A 13C-Análise de Fluxos Metabólicos (13C-MFA) tornou-se uma técnica de alta precisão para estimar fluxos metabólicos e obter informações importantes sobre o metabolismo. Este método consiste em procedimentos experimentais, técnicas de medição e em cálculos para análise de dados. Neste contexto, os grupos de pesquisa de engenharia metabólica necessitam de ferramentas computacionais precisas e adequadas aos seus objetos de estudo. No presente trabalho, foi construída uma ferramenta computacional na plataforma MATLAB que executa cálculos de 13C-MFA, com balanços de metabólitos e cumômeros. Além disso, um módulo para estimar os fluxos metabólicos e um módulo para quantificar as incertezas das estimativas também foram implementados. O programa foi validado com dados presentes na literatura e aplicado a estudos de caso. Na estimação de fluxos de Pseudomonas sp. LFM046, identificou-se que esse micro-organismo possivelmente utiliza a Via das Pentoses em conjunto com a Via Entner-Doudoroff para a biossíntese de Polihidroxialcanoato (PHA). No design ótimo de experimentos para uma rede genérica de Pseudomonas, identificou-se a glicose marcada no átomo cinco como um substrato que permitirá determinar o fluxo na Via das Pentoses com menor incerteza. / 13C-Metabolic Flux Analysis (13C-MFA) has become a high-precision technique to estimate metabolic fluxes and get insights into metabolism. This method consists of experimental procedures, measurement techniques and data analysis calculations. In this context, metabolic engineering research groups demand accurate and suitable computational tools to perform the calculations. A computational tool was implemented in MATLAB platform that performs 13C-MFA calculation, using metabolite and cumomer balances, as well as a module to estimate the fluxes and a module to quantify their uncertainty. The program was validated with some classical cases from literature. From the flux estimates of Pseudomonas sp. LFM046, it was identified that the microorganism possibly uses the Pentose Phosphate Pathway along with the Entner-Doudoroff Pathway for Polyhydroxyalkanoate (PHA) biosynthesis. From the optimal experimental design for a generic Pseudomonas network, it was possible to conclude that glucose labeled at atom five is the best option to determine the flux in the Pentose Phosphate Pathway with smaller uncertainty.

Submitted by Eduarda Figueiredo (eduarda.ffigueiredo@ufpe.br) on 2015-03-13T14:43:54Z No. of bitstreams: 2 Walane Maria Pereira de Mello Ivo-Doutorado PROTEN-UFPE.pdf: 1522136 bytes, checksum: 50c604e8c1a38c831708f21dd0122461 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T14:43:54Z (GMT). No. of bitstreams: 2 Walane Maria Pereira de Mello Ivo-Doutorado PROTEN-UFPE.pdf: 1522136 bytes, checksum: 50c604e8c1a38c831708f21dd0122461 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-08-21 / As mudanças de uso do solo afetam a dinâmica do carbono (C), podendo causar diminuição significativa do estoque deste elemento no solo, refletindo-se em aumento de emissões de gases de efeito estufa e diminuição da fertilidade. Desta forma, o desenvolvimento de sistemas de produção da cana-de-açúcar que permitam evitar a perda de C do solo, ou até sequestrar C, deverá aumentar a competitividade agrícola e ambiental desta cultura. O objetivo deste trabalho foi estudar a dinâmica da matéria orgânica dos solos dos tabuleiros costeiros do Nordeste cultivados com cana-de-açúcar colhida crua e queimada. De forma mais específica, buscou-se: (1) Quantificar o estoque do carbono orgânico do solo em cronossequência de cultivo de cana-de-açúcar colhida queimada e do solo sob o ecossistema florestal de Mata Atlântica, determinando o tempo de residência média (TRM) do carbono orgânico originário da floresta; (2) Quantificar a variação do estoque da carbono orgânico do solo em sistemas de produção da cana-de-açúcar submetida à colheita crua e queimada; (3) Quantificar as variações de alguns atributos do solo decorrentes da mudança do manejo da colheita de cana queimada para cana crua; (4) Quantificar a entrada de material orgânico (palhada e raízes) e a taxa de decomposição destes em sistemas de produção da cana-de-açúcar submetida à colheita com e sem despalha a fogo; (5) Determinar a emissão de C-CO2 do solo e construir um balanço anual de carbono para a socaria da cana-de-açúcar. Para este estudo, foi utilizada uma