The glpX gene of Escherichia coli encodes fructose 1,6-bisphosphatase II (FBPase II), an enzyme that would appear to be redundant with FBPase I, encoded by fbp. However, glpX mutants have no apparent phenotype, while fbp mutants are unable to grow on gluconeogenic substrates as sole carbon sources, suggesting that GlpX function is insufficient for growth of fbp mutants under these conditions. To gain insight into the physiological functions of the FBPases, regulation of glpX expression was investigated. It was found that glpX is transcribed as part of a complex glpFKX operon containing promoters upstream of glpF, glpK and glpX (PglpF, PglpK, PglpX, respectively). Transcription start sites of PglpX were found at -24 and -41 relative to the ATG translation initiation site using primer extension analysis. Unlike PglpF, these newly found promoters were not subject to regulation by GlpR or cAMP-CRP. Cra (Catabolite Repressor/Activator) positively regulated expression from PglpK and PglpX by increasing transcription approximately 2 fold. Western analysis using GlpX polyclonal antibodies revealed that GlpX levels were higher in cultures grown on glycerol compared with levels in maltose- or glucose-grown cultures (glycerol>maltose>glucose). Various strains and growth conditions were used to show that GlpX levels are regulated by GlpR, suggesting that PglpF can give rise to expression of glpX. GlpX protein was present in a strain containing a polar insertion in glpK, indicating that PglpX can also give rise to expression of glpX. Strains deficient in FBPase I or CsrA (carbon starvation regulator) did not reveal any difference in GlpX levels with respect to the wild type. All of these data indicate that glpX expression is achieved by its own promoter as well as the operon promoter, PglpF. Finally, the results show that the delta-fbp phenotype is not due to the absence of GlpX. / Ph. D.

In Escherichia coli, the utilization of glycerol and sn-glycerol 3-phosphate is mediated by gene products of the glp regulon. The regulon encompasses five operons, including the glpFKX operon. Although glpF and glpK encode glycerol diffusion facilitator and glycerol kinase,respectively, the function of glpX was unknown. In the present work, we show that glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 μM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP, ATP and phosphate and activated by PEP. The attributes of the glpX-encoded FBPase were different from those of the previously characterized E. coli FBPase encoded by fbp. Mutants deleted in fbp (Δfbp) display a growthnegative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal glpX+ to complement Δfbp. However, a Δfbp mutation was complemented by overexpression of glpX+. In contrast, a glpX mutant exhibited a growth-positive phenotype on glycerol, glucose or fructose media. Surprisingly, a double mutant strain glpX pfkA (6-phosphofructokinase I) was more inhibited in growth on glucose and glycerol media than the pfkA parent. Carbohydrate metabolism in the pfkA background may be affected by the glpXmediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF. / Master of Science

Yes / The geographically restricted Mycobacterium africanum lineages (MAF) are primarily found in West Africa, where they account for a significant proportion of tuberculosis. Despite this phenomenon, little is known about the co-evolution of these ancient lineages with West Africans. MAF and M. tuberculosis sensu stricto lineages (MTB) differ in their clinical, in vitro and in vivo characteristics for reasons not fully understood. Therefore, we compared genomes of 289 MAF and 205 MTB clinical isolates from the 6 main human-adapted M. tuberculosis complex lineages, for mutations in their Electron Transport Chain and Central Carbon Metabolic pathway in order to explain these metabolic differences. Furthermore, we determined, in silico, whether each mutation could affect the function of genes encoding enzymes in these pathways. We found more mutations with the potential to affect enzymes in these pathways in MAF lineages compared to MTB lineages. We also found that similar mutations occurred in these pathways between MAF and some MTB lineages. Generally, our findings show further differences between MAF and MTB lineages that may have contributed to the MAF clinical and growth phenotype and indicate potential adaptation of MAF lineages to a distinct ecological niche, which we suggest includes areas characterized by low oxygen tension. / European Research CouncilINTERRUPTB starting grant nr. 311725 (to BdJ, FG, CM, LR, BO, MA) and The UK Medical Research Council and the European & Developing Countries Clinical Trials Partnership (EDCTP) Grant No. CB. 2007. 41700.007. / Research Development Fund Publication Prize Award winner, January 2020.

