudiées fut exécutée avec l'acide phosphorique (H[indice inférieur 3]PO[indice inférieur 4]). Les conditions d'opération de l'unité jugées optimales ont été évaluées, a priori, par des simulations cinétiques. Des travaux préliminaires effectués sur la régénération et la réutilisation de ces catalyseurs hétérogènes ont démontré l'intérêt de poursuivre la recherche dans cette direction. Des rendements molaires et sélectivités élevés atteignant plus de 90 % du furfural potentiel furent obtenus. Un exercice de dimensionnement, présenté en Annexe A, a permis de démontrer que la conception d'un tel réacteur était réalisable à l'échelle industrielle.