cronossequência com mata nativa e cana-de-açúcar colhida queimada por cinco, 15 e 35 anos, fazendo-se uso da determinação da abundância natural do 13C para o cálculo do TRM. Áreas sob colheita de cana crua por três e oito anos também foram estudadas. O cultivo da cana modificou alguns atributos do solo, provocando aumento da densidade do solo e do fósforo disponível, redução dos teores de cálcio e magnésio, carbono orgânico e capacidade de troca de cátions do solo. O estoque de C do solo, até 100 cm de profundidade, foi reduzido de 259 Mg ha-1, sob mata, para 148 Mg ha-1, sob cana queimada, depois de 35 anos de cultivo; com taxa de perda anual de C de 3,0% (k = 0,308 ano-1) para os primeiros 20 cm do solo e de 1,9% (k = 0,019 ano-1) para todo o perfil (100 cm). Os TRM do C da mata nos Argissolos dos tabuleiros costeiros, cultivados com cana-de-açúcar, situaram-se nas faixas de: (1) 20,1 a 28,8 anos para as camadas de 0-5 a 20-30 cm; (2) 43,7 a 73,7 anos para as de 30-40 a 60-80 cm; (3) e na casa dos milhares de anos na camada de 80-100 cm. Para os primeiros vinte centímetros do perfil, este tempo foi 1,6 vezes menor que o tempo médio que o C reside nos solos cultivados com cana, na região Sudeste do Brasil. A implementação da colheita da cana crua alterou alguns atributos do solo, levando à diminuição da densidade e da temperatura do solo, aumento do potássio trocável, do diâmetro médio ponderado e da estabilidade de agregados e da umidade na superfície do solo. Também modificou a estrutura da comunidade microbiana, associando este tipo de colheita aos ambientes com menor grau de estresse microbiano. Em relação ao solo sob cana colhida queimada, o aumento do estoque de C nos Argissolos Amarelos dos tabuleiros costeiros, cultivados com cana colhida crua, foi de 10,6 Mg ha-1, com taxas anuais de sequestro de C iguais a 1,3 Mg ha-1 ano-1. Os balanços de C negativos estimados sob cana colhida queimada (-2,9 e -2,2 Mg de C ha-1 ano-1) sugeriram que, para as socarias, o solo sob este tipo de colheita atuou como uma fonte de C-CO2. Por outro lado, os balanços foram positivos sob cana crua (+1,1 e + 0,7 Mg de C ha-1 ano-1), com o solo apresentando-se como um possível sumidouro de C. Estes resultados mostraram que os estoques de C no solo sob Mata Atlântica são altos e muito susceptíveis ao cultivo do solo, uma vez que a estabilização do carbono nestes solos arenosos reside, principalmente, em mecanismos de proteção física. Os resultados também demonstram que algumas práticas de manejo do solo podem aumentar os níveis de carbono, mediante a adição de grandes quantidades de biomassa e a minimização do revolvimento do solo.

Submitted by Alice Araujo (alice.caraujo@ufpe.br) on 2018-06-20T17:52:58Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Kennedy Nascimento de Jesus.pdf: 2821633 bytes, checksum: ad7413c76f1a1fd792f8d226e55211d0 (MD5) / Made available in DSpace on 2018-06-20T17:52:58Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Kennedy Nascimento de Jesus.pdf: 2821633 bytes, checksum: ad7413c76f1a1fd792f8d226e55211d0 (MD5) Previous issue date: 2017-02-02 / CAPES / A conversão de áreas de vegetação nativa em áreas destinadas à exploração agropecuária é apontada como um dos principais fatores responsáveis pelas emissões de Gases de Efeito Estufa para a atmosfera. Essas mudanças no uso da terra causam na paisagem um mosaico de áreas com diferentes usos e coberturas do solo. Porém, ainda é limitado o conhecimento da dinâmica do carbono (C) e do Nitrogênio (N) do solo nesses sistemas, principalmente na região Nordeste do Brasil. Nesse sentido, o objetivo desse trabalho foi determinar as alterações dos estoques e da dinâmica do C e N no solo devido às mudanças de uso da terra nas diferentes regiões fisiográficas e nas principais classes de solo de Pernambuco. Foram realizados três estudos, que auxiliaram no alcance do objetivo proposto: 1) no primeiro, foi realizado um levantamento e compatibilização da literatura sobre os estoques de C na camada superficial (0-30 cm) dos solos de Pernambuco. 