Brettanomyces bruxellensis est une levure particulièrement redoutée des vinificateurs pour ses capacités d’altération organoleptique des vins. Elle est également associée à de nombreux produits fermentés et présente une importante diversité génétique en lien avec son origine écologique. L’analyse des profils microsatellites d’une collection importante d’individus (1318) d’origines géographiques variées montre une diversité génétique importante parmi les isolats de vin. Elle met notamment en évidence la coexistence d’individus diploïdes et triploïdes dans différentes régions du monde ainsi qu’à l’échelle d’un chai et d’un vin. La présence de certains génotypes dans plusieurs régions à travers le monde suggère la dispersion de cette espèce et une adaptation importante au milieu difficile qu’est le vin.La relation entre diversité génétique, matrice d’origine et traits physiologiques a été explorée. La nature des sucres utilisables pour supporter la croissance ainsi que les capacités de production de phénols volatils sont peu variables entre les souches étudiées, indépendamment de leur niveau de ploïdie ou de leur origine écologique. Néanmoins, les profils de croissance et de production de phénols volatils (vitesses et rendements) varient et traduisent des différences dans l’adaptation des souches au milieu et aux conditions d’oxygénation. Nos données suggèrent notamment une adaptation plus importante des souches triploïdes aux conditions physico-chimiques du vin. D’un point de vue pratique, l’influence de certains facteurs physico-chimiques, tels que les sucres et la température, sur le développement de B. bruxellensis dans les vins a été étudiée. Dans les vins rouges, la composition en sucres résiduels ne peut pas être considérée comme un outil de diagnostic du risque « Brett ». Néanmoins, les variations importantes de température observées dans les chais, jusqu’alors sous-estimées, pourraient expliquer en partie les phénomènes d’altération de vins rouges fréquemment observés au cours du premier été d’élevage en barrique. / The yeast species Brettanomyces bruxellensis is the most dreaded wine spoilage microorganism because of its repercussions on wine organoleptic wine alteration. It is also present in numerous fermented beverages and its high genetic diversity is partly associated with its ecological origin. Microsatellite analysis of a large collection of isolates (1318) from various geographical origins shows the species’ high genetic diversity, namely among wine strains. Notably, it highlights the coexistence of diploid and triploid individuals worldwide as well as at the region, winery and wine level. Isolation of some of the genotypes in several wine regions in the world suggests this species’ dispersion as well as the putative adaptation of these individuals to the harsh wine environment.The relationship between genetic diversity, matrix type, and physiological traits was further explored. The type of consumable sugars in relation to growth and phenol volatile production capacities of the studied strains, are independent from the ploidy level or ecological origin of the latter. Nevertheless, growth and phenol volatile production profiles (rates and yields) vary, highlighting differences in strains’ growth capacity in different media and aeration conditions. In particular, our data suggests an important adaptation of triploid strains to wine-type environment. From a practical point of view, influence of physicochemical parameters (such as sugars and temperature) on B. bruxellensis’ development in wine has been investigated. In red wine, residual sugar profiles don’t seem to be a relevant tool to estimate the risk associated with “Brett” spoilage. However, the important temperature variations occurring in wine cellars could be a possible explanation for contamination frequency during the first summer of barrel-ageing.

Les relations entre l'activité de la source et l'activité du puits contrôlent en grande partie la croissance des plantes. Ces activités varient au cours du développement, mais aussi en réponse à des changements des conditions environnementales. Notre étude avait pour but d'identifier le rôle du métabolisme carboné dans la réponse de la croissance d'E. americanum à la modulation des activités de la source et du puits. Dans une première partie, l'activité du puits est modulée par la température de croissance. Aux fortes températures, l'activité du puits est plus élevée, alors que sa capacité est réduite. Ces effets, dus à la modulation du métabolisme du saccharose, mènent à une saturation précoce en amidon des bulbes à forte température. Par la suite, la baisse de la demande en carbone du puits induit un rétrocontrôle négatif de l'activité photosynthétique et finalement, la sénescence foliaire. À l'inverse, l'activité du puits à faible température est en rythme avec l'accroissement de la capacité, menant à une biomasse supérieure du bulbe en fin de croissance épigée. Dans une seconde partie, l'activité de la source est modulée en changeant la concentration en CO2 et en O3. Malgré la stimulation de la source sous fort CO2 et son inhibition sous fort O3, l'accumulation d'amidon et la biomasse du bulbe ne sont pas affectées. En effet, le surplus de carbone parvenant au puits est brûlé par la voie alternative de la respiration, celle-ci étant stimulée par l'activité de l'enzyme malique. La voie alternative de la respiration évite ainsi une saturation hâtive en amidon et éventuellement, une sénescence foliaire précoce. Dans une dernière partie, l'activité de la source est modulée par l'irradiance et la photopériode. L'accumulation d'amidon varie en fonction de la photopériode alors que l'irradiance n'a aucun effet. De plus, l'activité photosynthétique est inhibée très précocement sous longue photopériode. Cette inhibition semble due à un déséquilibre entre la quantité totale de carbone fixé par jour et son utilisation suite à son transfert au sein du bulbe. Nous pouvons donc conclure que les régulations du métabolisme carboné permettent d'ajuster l'activité du puits à la capacité de celui-ci chez l’E. americanum / Relationships between source and sink activities largely control the growth of plants. Both activities vary during development as well as in response to changes in environmental conditions. The aim of our study was to identify the role carbon metabolism plays in the response of E. americanum growth to changes in source and sink activities. Firstly, sink activity is modulated by changing growth temperature. Sink activity is higher at higher temperatures, whereas sink capacity is more restricted. These effects, due to the modulation of sucrose metabolism, lead to an earlier starch saturation of bulbs at higher temperature. Thereafter, the reduction in carbon sink demand induces a feedback inhibition of photosynthetic activity and finally, leaf senescence. In contrast, sink activity at low temperature is more in rhythm with the increasing sink capacity, leading to larger bulbs at the end of the epigeous growth season. Secondly, source activity is modulated by changing CO2 and O3 concentrations. Despite a stimulation of the source activity under high CO2 and its inhibition under high O3, neither plant growth nor starch accumulation was affected. Indeed, excess carbon translocated within the sink is burned by the alternative respiratory pathway. This pathway is stimulated by malic enzyme. Alternative respiratory pathway thus avoids an early starch saturation of the bulb and eventually, an early leaf senescence. Finally, source activity is also modulated by changing irradiance and photoperiod regimes. Starch accumulation changes in response to photoperiod but not to irradiance. Furthermore, photosynthetic activity is inhibited early in the season under long photoperiod. This inhibition seems due to an imbalance between the total amount of carbon fixed per day and its utilisation following translocation to the bulb. We can thus conclude that regulation of carbon metabolism allow to adjust sink activity to sink capacity in E. americanum.

Since a proper balance between anabolic and catabolic reactions is essential for all eukaryotes, the basic mechanisms for regulation of the energy and carbon metabolism have been conserved throughout evolution. The moss Physcomitrella patens, which belongs to one of the basal clades among land plants, has many unique properties that make it an excellent plant model system. We have used a yeast two-hybrid system to identify novel possible regulators or targets of the moss Snf1-related kinases, previously shown to regulate energy homeostasis. The function of the identified interactors PpSki1 and PpSki2 was analyzed in order to better understand the biological role of plant Snf1-related kinases. The recently completed genome sequence of Physcomitrella was used in a comparative approach to study to what extent key enzyme and gene families involved in transport and metabolism of sugars and in regulation of the energy and carbon metabolism are conserved between mosses and vascular plants. It has long been known that transformed DNA can replicate episomally in Physcomitrella. We have now shown that such DNA can be rescued back into E. coli. Surprisingly, we found that the original plasmid can be recovered from moss transformants obtained with circular DNA. Plasmids rescued from transformants obtained with linearized DNA had been repaired either by homologous recombination or by cohesive end re-ligation. These findings suggest that methods using shuttle plasmids are feasible in Physcomitrella. Hexokinase, a key enzyme in the carbon metabolism, catalyzes the first step in hexose metabolism, but is also involved in sugar sensing and signaling. We have now made an initial characterization of the complete hexokinase family in Physcomitrella which is encoded by 11 genes. Two new types of plant hexokinases, types C and D, were found in addition to the previously described types A and B.