2) no segundo estudo, foram quantificados os estoques de carbono (in situ) e sua distribuição nas camadas ao longo do perfil do solo (0-100 cm) em áreas de vegetação nativa, agrícolas e pasto, nas principais regiões fisiográficas e classes de solo do Estado; e 3) no terceiro, foram avaliados a dinâmica do C e do N, através da abundância natural de δ13C e δ15N e das concentrações de C e N em perfis (0-100 cm) de Argissolos ao longo de um gradiente climático em Pernambuco em áreas representativas de floresta nativa, de pasto e agrícolas. Em todos os estudos, as áreas de vegetação nativa foram tomadas como condição original do solo de acordo com cada região estudada. Nos estudos 2 e 3 quantificou-se as concentrações de C e N por meio de analisador elementar CHN e no estudo 3 foram quantificadas a abundância natural de δ 13C e δ 15N nos Argissolos do Estado, por meio de espectrômetro de massa. Aproximadamente 368 Tg de C estão estocados nos solos de Pernambuco de 0-30 cm. De uma forma geral, os maiores estoques de carbono total ocorreram nas vegetações nativas densas e decresceram da região úmida à semiárida Oeste, com grandes variações entre as classes de solos. A substituição da vegetação nativa para implantação de áreas de pastagens ou agrícolas acarretou mudanças no sinal δ13C do solo, sendo maiores nas áreas úmidas e subúmidas do estado em relação às áreas semiáridas. O enriquecimento de δ15N encontrado na superfície do solo das regiões semiáridas em relação às áreas úmidas e subúmidas estão associados as menores concentrações de C e N presentes nos Argissolos dessa região, em decorrência das maiores temperaturas e menores precipitações pluviais. Fica evidente que a intervenção humana nessas áreas através de práticas agropecuárias, reduz os estoques e modificam a dinâmica do C e do N no solo, e para se contrapor a esta situação no Estado, são necessárias medidas mitigatórias, como a adoção de sistemas conservacionistas do solo e da água, através de políticas públicas de incentivo às práticas conservacionistas em substituição à agricultura de baixo C. / The conversion of native vegetation areas into agricultural areas is pointed out as one of the main factors responsible for the emissions of greenhouse gases to the atmosphere. Changes in land use create a mosaic of areas in the landscape with different soil coverages and knowledge of the dynamics of C and N in these systems is limited. The objective of this work was to determine the changes in soil carbon (C) and nitrogen (N) stocks and dynamics due to changes in land use in the different physiographic regions and in the main soil classes of Pernambuco state. Three studies were carried out: 1) the first was a survey and compatibilization of the literature on C stocks in the surface layer (0-30 cm) of the soils of Pernambuco; 2) the second study was a quantification of the C stocks along the different layers of the soil profile (0-100 cm) in areas under native vegetation, agriculture and pasture, in the main physiographic regions and soil classes of Pernambuco; and 3) the third study evaluated soil C and N dynamics through the natural abundance of δ13C and δ15N and the concentrations of C and N in profiles of Ultisols (0-100 cm) along a climatic gradient in Pernambuco. In all studies, dense native vegetation areas were taken as the original, reference soil condition. In studies 2 and 3 the concentrations of C and N were quantified using CHN elemental analyzer. In study 3 the natural abundance of δ13C and δ15N were quantified by mass spectrometer. Approximately 368 Tg of carbon are stored in the soils of Pernambuco in the 0-30 cm layer. The largest total carbon stocks occurred under dense native vegetation and decreased from the humid region towards the semi-arid western state region, with large variations among soil classes. The replacement of the native vegetation by agriculture or pasture caused changes in the soil δ13C signal, being greater in the humid areas and subhumid regions than in the semi-arid region. The enrichment of δ15N in the soil surface layer of the semi-arid regions in relation to the humid and subhumid regions is associated with the lower concentrations of C and N in these regions, due to their higher temperatures and lower rainfall. It is evident that Human intervention in these areas through conventional farming practices reduces soil carbon stocks at levels lower than those found in native areas, and modifies the C and N dynamics in the soil in these regions. To counteract this situation in the State, mitigating measures are necessary, such as the adoption of soil and water conservation systems, through public policies to encourage conservation practices in place of low carbon agriculture.