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No. of bitstreams: 1 DissJRFN.pdf: 1477527 bytes, checksum: cb09da4bfdd14f1e2c076f57d365b7a1 (MD5) Previous issue date: 2017-02-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / In recent years, the application of attenuated Salmonella spp. has been investigated for development of several biotechnological products, mainly vaccines. However, the implementation of industrial processes to obtain these products depends on the development of strategies for this microorganism high-cell density cultures (HCDC). One of the HCDC’s greatest challenges is overcoming Salmonella’s metabolic limitations, as it presents a high organic acids production (mainly acetic acid) that inhibits biomass formation. In this context, this work proposes two approaches to deal with this problem and implement Salmonella’s HCDC: studying the cultivation of S. typhimurium using glycerol as carbon source to reduce the generation of organic acids; studying the cultivation of a recombinant strain of S. typhimurium expressing the enzyme acetyl-CoA synthetase (ACS) from E. coli, for improved acetate assimilation). Initially, cultures were grown in agitated flasks in minimal media for two carbon sources (glucose or glycerol) for the wild-type and the recombinant strain. After the preliminary experiments, the recombinant strain was cultivated in bioreactor operated in batch mode with minimal medium formulated with glycerol, glucose or acetic acid as carbon source, to evaluate acetate production and assimilation. The wild-type strain was cultivated in a continuous-mode bioreactor on minimal medium with glycerol at D=0.10; 0.17 and 0.22 h-1 to evaluate the S. typhimurium glycerol metabolism. During the cultivations, samples were collected and analyzed by high-performance liquid chromatography to quantify the production of organic acids and substrate consumption. To quantify the concentration of biomass, optical density (600 nm) readings of culture broth and dry cell weight measurements were performed. Agitated flasks and batch cultivations results indicated that the acetate production is reduced in medium with glycerol for both strains, and that the genetically modified cells present a lower acetate accumulation phenotype compared to the wild-type. Continuous cultures of the wild-type strain showed no acetate accumulation for a 0.1 h-1 dilution rate. At rates of 0.17 h-1 and 0.22 h-1 acetate accumulation was observed, but acetate flux was 2-fold lower than the flux reached in chemostat with glucose-formulated medium. Simulations were performed with the STM_v1.0 model using as input data the glycerol and oxygen fluxes estimated from the experimental results. Good predictions were obtained for the biomass, CO2 and acetate fluxes at the higher dilution rates. The results suggest that fed-batch culture using glycerol as carbon source along with an exponential feed to maintain a 0.1 h-1 specific growth rate as a promising strategy to obtain high cellular concentrations of wild-type S. typhimurium. The efficient acetate uptake observed for the recombinant S. typhimurium cells may allow higher values of specific growth rate to be used for this strain, resulting in a higher productivity of biomass. / Nos últimos anos, a aplicação de linhagens atenuadas de Salmonella spp. vem sendo amplamente investigada para o desenvolvimento de diversos produtos biotecnológicos, principalmente vacinas. No entanto, a implementação de processos industriais para a obtenção destes produtos depende do desenvolvimento de estratégias para o cultivo em alta densidade celular (CADC) deste microrganismo. Para isso, um dos grandes desafios a ser superado se refere às limitações metabólicas da Salmonella, uma vez que esta apresenta elevada produção de ácidos orgânicos (principalmente ácido acético) que inibem a formação de biomassa. Neste contexto, este trabalho propõe duas abordagens para lidar com este problema e implementar CADC de Salmonella: estudar o crescimento de Salmonella typhimurium em glicerol avaliando seu metabolismo como uma fonte de carbono menos propensa à geração de ácidos orgânicos; e estudar a produção destes ácidos por uma cepa de S. typhimurium geneticamente modificada para diminuir o acúmulo de acetato (super-expressão do gene acs, de Escherichia coli, que codifica a enzima acetil-CoA sintetase (ACS) responsável pela assimilação de acetato). Inicialmente foram realizados cultivos em frascos agitados em meio mínimo para duas fontes de carbono (glicose ou glicerol), para a cepa selvagem e para a cepa recombinante. Após os experimentos preliminares, foram realizados cultivos em biorreator operado em modo batelada com a cepa modificada em meio mínimo formulado com glicerol, glicose ou ácido acético, como fonte de carbono, a fim de avaliar a produção e a assimilação de acetato. Foram realizados cultivos em biorreator em modo contínuo com a cepa selvagem em meio mínimo com glicerol com D=0,10; 0,17 e 0,22 h-1 para avaliar o metabolismo do glicerol pela S. typhimurium. Ao longo dos cultivos foram coletadas amostras do caldo e analisadas por cromatografia líquida de alta eficiência para quantificar a produção de ácidos orgânicos e o consumo de substrato. Para quantificar a biomassa produzida, foram realizadas medidas de densidade ótica (600 nm) e de massa seca. Os resultados dos cultivos em frascos agitados e das bateladas indicam que, para ambas as cepas, a produção de acetato é reduzida em meio formulado com glicerol, e que as células modificadas geneticamente apresentam um fenótipo de menor acúmulo de acetato comparadas à linhagem selvagem. Os cultivos contínuos realizados com a cepa selvagem mostraram que não houve acúmulo de acetato para a taxa de diluição de 0,1 h-1. Já nas taxas de 0,17 h-1 e 0,22 h-1, apesar de haver acúmulo, o fluxo de produção de acetato foi cerca de 2 vezes menor que o observado em quimiostato em meio formulado com glicose. Foram realizadas simulações com o modelo STM_v1.0, tendo como dados de entrada os fluxos de glicerol e de oxigênio estimados a partir dos dados experimentais. O modelo descreveu bem os fluxos de biomassa, CO2 e acetato para as taxas de diluição mais altas. Os resultados obtidos sugerem o cultivo em batelada alimentada com glicerol como fonte de carbono, e alimentação exponencial definida para manter a velocidade específica de crescimento em 0,1 h-1, como uma estratégia promissora para obter altas concentrações celulares de S. typhimurium selvagem. Para a S. typhimurium recombinante, devido à sua eficiente assimilação de acetato, valores ainda maiores de velocidade específica de crescimento poderiam ser impostos, com elevado aumento de produtividade em biomassa.

Made available in DSpace on 2017-07-10T17:40:49Z (GMT). No. of bitstreams: 1 Cristiane_Claudia_Meinerz.pdf: 1140657 bytes, checksum: 515f23cafb30196ea1e4641ed2debc7d (MD5) Previous issue date: 2013-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Induced resistance involves the activation of latent defense mechanisms existing in plants in response to treatment with biotic or abiotic agents. The use of plant extracts in order to induce resistance mechanisms is an attractive alternative to chemical control, however, these extracts may occur the presence of inducer, as well ass the presence of suppressor. This study aimed to evaluate biochemical, physiological and growth of sorghum (Sorghum bicolor L.) treated with extracts of Rosmarinus officinalis, Curcuma longa and Pycnoporus sanguineus. It was evaluated the induction of phytoalexins deoxiantocianidinas, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-1,3 glucanase and chitinase, carbon metabolism (photosynthesis and respiration) and growth of sorghum plants. Mesocotyls sorghum were treated with the fungal and plant extracts, and acibenzolar-S-methyl (ASM) (125 mg L-1 elicitor would like reference) and distilled water. Sorghum seedlings were treated with the inducers and at 24, 48, 96 and 144 hours samples were taken for analyzes of defense enzymes. Evaluations of gas exchange were conducted periodically over 7 days with three days interval of application of foliar treatments. Were determined rate of net CO2 assimilation (A, CO2 mol m-2s-1), stomatal conductance (gs mmol H2O m-2s-1), sweating (E, H2O mmol m-2s-1) efficiency Water use (U.S. mol m-2s-1) and intrinsic efficiency of the use of water (EIUA, mol m-2s-1). For growth analysis, carried out 120 days after sowing, the parameters evaluated were: fresh leaves and roots, dry weight of leaves and roots and root volume. The parameters for production analysis were the number of panicle, seed number, mean grain mass and total production. Biotic inducers rosemary, turmeric and Pycnoporus sanguineus induced phytoalexins in mesocotyls and biochemical activities in different cultivars of sorghum, may note that the results reveal an important target for the action of elicitors in these extracts. Overall, rosemary extract caused increase in peroxidase, polyphenol oxidase and phenylalanine ammonia lyase activities, the turmeric extract induced phenylalanine ammonia-lyase activity and the extract of P. sanguineus polyphenol oxidase and β-1,3 glucanase activities. ASM interfere with the metabolism of BRS 610 sorghum in relation to the efficiency of water use (U.S.) and intrinsic efficiency of water use (EIUA), without interfering with productivity, improving the quality of production and improving markedly the number of panicle, panicle, grain number and grain weight. The fungal and plant extracts did not affect these parameters. It was possible to induce defense mechanisms in sorghum by application of extracts of rosemary, turmeric and P. sanguineus, which may allow the obtention of new molecules, and the development of alternative methods for controlling plant diseases / A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. A aplicação de extratos vegetais visando à indução de mecanismos de resistência é uma alternativa interessante ao controle químico, entretanto, nestes extratos pode ocorrer além da presença de indutores, a presença de supressores. Este trabalho teve por objetivo avaliar aspectos bioquímicos, fisiológico e de crescimento de sorgo (Sorghum bicolor L.) tratado com extratos de Rosmarinus officinalis, Curcuma longa e Pycnoporus sanguineus. Foram avaliados a indução de fitoalexinas deoxiantocianidinas, enzimas peroxidase, polifenoloxidase, fenilalanina amônia-liase, β-1,3 glucanase e quitinase, metabolismo de carbono (fotossíntese e respiração) e crescimento de plantas de sorgo. Mesocótilos de sorgo foram tratados com os extratos vegetais e fúngico, além de acibenzolar-S-metil (ASM) (125 mg L-1 do i.a. como elicitor de referência) e água destilada. Plântulas de sorgo foram tratadas com os indutores e às 24, 48, 96 e 144 horas foram retiradas amostras para as análises das enzimas de defesa. As avaliações de trocas gasosas foram realizadas periodicamente no período de 7 dias, com três dias de intervalo da aplicação dos tratamentos via foliar. Foram determinados: taxas de assimilação líquida de CO2 (A, μmol CO2 m-2s-1), condutância estomática (gs, mmol H2O m-2s-1), transpiração (E, mmol H2O m-2s-1), eficiência do uso de água (EUA, mol m-2s-1) e eficiência intrínseca do uso de água (EIUA, mol m-2s-1). Para análise de crescimento, realizada 120 dias após a semeadura, os parâmetros avaliados foram: massa fresca das folhas e raízes; massa seca das folhas e raízes e volume de raiz. Os parâmetros avaliados na análise de produção foram: número de panícula, número de sementes; massa média de grão e produção total. Os indutores bióticos alecrim, cúrcuma e Pycnoporus sanguineus induziram fitoalexinas em mesocótilos e atividades bioquímicas nas diferentes cultivares de sorgo, podendo ressaltar que os resultados revelam um importante alvo de ação de elicitores presentes nesses extratos. De forma geral, o extrato de alecrim causou incremento na atididade de peroxidase, polifenoloxidase e fenilalanina amônia-liase; o extrato de cúrcuma induziu a atividade de fenilalanina amônia-liase e o extrato de P. sanguineus as atividades de polifenoloxidase e β-1,3 glucanase. O indutor ASM interferiu no metabolismo da cultivar BRS 610 de sorgo em relação à eficiência do uso da água (EUA) e eficiência intrínseca do uso da água (EIUA), sem interferir na produtividade, melhorando a qualidade da produção e melhorando acentuadamente o número de panícula, massa de panículas, número de grãos e massa de grãos. Os extratos vegetais e fúngico não interferiram nesses parâmetros. Foi possível induzir mecanismos de defesa em sorgo pela aplicação dos extratos de alecrim, cúrcuma e P. sanguineus, o que pode permitir a obtenção de novas moléculas e o desenvolvimento de métodos alternativos para controle de doenças em plantas

Au vignoble, des nuits froides peuvent se produire pendant la floraison et en particulier au moment de la méiose ovulaire dans les fleurs. La vigne est particulièrement vulnérable puisque la méiose ovulaire se produit lors de la transition du métabolisme de la plante d'un état hétérotrophe à un état autotrophe. Bien que la feuille soit l'organe photosynthétique principal, la jeune inflorescence de vigne possède un statut particulier et participe activement à l'effort reproducteur en exportant la majorité du carbone qu'elle assimile, permettant ainsi le développement des feuilles. Par conséquent, le métabolisme carboné de l'inflorescence lors du développement reproducteur peut être impliqué dans le futur rendement. Chez la vigne, le phénomène de coulure, propre à chaque cépage, peut engendrer des pertes de rendement importantes lorsque la plante est exposée à un stress. Le Pinot noir (PN) est considéré comme un cépage relativement « résistant » à la coulure alors que le taux d'abscission chez le Gewurztraminer (GW) augmente considérablement lors de conditions climatiques défavorables. Chez le PN, l'inflorescence effectue une photosynthèse inférieure et une respiration de nuit supérieure à celle de la feuille. L'activité et la régulation des deux photosystèmes sont très différentes entre ces deux organes lors des premiers stades de développement et les activités des deux photosystèmes sont supérieures au niveau de l'inflorescence. Néanmoins, la protection de la chaîne photosynthétique contre l'excès d'énergie est plus efficace dans la feuille. Contrairement à la feuille, l'activité photosynthétique de l'inflorescence évolue au cours de son développement. En effet, l'activité de la chaîne photosynthétique ainsi que la photosynthèse nette diminuent progressivement au cours de la floraison et le flux cyclique des électrons se met en place pour être finalement supérieur à celui de la feuille. L'activation de ce flux peut alors permettre une synthèse accrue d'ATP, une protection de la chaîne photosynthétique contre les dommages provoqués par un excès d'énergie ou encore la réparation des photosystèmes. Lorsque la nuit froide survient lors de la méiose ovulaire, le métabolisme carboné de l'inflorescence de PN est différemment modifié selon l'intensité du stress. Ainsi, après une nuit à 4°C, la modification de l'activité photosynthétique de l'inflorescence est due à des limitations de nature non-stomatique alors qu'après une nuit à 0°C, cette modification est due à des limitations de nature stomatique. Une nuit à -3°C altère profondément l'activité photosynthétique de l'inflorescence. Ces nuits froides induisent également une accumulation de glucides. Lors du développement floral en conditions optimales, le PN et le GW présentent une activité photosynthétique et un métabolisme carboné différents. La régulation des flux linéaire et cyclique des électrons est également différente. Ce dernier semble avoir une fonction différente chez ces deux cépages avec notamment une possible implication dans la réparation du PSII et/ou dans une synthèse d'ATP accrue à la fin du processus de floraison chez le PN. La chaîne photosynthétique du GW semble mieux protégée ce qui peut expliquer le rendement supérieur de ce cépage en conditions optimales. Néanmoins, l'exposition à une nuit froide entraine des modifications différentes de l'activité de l'inflorescence chez ces cépages avec une perturbation plus importante chez le GW. En effet, chez ce cépage, seule la photosynthèse nette est perturbée suite à la nuit froide alors que chez le PN, les processus de photosynthèse et de respiration sont modifiés. L'activité de la chaîne photosynthétique ainsi que l'activité métabolique de l'inflorescence de GW est également davantage affectée. De manière intéressante, nos résultats suggèrent que ces différentes perturbations de l'activité de l'inflorescence sont dues à des régulations différentes. / In the vineyard, cold night can occur during flowering and particularly at time of female meiosis in flowers. In grapevine, stress vulnerability is enhanced because female meiosis occurs when the whole plant physiology is switching its carbon nutrition from mobilization of wood reserves to photosynthesis in the leaves. Nevertheless, although leaf is the major photosynthetic organ, in grapevine, the young inflorescence has a particular status and takes part in the reproductive effort by exporting the majority of assimilated carbon, allowing in particular the leaves development. Consequently, the inflorescence metabolism during this phase can ultimately determines yield. In grapevine, coulure phenomenon, differing according to the cultivar, can generate important yield losses when a stress occurs. Pinot noir (PN) is considered as a cultivar relatively “resistant” to coulure phenomenon whereas Gewurztraminer (GW) abscission rate considerably increases under environmental stress.In PN, inflorescence performs a lower photosynthesis and a higher dark respiration than leaves. Functioning and regulation of PSI and PSII are very different between inflorescence and leaf during the first developmental stages and activities of these photosystems are higher in the inflorescence. Nevertheless, the photosynthetic chain against excess energy is more efficient in the leaf. Contrary to the leaf, the inflorescence photosynthetic activity evolves during the floral development. Indeed, photosynthetic chain activity and net photosynthesis progressively decrease and the cyclic electron flow appears and is higher than in leaf. This activation could provide ATP, protection against photodamage or repair of the photosystems.When cold night occurs at the female meiosis stage, carbohydrate metabolism of the PN inflorescence is differently modified according to the intensity of the cold stress. At 4°C, photosynthesis in the inflorescence is impaired through non-stomatal limitations, whereas at 0°C it is affected through stomatal limitations. A freezing night (-3°C) severely deregulates photosynthesis in the inflorescence. Cold nights also induce accumulation of sugars.Comparing PN and GW, different photosynthetic activity and carbohydrate metabolism have been showed during the floral development under optimal conditions. Regulations of the linear and cyclic electron flow are also different and the cyclic electron flow seems to have a different aim with particularly an implication in the recovery of PSII and ATP synthesis at the end of the flowering process in PN. GW could have higher protection of the photosynthetic chain and consequently gets a higher yield under optimal conditions. Nevertheless, chilling night impacts differently the activity of the inflorescences of both cultivars with higher modification in GW inflorescence. Indeed, in this cultivar, only the net photosynthesis is altered whereas in PN, both net photosynthesis and respiration processes are modified. The photosynthetic chain activity and metabolical activity of the inflorescence are also more affected by the cold night in GW. Interestingly, our results suggest that the different fluctuation of the inflorescence activities as response to the chilled night is due to different regulations.

ABSTRACT It has been widely recognized that microRNAs are involved in nearly all cellular processes that have been investigated and contribute to a variety of diseases including cancer. Our prior studies demonstrated the depletion of one-carbon metabolism related B-vitamins, including folate, vitamin B2, B6 and B12, induced a genomic DNA hypomethylation and an elevation of the tumorigenic Wnt signaling in mouse colonic epithelium. The present study aimed to define whether microRNAs serve as mediators between these B-vitamins and the Wnt signaling, and thereby influence intestinal tumorigenesis. MicroRNA expression profiles were measured using miRNA microarray and real-time PCR on colonic epithelial cells from Apc1638N mice fed with diets deplete or sufficient in those B-vitamins. In silico bioinformatic analysis were performed to predict microRNA gene targets within the Wnt signaling cascade. Out of 609 microRNA examined, 18 microRNAs were found to be either significantly (p < 0.05) or mildly (p < 0.10) differentially expressed in the colonic epithelium of mice fed the depleted diet when compared to the counterpart. Bioinformatic prediction of microRNA gene targets identified 40 genes within the Wnt pathway to have homology with microRNA seed sequences within their 3’-UTR or protein coding sequence. Of the 6 genes tested for experimentally target validation, the expression of Sfrp1 was shown to be significantly inhibited (p < 0.05) whereas β-catenin was shown to be significantly elevated (p < 0.05) with alterations of others in a fashion indicating the activation of Wnt signaling. These findings indicate that microRNAs may constitute a mechanism by which one-carbon B-vitamin depletions regulate the Wnt signaling pathway and thereby inform intestinal tumorigenesis.