Les estuaires, de par leur position stratégique à l'interface entre les eaux continentales et les eaux marines jouent un rôle écologique de première importance et sont le siège de nombreuses activités humaines. L’estuaire de Seine est caractéristique des grands estuaires anthropisés. Le management à long terme de ces écosystèmes soumis à des pressions croissantes réside notamment en une meilleure connaissance de la dynamique spatiale et temporelle des réseaux trophiques estuariens. Le phytoplancton et le microphytobenthos sont les principaux contributeurs de la production primaire (PP) dans ces écosystèmes et sont à la base des réseaux trophiques. Ces compartiments sont souvent réduits à la teneur en chlorophylle du milieu et leur productivité n’a jamais été mesurée à l’échelle de l’estuaire de Seine. L’objectif de ces travaux a été d’estimer la PP de ces compartiments le long du gradient halin. Afin d’accéder à des mesures à une haute résolution spatiale, des mesures de fluorescence modulée (PAM) à haute fréquence ont été couplées à des mesures d’incorporation de carbone (13C) à basse fréquence. Les mesures de production primaire ont été mises en perspective avec la dynamique des paramètres physico-chimiques du milieu et la structure communautés phytoplanctoniques déterminés par différentes techniques (microscopie, cytométrie, biologie moléculaire). La dynamique du carbone excrété sous forme de TEP (Transparent Exopolymeric Particules) et EPS (Exopolymeric substances) a également été étudiée pour chacun des compartiments. Au-delà des méthodologies innovantes mises en place qui montre l’intérêt des mesures à haute fréquence dans ces écosystèmes très dynamiques, ce travail apporte une nouvelle vision de la dynamique du phytoplancton et de la richesse spécifique du microbiome estuarien et apporte une estimation fiable de la production primaire. / The estuaries play an important ecological role and are the site of many human activities because of their strategic position at the interface between continental and marine waters. Seine estuary is characteristic of large anthropized estuaries. Long-term management requires better knowledge of the spatial and temporal dynamics of the estuarine food webs. Phytoplankton and microphytobenthos are the main contributor of primary production (PP) in these ecosystems and are at the basis of trophic food webs. These compartments are often reduced to the chlorophyll concentration and their productivity has never been measured along the Seine estuary. The objective of this study was to estimate the PP of these compartments along the salinity gradient. In order to access to measurements at high spatial resolution, high-frequency measurements of modulated fluorescence (PAM) were coupled to low-frequency carbon (13C) incorporation measurements. The primary production measurements have been put into perspective with the dynamics of the physical and chemical parameters and the structure of phytoplankton communities determined by different techniques (microscopy, cytometry, molecular biology). The dynamics of carbon excreted as TEP (Transparent Exopolymeric Particles) and EPS (Exopolymeric substances) were also studied for each compartment. Beyond the innovative methodologies which demonstrate the interest of high-frequency measurements in these highly dynamic ecosystems, this work provides a new insight into the phytoplankton dynamics and the specific richness of the estuarine microbiome and provides a reliable estimate of primary production.

No description available.

<p>Plants naturally deposit a significant amount of carbon towards lignin, a polymer that imparts mechanical strength to cell walls but impedes our utilization of the polysaccharides in lignocellulosic biomass. Genetic engineering of lignin has demonstrated profound success in improving the processing of the biomass. Lignin is derived from the phenylpropanoid pathway, the architecture of which is well understood based upon the biochemical and genetic studies conducted to date. In contrast, we lack a systematic and quantitative view of the factors that determine carbon flux into and within this branched metabolic pathway in plants. To explore the control of carbon allocation for phenylalanine and lignin biosynthesis, we have developed a kinetic model of the pathway in Arabidopsis to test the regulatory role of several key enzymatic steps. We first established a ¹³C isotope feeding system for the measurement of flux using excised wild-type Arabidopsis stems. The excised stems continued to grow and lignify in our feeding system. When ring ¹³C₆-labeled phenylalanine ([¹³C₆]-Phe) was supplied to excised stems, isotope label was rapidly incorporated into soluble intermediates and lignin. Using this approach, we then analyzed metabolite pool sizes and isotope abundances of the pathway intermediates in a time course from stems fed with [¹³C₆]-Phe of different concentrations, and used these data to parameterize a kinetic model constructed with Michaelis-Menten kinetics. Our model of the general phenylpropanoid pathway captured the dynamic trends of metabolite pools <i>in vivo</i>and predicted the metabolic profiles of an independent feeding experiment. Based on the model simulation, we found that subcellular sequestration of pathway intermediates is necessary to maintain lignification homeostasis when metabolites are over-accumulated. Both the measurements and simulation suggested that theavailability of substrate Phe is one limiting factor for lignin flux in developing stems. This finding indicates new gene targets for lignin manipulation in plants. To extend our kinetic model to simulate flux distribution in response to genetic perturbations, we conducted an RNA-sequencing experiment in wild type and 13 plants with modified lignification, and integrated the transcriptional data with the metabolic profiles. We found that the biosynthesis of Phe and lignification are tightly coordinated at transcriptional level. The coregulation of the shikimate and phenylpropanoid pathways involves transcriptional and post-translational regulatory mechanisms to maintain pathway homeostasis. Our results also indicate that induction of Phe supply and enhancement of PAL activity are both effective strategies to increase carbon flux into the phenylpropanoid network.</p><p>In this interdisciplinary project, we have taken various system biology approaches to understand metabolic flux towards lignin, the second most abundant carbon sink in nature. We have combined isotope labeling aided flux measurements and mathematical simulation, and have integrated metabolome data with transcriptome profiles. The experiments and analysis have been conducted in both wild-type Arabidopsis and those with perturbed lignification. The novel work not only provides insight into our knowledge of phenylpropanoid metabolism, but also creates a framework to systematically assemble gene expression, enzyme activity, and metabolite accumulation to study metabolic fluxes, the ultimate functional phenotypes of biochemical networks.</p>

Arctic soils contain a large fraction of our planets terrestrial carbon (C) pool. When tundra soils become warmer and permafrost thaws, non-native geoengineering earthworms can enter these soils and ingest organic matter accumulated over long timescales. Previous studies have found that earthworms increase mineralization rates of soil organic matter into carbon dioxide (CO2) when introduced. Yet, this initial mineralization boost seems transient with time and it has been hypothesized that earthworms stimulate the formation of persistent C forms. In this study, I investigated how non-native, geoengineering earthworms affected the relative proportions of seven carbon forms in the O and A1 horizon of tundra soil and if their effect induced a change in pH. I used Nuclear Magnetic Resonance (NMR) spectroscopy to understand what happens to soil carbon compounds in two different tundra vegetation types (heath and meadow), that had been subjected to earthworm treatment for three summers. I found that O-aromatic C increased from 7.22% ± 0.24 (mean ± stderr) in the meadow soil lacking earthworms to 8.98% ± 0.30 in the meadow exposed to earthworms, and that aromatic C increased from 8.71% ± 0.23 to 9.93% ± 0.25. In similar, the result suggested that alkyl C decreased in this vegetation type from 20.43% ± 0.38 to 18.70% ± 0.25 due to earthworm activities. I found no effect on the chemical properties in the heath. I conclude that geoengineering earthworms affect the two vegetation types differently and that earthworms seem to enhance the accumulation of recalcitrant aromatic C forms.

Cellular metabolism is one of the most fundamental systems for any living organisms, involving thousands of metabolites and reactions that forms large interconnected metabolic networks. Proper and comprehensive understanding of the metabolism in human cells has been a field of research for a long time. One of the key parameters in understanding the metabolism are the metabolic fluxes, which are the rates of conversion of metabolic intermediates. Currently, one of the main approaches for determining these fluxes is metabolic flux analysis (MFA), in which isotope-labelled compounds are introduced into the system and measured. Mathematical models are then used to calculate a prediction of the systems flux configuration. However, the current paradigm of MFA lack established methods for validating that a model can accurately predict quantities for which there are no experimental data. In this study, a model for the central human metabolism was created and evaluated with regards to the model’s ability to predict a validation dataset. Further, an uncertainty analysis of these predictions were performed with a prediction profile likelihood analysis. This study has conclusively shown that MFA models can be validated against experimental data that the model has never seen before. Additionally, such model predictions were shown to be observable with a well determined prediction uncertainty. These results shows that a systematic validation of MFA models is possible. This in turn allows for a greater trust to be placed in the models, and in any conclusions that are based on such models.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23950号 / 農博第2499号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5385(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)准教授 岡田 直紀, 教授 井鷺 裕司, 教授 柴田 昌三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM