Adequate nutrition plays a key role in the phytoextraction of soil contaminants and may influence the response of plants to heavy metal toxicity. Nevertheless, there is no information on the influence of nitrogen (N), particularly in the forms of nitrate (NO3-) and ammonium (NH4+), on tanzania guinea grass (Panicum maximum cv. Tanzania) under cadmium (Cd) stress. Thus, in the present work, it was aimed to study the effect of these ions on the mitigation of Cd toxicity in this grass. Plants were grown in a greenhouse, using plastic pots containing nutrient solution and ground quartz as substrate. The experiment was laid out in a randomized complete block design, in a 3 × 3 factorial arrangement, with six replications. The factors tested were NO3-/NH4+ ratios (100/0, 70/30 and 50/50) and Cd rates (0.0, 0.5 and 1.0 mmol L-1). Morphological, physiological and biochemical changes in the plants were studied throughout two growth periods. In addition to causing oxidative stress and visual toxicity symptoms, Cd exposure resulted in reduced chlorophyll content and photosynthetic activity, negatively affecting shoot and root growth. Moreover, it altered N metabolism and induced the accumulation of NO3- and NH4+ mainly in shoots, increasing plant total N concentration. The exclusive use of NO3- mitigated toxicity symptoms by favoring Cd accumulation in roots, maintaining normal N metabolism and increasing guaiacol peroxidase activity. On the other hand, although the simultaneous supply of NO3- and NH4+ increased the uptake, transport and accumulation of this metal by the grass, it also increased the photosynthetic capacity along with the synthesis of proline and protein. The use of these N forms at a 50/50 ratio increased the tolerance of the plants to Cd by inducing high superoxide dismutase and glutathione reductase activities in shoots and roots, respectively, maintaining cellular homeostasis and reducing oxidative stress. Cd uptake and accumulation in tanzania guinea grass are strongly related to the form of N available, and the negative effects of this metal on photosynthesis and on the balance between oxidants and antioxidants are attenuated by the partial replacement of NO3- by NH4+ in the nutrient solution. / A nutrição adequada desempenha papel fundamental na fitoextração de contaminantes do solo e pode influenciar a resposta das plantas à toxidez por metais pesados. No entanto, não há informações sobre a influência do nitrogênio (N), particularmente nas formas de nitrato (NO3-) e amônio (NH4+), no capim tanzânia (Panicum maximum cv. Tanzânia) sob estresse por cádmio (Cd). Assim, no presente trabalho, objetivou-se estudar o efeito desses íons na mitigação da toxidez por Cd nesse capim. As plantas foram cultivadas em casa de vegetação, utilizando vasos plásticos contendo solução nutritiva e quartzo moído como substrato. O experimento foi arranjado em delineamento de blocos completos ao acaso, em esquema fatorial 3 × 3, com seis repetições. Os fatores testados foram proporções de NO3-/NH4+ (100/0, 70/30 e 50/50) e doses de Cd (0,0, 0,5 e 1,0 mmol L-1). Foram estudadas alterações morfológicas, fisiológicas e bioquímicas nas plantas ao longo de dois períodos de crescimento. Além de causar estresse oxidativo e sintomas visuais de toxidez, a exposição ao Cd resultou em baixos teor de clorofila e atividade fotossintética, afetando negativamente o crescimento da parte aérea e das raízes. Além disso, ela alterou o metabolismo do N e induziu o acúmulo de NO3- e NH4+ principalmente na parte aérea, aumentando a concentração de N total na planta. O uso exclusivo do NO3- mitigou sintomas de toxidez favorecendo o acúmulo de Cd nas raízes, mantendo o metabolismo normal do N e aumentando a atividade da guaiacol peroxidase. Por outro lado, embora o suprimento simultâneo de NO3- e NH4+ tenha aumentado a absorção, o transporte e o acúmulo desse metal pelo capim, também aumentou a capacidade fotossintética juntamente com a síntese de prolina e proteína. O uso dessas formas de N na proporção de 50/50 aumentou a tolerância das plantas ao Cd induzindo altas atividades da superóxido dismutase e da glutationa redutase na parte aérea e nas raízes, respectivamente, mantendo a homeostase celular e reduzindo o estresse oxidativo. A absorção e o acúmulo de Cd no capim tanzânia estão fortemente relacionados à forma de N disponível, e os efeitos negativos desse metal na fotossíntese e no balanço entre oxidantes e antioxidantes são atenuados pela substituição parcial de NO3- por NH4+ na solução nutritiva.

Made available in DSpace on 2016-04-28T14:58:31Z (GMT). No. of bitstreams: 1 2003- Daniele Cristina Costa Sabino.pdf: 534915 bytes, checksum: c4045f2c9f540e49bb6c91877c6845ff (MD5) Previous issue date: 2003-03-26 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Rice is colonized by diazotrophic bacteria that can act as root growth promoting, as well as to increase plant nutrition throw the biological nitrogen fixation process. These associations can modify the plant physiology since they compete for carbohydrate and other compounds necessary to host plants. The aim of this work was to evaluate the influence of the endophytic diazotrophic bacteria, Burkholderia brasilensis and Herbaspirillum seropedicae on nitrogen metabolism of two rice cultivars (IR42 and IAC4440). The experiment was carried out in pots containing 3 kg of soil, fertilized with 3 nitrogen levels (0, 40, and 80 kg N ) as (NH4)2SO4, and three inoculation treatments (H. seropedicae (ZAE94), B. brasilensis (M130) and the mixture of both). The plants were harvested at vegetative (45 days of inoculation) and flowering stages (according to the cycle of each cultivar). Bacterial counting, dry matter, N content and the concentration of nitrogen compounds were measured at each harvest. The results showed no statistical differences on bacterial counting. The number of root bacteria was higher than in shoots. A population reduction occurred on the flowering stage as compared to vegetative one. Cultivar IR42 responded to inoculation more noticiable than IAC4440. The dry matter accumulation of the IR42 cultivar at the vegetative stage varied with inoculation and nitrogen dose applied. This cultivar accumulated higher amounts of nitrogen when inoculated with ZAE94 strain, independent of the nitrogen applied. Concentrations of nitrate, free amino acid and soluble sugar in the aerial parts were higher in plants fertilized with 40kgN/ha and inoculated with the mixture of bacteria. These results suggested that the diazotrophic bacteria inoculation could modify the plants nitrogen metabolism and theses effects were variable according to the rice cultivars and nitrogen fertilization level applied. / O arroz (Oryza sativa L.) ? colonizado por bact?rias diazotr?ficas que, podem atuar como promotoras de crescimento das ra?zes, assim como melhorar a nutri??o da planta atrav?s do N2 fixado biologicamente. No entanto, estas associa??es podem modificar a fisiologia das plantas, uma vez que as bact?rias diazotr?ficas para fixarem o nitrog?nio necessitam de carboidratos e outros componentes, competindo, deste modo, com as plantas hospedeiras. Assim sendo, este trabalho teve como objetivo avaliar a influ?ncia da inocula??o de bact?rias diazotr?ficas endof?ticas Burkholderia brasilensis e Herbaspirillum seropedicae no metabolismo de nitrog?nio de duas cultivares de arroz adubadas ou n?o com nitrog?nio mineral. Um experimento em condi??es de vasos foi realizado na ?rea experimental da Embrapa Agrobiologia com as cultivares de arroz IR42 e IAC4440. A inocula??o consistiu no uso das estirpes ZAE94 de Herbaspirillum seropedicae, e M130 de Burkholderia brasilensis , a mistura destas duas estirpes e da testemumha (controle n?o inoculado). A aduba??o na forma de (NH4)2SO4, foi aplicada nas concentra??es de 0, 40 e 80 kgN/ha. As coletas foram realizadas no per?odo vegetativo (45 dias) e no florescimento (de acordo com o ciclo de cada cultivar). Os par?metros avaliados foram a presen?a das bact?rias nos tecidos das plantas, o ac?mulo de mat?ria seca da parte a?rea, o ac?mulo de N, e a concentra??o dos compostos nitrogenados na planta. Os resultados mostraram que n?o houve diferen?as nas popula??es das bact?rias em fun??o da inocula??o, por?m, o n?mero de bact?rias na raiz foi ligeiramente maior do que na parte a?rea. Foi tamb?m observado uma redu??o na popula??o no per?odo de florescimento, em rela??o ao per?odo vegetativo. De um modo geral, a cultivar IR42 apresentou maiores respostas ? inocula??o, quando comparada com a cultivar IAC4440. O ac?mulo de mat?ria seca da cultivar IR42 no per?odo vegetativo, variou com a inocula??o e a dose de nitrog?nio aplicada. Por?m, a percentagem de nitrog?nio (%N) acumulada na parte a?rea destas plantas, foi maior nos tratamentos inoculados com a estirpe ZAE94, independente da dose de nitrog?nio aplicada. As plantas que receberam 40 kgN/ha e foram inoculadas com a mistura, apresentaram as maiores concentra??es de nitrato, amino?cidos livres e a??cares sol?veis na parte a?rea. Os resultados obtidos sugerem que a inocula??o das bact?rias diazotr?ficas modifica o metabolismo de nitrog?nio e que este efeito foi diferenciado em fun??o da variedade e da dose de nitrog?nio aplicada.

Made available in DSpace on 2017-07-10T17:48:33Z (GMT). No. of bitstreams: 1 Debora_Cristiane_Freitag.pdf: 951246 bytes, checksum: 2fcc6daede28dcf8dac6f7f4dc54f30d (MD5) Previous issue date: 2012-08-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study aimed to evaluate the reduction of crude protein ration on nitrogen balance, blood parameters, and physiological urinary pH of barrows in the initial phase in stress condition (28,77 ˚C) and thermal comfort (18,13 ˚C). For the experiment we used 32 barrows in the initial phase, with initial mean body mass of 18,5 ± 0,73 kg. The animals were divided into two experiments in two environmental conditions (stress and thermal comfort) in a randomized complete block with four decreasing levels of crude protein (20,5; 19,4; 18,3 and 17,2%) with four replications, totaling 16 plots in each experiment. Each plot consisted of an animal, totaling 32 animals. The parameters analyzed were ingested nitrogen, excreted in feces and urine, absorbed, retained, retained/absorbed, total excretion, net protein utilization (ULP), cholesterol, triglycerides, glucose, urea, creatinine in blood plasma, urine pH, respiratory rate, rectal temperature and body. In heat stress condition, reducing the crude protein content of the diet decreased the nitrogen excreted in the urine, the total excretion and plasma triglyceride in growing pigs. For thermal comfort condition in growing pigs had higher nitrogen ingested, absorbed, retained and, of plasma urea. The response surface for rectal temperature, body temperature and respiratory rate increased, in function to ITGU and different times of observation, being more evident in thermal comfort condition / Este trabalho teve por objetivo estudar a redução da proteína bruta de rações sobre o balanço de nitrogênio, parâmetros sanguíneos, fisiológicos e pH urinário de suínos machos castrados na fase inicial, em condição de estresse (28,77˚C) e conforto térmico (18,13˚C). Para o experimento foram utilizados 32 suínos machos castrados em fase inicial, com massa corporal média inicial de 18,5 ± 0,73kg. Os animais foram distribuídos em dois experimentos em duas condições de ambiente (estresse e conforto térmico) em um delineamento experimental de blocos com quatro níveis decrescentes de proteína bruta (20,5, 19,4, 18,3 e 17,2%) com quatro repetições, totalizando 16 parcelas em cada experimento. Cada parcela experimental constou de um animal, totalizando 32 animais. Os parâmetros analisados foram nitrogênio ingerido, excretado nas fezes e na urina, absorvido, retido, retido/absorvido, excreção total, utilização líquida de proteína (ULP), colesterol, triglicerídeos, glicose, uréia, creatinina no plasma sanguíneo, pH da urina, frequência respiratória, temperatura retal e corporal. Em condição de estresse térmico, a redução do teor de proteína bruta da ração diminuiu o nitrogênio excretado na urina, a excreção total e o triglicerídeo plasmático de suínos em crescimento. Para condição de conforto térmico os suínos em crescimento apresentaram maiores valores de nitrogênio ingerido, absorvido, retido, bem como, de uréia plasmática. A superfície de resposta para temperatura retal, temperatura corporal e frequência respiratória aumentaram em função do ITGU e diferentes horários de observação, sendo mais evidente em condição de conforto térmico

Made available in DSpace on 2016-04-26T19:39:32Z (GMT). No. of bitstreams: 1 2009 - Leandro Azevedo Santos.pdf: 3069700 bytes, checksum: dbd1726a5b683e7f290e1829143eacac (MD5) Previous issue date: 2009-06-03 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / To improve nitrogen usage efficiency in plants the rice transcriptional factor OsDof25 was identified and cloned, whose probably orthologe is the maize ZmDof1, already identified and partially characterized. The ZmDof1 was also cloned for comparative analysis with OsDof25, in order to confirm this last one as ZmDof1 orthologe in rice. The constructions for Arabidopsis superexpression of these transcriptional factors were made using the cloning system of gateway technology (Invitrogen), to obtain the expression vectors 35S:ZmDof1:HA and 35S:OsDof25:HA. Lineages with different expression levels of these genes were obtained, but with only one inserted copy. These transgenic lineages when grown in a half strength of MS medium (10mM of NH4 + and 20mM of NO3 -) showed phenotypes with chloroses and growth difficulty; although when they were cultured in soil they showed great vegetative development and delay in the inflorescence emission. When analyzed the gene expression changes induced by the superexpression of these transcriptional factors, it was observed that both genes produced an increase in the expression levels of high and low affinity ammonium transporters (AMT1.1 and AMT2.1, respectively), indicating that these phenotypes may be due to the toxic effect of an excess of ammonium uptake. We also verified an increase of expression for pyruvate kinase (PK1 and PK2), and phosphoenolpyruvate carboxylase (PEPC1 and PEPC2). Pyruvate kinase converts phophoenolpyruvate (PEP) to pyruvate, and phosphoenolpyruvate carboxylase converts PEP to oxalacetate, which is substrate for malate dehydrogenase to form malate. Both pyruvate and malate may feed the Krebs cycle. In addition, there was an increase in the expression of isocitrate dehydrogenase, which is present in the citosol and mitochondria, needed for converting isocitrate to 2- oxoglutarate. Thus, it was hypothesized that the increase of expression levels of these carbon metabolism enzymes was necessary to increase the production of 2-oxoglutarate and, consequently, to reduce the toxic effect of ammonium uptaked. Besides, it was observed an increase of expression levels and activity of glutamate dehydrogenase (GDH). This enzime may work as much in the direction of glutamate amination as in deamination, when the plants were submitted to ammonium excess or carbon limitation conditions, respectively. / Com o objetivo de aumentar a efici?ncia de uso de nitrog?nio (EUN) em plantas, foi identificado e clonado o fator de transcri??o OsDof25 de arroz, cujo prov?vel ort?logo ? o ZmDof1 de milho, j? identificado e parcialmente caracterizado. Tamb?m foi clonado o ZmDof1 para an?lises comparativas com o OsDof25, a fim de comprovar que este ?ltimo ? realmente ort?logo do ZmDof1. As constru??es para superexpress?o destes fatores de transcri??o em Arabidopis foram feitas utilizando o sistema gateway de clonagem para obten??o dos vetores de express?o 35S:ZmDof1:HA e 35S:OsDof25:HA. Foram obtidas linhagens com diferentes n?veis de express?o destes genes, mas com apenas uma inser??o. As linhagens transg?nicas obtidas quando crescidas em meio MS ? for?a i?nica (10mM de NH4 + e 20mM de NO3 -) apresentaram fen?tipos como clorose e dificuldade de desenvolvimento, ao passo que quando cultivadas em solo mostraram desenvolvimento vegetativo mais intenso e atraso para emiss?o da infloresc?ncia. Quando analisadas as modifica??es de express?o g?nica causadas pela superexpress?o destes fatores de transcri??o, observou-se que ambos os fatores de transcri??o provocaram aumento de express?o dos transportadores de am?nio de alta e baixa afinidades (AMT1.1 e AMT2.1 respectivamente), indicando que o fen?tipo observado pode ser devido ao efeito t?xico do excesso de am?nio absorvido. Verificou-se tamb?m aumento de express?o das enzimas piruvato quinase (PK1 e PK2) e fosfoenolpiruvato carboxilase (PEPC1 e PEPC2). A piruvato quinase converte o fosfoenolpurato (PEP) a piruvato, enquanto a fosfoenolpiruvato carboxilase converte o PEP a oxalacetato (OAA) que pode sofrer a??o da malato desidrogenase originando o malato. Ambos os metab?litos, piruvato e malato, alimentam o ciclo de Krebs. Houve tamb?m aumento de express?o da isocitrato desidrogenase, enzima presente na mitoc?ndria (ciclo de Krebs) e no citosol que converte isocitrato a 2-oxoglutarato (2-OG). Assim, ? prov?vel que o aumento da express?o destas enzimas do metabolismo de carbono foi necess?rio para aumentar a produ??o de 2-OG e, por conseguinte, diminuir o efeito t?xico do excesso de am?nio absorvido. Al?m disso, observou-se aumento de express?o e atividade da glutamato desidrogenase (GDH). Essa enzima pode atuar tanto na dire??o da amina??o, quanto na dire??o da desamina??o, em condi??es de excesso de am?nio e/ou sob condi??es de limita??o de carbono nas plantas, respectivamente.

Porteur redox intervenant dans nombre de processus métaboliques, le NAD (nicotinamide adénine dinucléotide) est central pour les cellules vivantes. Outre son importance dans le métabolisme oxydoréductif, des données récentes suggèrent fortement d’autres rôles importants pour le NAD dans la signalisation cellulaire. Un système inductible d’enrichissement en NAD en surproduisant la quinolinate phosphoribosyltransférase (QPT) d’Escherichia coli chez Arabidopsis thaliana a permis de mettre en évidence l’implication du NAD dans les mécanismes spécifiques de défenses qui régissent les interactions plante-pathogène. Par ailleurs, une dérégulation de la synthèse de NAD sur l’étape enzymatique catalysée par la QPT endogène d’Arabidopsis thaliana souligne le rôle critique du NAD dans la balance C/N des plantes, en particulier en bouleversant l’assimilation de l’azote en conditions photorespiratoires. Ces travaux nous ouvrent à une nouvelle compréhension des mécanismes de signalisation impliquant le NAD dans les grandes fonctions métaboliques des plantes. / Plant development and functions are underpinned by redox reactions which depend on cofactors such as pyridine nucleotides as nicotinamide adenine dinucleotide (NAD). Beside its redox properties, NAD has recently been implicated in cellular signalling. An inducible system based on Escherichia coli quinolinate phosphoribosyltransferase (QPT) overproduction in transgenic Arabidopsis thaliana was set up as a convenient experimental technique to raise NAD content. This build-up highlights the involvement of NAD in plant-pathogen specific defense mechanisms. Furthermore, manipulating endogenous Arabidopsis thaliana QPT levels was used to deregulate NAD production. Such an approach points out the critical role of NAD in C/N interactions by shaking up nitrogen assimilation upon photorespiratory conditions. These results pave the way for a new understanding of signalling mechanisms involving NAD in plants major metabolic functions.

A fase de desenvolvimento é um importante fator a ser considerado em pesquisas sobre nutrição de bromélias. O hábito de vida dessas plantas pode mudar de: atmosférica (com folhas sem formar um tanque) para o com tanque ao longo do seu desenvolvimento. Algumas pesquisas mostraram que o conteúdo de nitrogênio foliar ou capacidade fotossintética são significantemente influenciados pela fase de desenvolvimento, porém não há registros de que a nutrição e o metabolismo do nitrogênio diferem entre bromélias jovens ou adultas. O objetivo principal deste projeto foi verificar se existem diferenças na dinâmica do metabolismo do nitrogênio (absorção, transporte e assimilação), decorrente da utilização de fontes de distintas (amônio, nitrato ou ureia), entre bromélias nas fases atmosférica ou adultas com tanque desenvolvido. Para tanto, plantas de Vriesea gigantea foram regadas com uma solução nutritiva que conteve 5mM de N total, disponível nas formas: 15NH4+ ou 15NO3- ou 15N-ureia. Foram feitas coletas temporais das raízes e de duas diferentes porções da folha (ápice e base) das bromélias jovens e de três regiões foliares (ápice, mediana e base) das folhas das bromélias adultas com tanque. Todas as amostras vegetais foram utilizadas na avaliação das atividades da: urease, redutase do nitrato, sintetase da glutamina e desidrogenase do glutamato; e da quantificação da abundância isotópica do 15N. Segundo os resultados, o nitrato foi considerado a fonte de nitrogênio absorvida em concentrações menores quando comparada com a ureia e o amônio pelas bromélias de ambas as fases de desenvolvimento. Entretanto, as bromélias atmosféricas mostraram ser capazes de capturar essa fonte inorgânica de nitrogênio mais eficientemente do que as bromélias com tanque, uma vez que o nitrato foi absorvido, transportado e assimilado rapidamente na 1ª hora após o fornecimento dessa fonte. Já para as bromélias adultas, a absorção do nitrato foi lenta e ocorreu, principalmente, no final do experimento (12ª e 24ª hora). O amônio e a ureia foram as fontes absorvidas em maiores concentrações tanto pelas bromélias jovens quanto pelas adultas. Entretanto, as bromélias atmosféricas foram capazes de captar e metabolizar maiores concentrações de nitrogênio proveniente do amônio, enquanto que as da fase adulta com tanque foram mais aptas a absorver e assimilar maiores concentrações de ureia em seus tecidos. A bromélia V. gigantea pode mudar a sua morfologia e fisiologia ao longo de seu desenvolvimento, tornando-se apta a captar as fontes de nitrogênio que, talvez, sejam mais abundantes em cada fase de seu desenvolvimento. A água da chuva que contém, principalmente, fontes inorgânicas de nitrogênio diluídas, pode ser o principal meio por onde as bromélias jovens captam o nitrogênio. Ao desenvolverem um tanque, as bromélias podem mudar a sua fisiologia, capturando preferencialmente fontes de nitrogênio provenientes de matéria orgânica decomposta que se acumula no interior da cisterna. As raízes das bromélias atmosféricas também mostraram cumprir um papel fundamental na nutrição dessas plantas durante a fase juvenil, pois aumentaram a capacidade de absorção e assimilação de fontes de nitrogênio. Quando as bromélias iniciam o desenvolvimento de um tanque, as bases das folhas passaram a assumir a função do sistema radicular, enquanto que as raízes, talvez, começassem a diminuir sua capacidade de captar os nutrientes do meio ambiente. Os resultados bioquímicos demonstraram que existe uma forte sincronização de todas as etapas do metabolismo do nitrogênio (absorção, transporte e assimilação) envolvendo diferentes partes do corpo das bromélias (raízes, porções foliares da base, mediana ou ápice) de ambas as fases de desenvolvimento, sugerindo que nos tecidos vegetais dessas plantas, existe uma fina regulação de todos os processos fisiológicos e metabólicos que compreendem o metabolismo do nitrogênio. Essa regulação controlada seria necessária para que as bromélias atmosféricas ou com tanque desenvolvido consigam absorver, transportar e assimilar as fontes de nitrogênio rapidamente e com grande eficiência. Para finalizar, o novo termo \"bromélia epífita jovem sem tanque\" foi sugerido para se referir à bromélia V. gigantea na fase juvenil ao invés de \"bromélia epífita atmosférica\". As raízes dessa bromélia jovem demonstraram ter um papel fundamental nos processos de absorção e assimilação das fontes de nitrogênio, uma característica que geralmente não é atribuída para as raízes das bromélias com o hábito de vida atmosférico / The stages of ontogenetic development of bromeliad can be an important feature to be considered in the physiology studies because the young plants can be classified as atmospheric bromeliads, while the adult plants have a special structure formed by leaves called tank. Some studies showed that some physiological characteristics can be influenced by the stages of ontogenetic development in bromeliads as photosynthetic taxes or the total nitrogen (N) content in leaves. However, there are no records that nutrition and nitrogen metabolism differ between young and adult epiphytic bromeliads. The objective of this project was to verify the existence of differences in the dynamics of nitrogen metabolism (absorption, transportation and assimilation) arising from the use of distinct nitrogen sources (NH4+, NO3- or urea) in epiphytic bromeliad Vriesea gigantea with different stages of ontogenetic development (atmospheric or tank). A nutrient solution, consisting 5mM of total N, was offered to bromeliads. Three different forms of N sources were used: NH4+, NO3- or urea, enriched with 15N isotopes. Three distinct portions of leaf (apex, middle and base) of adult tank bromeliad and two different regions of leaf (apex and base) and roots of young bromeliads were harvested in six different times. All samples were used in enzymatic assays of urease, nitrate reductase, glutamate sinthetase and glutamate dehydrogenase and in the 15N isotope quantification. According to the results, the nitrate was considered the nitrogen source absorbed at lower concentration by young and adult bromeliads. The atmospheric bromeliads were able to capture nitrate more efficiently than the tank plants, since this inorganic nitrogen source was absorbed and assimilated quickly in the 1st hour of the experimental time while the tank bromeliads absorbed nitrate slowly at the end of the experiment (12th and 24th hour). Ammonium and urea sources were absorbed in higher concentrations by atmospheric and tank bromeliads. The young bromeliads were able to absorb and assimilate higher concentrations of nitrogen from ammonium, while tank bromeliad absorbed and assimilated higher concentrations of urea. In each development stage, the epiphytic bromeliad V. gigantea can absorb and assimilate the nitrogen sources which are more available in the environment. The atmospheric bromeliads get to absorb diluted nutrients as inorganic nitrogen sources mainly from rainwater. After the tank structure developed in the rosette, the morphology and/or physiology features changes in the adult bromeliads. The tank bromeliads get to absorb mainly organic nitrogen sources from decomposed organic matter which accumulates inside the tank. The roots of atmospheric bromeliads also showed an important role in the nutrition of the young plants since the atmospheric bromeliads get to improve the nitrogen sources uptake and nitrogen assimilation. When the bromeliads developed a tank, the bases of the leaves might assume the absorption function, whereas the roots, perhaps, might decrease its capacity to capture the nutrients from the environment. The biochemical results showed that there is a strong synchronization of all stages of nitrogen metabolism (uptake, transport and assimilation) involving different body parts of bromeliads (roots, leaf portions of the base, middle or apex) of both development stages, suggesting that there might have a thin regulation of all physiological and metabolic processes of nitrogen metabolism in the bromeliad\'s tissues. This controlled regulation might be important to the atmospheric or tank bromeliads are able to absorb, allocate and assimilate nitrogen sources quickly and with great efficiency. Finally, the terminology “atmospheric epiphytic bromeliad” might not be appropriated to refer to young plants since their roots showed an important role in the absorption and assimilation of nitrogen sources. This feature is not usually attributed to the roots of atmospheric bromeliads. Then, the new terminology “young epiphytic bromeliad without tank” was suggested to refer the bromeliad V. gigantea in the juvenile phase

Candida parapsilosis is an emerging human opportunistic pathogen causing a wide spectrum of potentially life-threatening infections in immunocompromised hosts. One of the most important virulence factors of Candida spp. is a production of secreted aspartic proteinases (Saps). Presented thesis is mainly focused on the study of secreted aspartic proteinase 1 (Sapp1p) of C. parapsilosis, its processing and secretion under variable conditions and by use of various experimental models. Sapp1p is secreted by C. parapsilosis cells into the extracellular space as a completely processed and fully active enzyme. Experiments studying the C. parapsilosis cell wall (CW) confirmed the prolonged presence of completely processed Sapp1p on the cell surface (CW- Sapp1p). Proteolytic activity assay performed with the intact cells showed that CW-Sapp1p is proteolytically active prior to its release into the extracellular space and is capable of substrate cleavage. Biotinylation experiments with consecutive MS analysis revealed that CW-Sapp1p biotinylation is incomplete but saturable process, leaving partially unlabelled molecules. The accessibility of individual lysine residues in the Sapp1p molecule varied, with exception of four residues that were labelled in all of our experiments performed. The final step of...

O suprimento de proporções de nitrato (NO3-) e amônio (NH4+) no meio de crescimento pode otimizar o metabolismo e o crescimento do Panicum maximum. A toxidez de cobre (Cu) causa estresse oxidativo nas plantas, afetando o metabolismo, a fotossíntese e a produção de massa. Não há pesquisas associando proporções de NO3-/NH4+ no alívio do estresse por Cu em plantas. O objetivo foi avaliar o efeito da toxidez de Cu no metabolismo, nutrição mineral, fotossíntese e produção do capim tanzânia, assim como o papel de proporções de NO3-/NH4+ no alívio dessa toxidez. O delineamento experimental foi de blocos ao acaso em arranjo fatorial 3×4 com seis repetições, sendo três destinadas à avaliação dos atributos metabólicos e fisiológicos e outras três aos atributos nutricionais e produtivos. Os fatores empregados foram: proporções de NO3-/NH4+ (100/0; 70/30 e 50/50) combinadas com doses de Cu (0,3; 250; 500 e 1000 μmol L-1) em solução nutritiva. Foram avaliados dois períodos de crescimento das plantas, sendo o primeiro com exposição ao Cu e o segundo sem exposição ao Cu. No primeiro corte as plantas supridas com 70/30 de NO3-/NH4+ e Cu de 1000 μmol L-1 apresentaram maior concentração de Cu nas folhas recém-expandidas (LR) e maior acúmulo de Cu na biomassa, maior concentração de NH4+ nas LR, maior acúmulo de NH4+ na parte aérea, maior atividade da glutamina sintetase nas LR, maior concentração de prolina nas LR, maior atividade da enzima superóxido dismutase (SOD) nas partes do capim e menor produção de biomassa. No capim suprido com 100/0 de NO3-/NH4+ e Cu de 1000 μmol L-1 foram obtidos menor concentração de Cu nas LR e menor acúmulo de Cu na parte aérea, mas a concentração e o acúmulo desse metal nas raízes foram incrementados mesmo nas mais baixas doses de Cu. As plantas crescidas com N na forma de NO3- ainda apresentaram maior concentração de NO3- nas LR, maior acúmulo de NO3- nas raízes, maior acúmulo de N total, maior atividade da nitrato redutase nas LR, maior condutância estomática e maior produção de biomassa. No capim recebendo 100/0 de NO3-/NH4+ também ocorreu menor atividade da SOD e menor concentração de prolina nas partes da planta. A concentração de malondialdeído foi menor na mais elevada dose de Cu, assim como a taxa de transpiração, a eficiência do fotossistema II, a taxa de transporte de elétrons e a taxa de assimilação de CO2. A atividade da catalase, guaiacol peroxidase, ascorbato peroxidase e glutationa redutase foram ativadas principalmente nas raízes, sendo maiores na mais elevada dose de Cu. A toxidez de Cu afeta negativamente o metabolismo, a nutrição mineral, a fisiologia e a produção do capim tanzânia, mas as proporções de NO3-/NH4+ alteram essa toxidez. O emprego de N na forma de NO3- é estratégia de maior potencial de uso em plantas com elevado estresse por Cu. Entretanto, a combinação de NO3- ao NH4+ no meio de crescimento em situação de moderado estresse por Cu é estratégica para maior fitoextração desse metal. / The supply of nitrate (NO3-) and ammonium (NH4+) proportions in the growth medium can optimize the metabolism and growth of Panicum maximum. Copper (Cu) toxicity causes oxidative stress in plants, affecting metabolism, photosynthesis and biomass production. There are no researchs associating proportions of NO3-/NH4+ for alleviating Cu stress toxicity. The objective was to evaluate the effect of Cu toxicity on metabolism, mineral nutrition, photosynthesis and dry matter production, as well as the role of NO3-/NH4+ proportions in alleviating such toxicity. The experimental was carried out in randomized complete block design in a 3×4 factorial with six replications. Three replications were used to evaluated metabolic and physiological attributes and other three to determine nutrition and productive attributes. The factors were three proportions of NO3-/NH4+ (100/0, 70/30 and 50/50) combined with four rates of Cu (0.3, 250, 500 and 1000 μmol L-1) in nutrient solution. Two plant growth periods evaluated, being the first with plants exposure to Cu and the second without the exposure to Cu. In the first cut, plants supplied with 70/30 of NO3-/NH4+ proportions and Cu rates of 1000 μmol L-1 showed high Cu concentration in recently fully expanded leaf laminae (LR), high Cu accumulation in the biomass, high NH4+ concentration in LR, high NH4+ accumulation in shoots, high glutamine synthetase activity in LR, higher proline concentration in LR, high superoxide dismutase (SOD) activity in parts of grass and low shoot dry matter production. In the grass supplied with 100/0 of NO3-/NH4+ and Cu of 1000 μmol L-1 it was found a low Cu concentration in LR and low Cu accumulation in shoots, but the concentration and accumulation of this metal in roots were increased even in lower Cu rate. Plants supply with 100/0 of NO3-/NH4+ also showed high NO3- concentration in LR, high NO3- accumulation in roots, high N accumulation in shoots, high nitrate reductase activity in LR, high stomatal conductance and high biomass production. In the grass supplied with 100/0 of NO3-/NH4+, there was low SOD activity and low proline concentration in shoots. The concentration of malondialdehyde was low at the high Cu rate, as well as the transpiration rate, photosystem II efficiency, electron transport rate and CO2 assimilation rate. Catalase, guaiacol peroxidase, ascorbate peroxidase and glutathione reductase activities were activated mainly in roots, being high in high Cu rate. Cu toxicity negatively affects metabolism, mineral nutrition, physiology and dry matter productions of tanzania guineagrass, but NO3-/NH4+ proportions change this toxicity. The only use of NO3- in the N supply is a strategy of great potential to be use in plants with of high Cu stress. However, combinations of NO3- with NH4+ in the growth medium when plants suffer moderate Cu stress is strategic for great metal phytoextraction.

O suprimento de proporções de nitrato (NO3-) e amônio (NH4+) no meio de crescimento pode otimizar o metabolismo e o crescimento do Panicum maximum. A toxidez de cobre (Cu) causa estresse oxidativo nas plantas, afetando o metabolismo, a fotossíntese e a produção de massa. Não há pesquisas associando proporções de NO3-/NH4+ no alívio do estresse por Cu em plantas. O objetivo foi avaliar o efeito da toxidez de Cu no metabolismo, nutrição mineral, fotossíntese e produção do capim tanzânia, assim como o papel de proporções de NO3-/NH4+ no alívio dessa toxidez. O delineamento experimental foi de blocos ao acaso em arranjo fatorial 3×4 com seis repetições, sendo três destinadas à avaliação dos atributos metabólicos e fisiológicos e outras três aos atributos nutricionais e produtivos. Os fatores empregados foram: proporções de NO3-/NH4+ (100/0; 70/30 e 50/50) combinadas com doses de Cu (0,3; 250; 500 e 1000 μmol L-1) em solução nutritiva. Foram avaliados dois períodos de crescimento das plantas, sendo o primeiro com exposição ao Cu e o segundo sem exposição ao Cu. No primeiro corte as plantas supridas com 70/30 de NO3-/NH4+ e Cu de 1000 μmol L-1 apresentaram maior concentração de Cu nas folhas recém-expandidas (LR) e maior acúmulo de Cu na biomassa, maior concentração de NH4+ nas LR, maior acúmulo de NH4+ na parte aérea, maior atividade da glutamina sintetase nas LR, maior concentração de prolina nas LR, maior atividade da enzima superóxido dismutase (SOD) nas partes do capim e menor produção de biomassa. No capim suprido com 100/0 de NO3-/NH4+ e Cu de 1000 μmol L-1 foram obtidos menor concentração de Cu nas LR e menor acúmulo de Cu na parte aérea, mas a concentração e o acúmulo desse metal nas raízes foram incrementados mesmo nas mais baixas doses de Cu. As plantas crescidas com N na forma de NO3- ainda apresentaram maior concentração de NO3- nas LR, maior acúmulo de NO3- nas raízes, maior acúmulo de N total, maior atividade da nitrato redutase nas LR, maior condutância estomática e maior produção de biomassa. No capim recebendo 100/0 de NO3-/NH4+ também ocorreu menor atividade da SOD e menor concentração de prolina nas partes da planta. A concentração de malondialdeído foi menor na mais elevada dose de Cu, assim como a taxa de transpiração, a eficiência do fotossistema II, a taxa de transporte de elétrons e a taxa de assimilação de CO2. A atividade da catalase, guaiacol peroxidase, ascorbato peroxidase e glutationa redutase foram ativadas principalmente nas raízes, sendo maiores na mais elevada dose de Cu. A toxidez de Cu afeta negativamente o metabolismo, a nutrição mineral, a fisiologia e a produção do capim tanzânia, mas as proporções de NO3-/NH4+ alteram essa toxidez. O emprego de N na forma de NO3- é estratégia de maior potencial de uso em plantas com elevado estresse por Cu. Entretanto, a combinação de NO3- ao NH4+ no meio de crescimento em situação de moderado estresse por Cu é estratégica para maior fitoextração desse metal. / The supply of nitrate (NO3-) and ammonium (NH4+) proportions in the growth medium can optimize the metabolism and growth of Panicum maximum. Copper (Cu) toxicity causes oxidative stress in plants, affecting metabolism, photosynthesis and biomass production. There are no researchs associating proportions of NO3-/NH4+ for alleviating Cu stress toxicity. The objective was to evaluate the effect of Cu toxicity on metabolism, mineral nutrition, photosynthesis and dry matter production, as well as the role of NO3-/NH4+ proportions in alleviating such toxicity. The experimental was carried out in randomized complete block design in a 3×4 factorial with six replications. Three replications were used to evaluated metabolic and physiological attributes and other three to determine nutrition and productive attributes. The factors were three proportions of NO3-/NH4+ (100/0, 70/30 and 50/50) combined with four rates of Cu (0.3, 250, 500 and 1000 μmol L-1) in nutrient solution. Two plant growth periods evaluated, being the first with plants exposure to Cu and the second without the exposure to Cu. In the first cut, plants supplied with 70/30 of NO3-/NH4+ proportions and Cu rates of 1000 μmol L-1 showed high Cu concentration in recently fully expanded leaf laminae (LR), high Cu accumulation in the biomass, high NH4+ concentration in LR, high NH4+ accumulation in shoots, high glutamine synthetase activity in LR, higher proline concentration in LR, high superoxide dismutase (SOD) activity in parts of grass and low shoot dry matter production. In the grass supplied with 100/0 of NO3-/NH4+ and Cu of 1000 μmol L-1 it was found a low Cu concentration in LR and low Cu accumulation in shoots, but the concentration and accumulation of this metal in roots were increased even in lower Cu rate. Plants supply with 100/0 of NO3-/NH4+ also showed high NO3- concentration in LR, high NO3- accumulation in roots, high N accumulation in shoots, high nitrate reductase activity in LR, high stomatal conductance and high biomass production. In the grass supplied with 100/0 of NO3-/NH4+, there was low SOD activity and low proline concentration in shoots. The concentration of malondialdehyde was low at the high Cu rate, as well as the transpiration rate, photosystem II efficiency, electron transport rate and CO2 assimilation rate. Catalase, guaiacol peroxidase, ascorbate peroxidase and glutathione reductase activities were activated mainly in roots, being high in high Cu rate. Cu toxicity negatively affects metabolism, mineral nutrition, physiology and dry matter productions of tanzania guineagrass, but NO3-/NH4+ proportions change this toxicity. The only use of NO3- in the N supply is a strategy of great potential to be use in plants with of high Cu stress. However, combinations of NO3- with NH4+ in the growth medium when plants suffer moderate Cu stress is strategic for great metal phytoextraction.

A series of experiments were conducted to investigate how dietary and ruminal factors regulate urea-N recycling in ruminants. In Experiments 1, 2, and 3, urea-N kinetics were measured using 4-d intra-jugular infusions of [15N15N]-urea. In Experiment 1, the objective was to determine how interactions between dietary ruminally-degradable protein (RDP) level and ruminally-fermentable carbohydrate (RFC) may alter urea-N transfer to the gastrointestinal tract (GIT) and the utilization of this recycled urea-N in rapidly-growing lambs fed high N diets. The dietary factors were: 1) dry-rolled barley (DRB) vs. pelleted barley (PB) as the principal source of RFC; and 2) dietary levels of RDP of 60 vs. 70% (% of CP). Nitrogen intake, fecal and urinary N excretion increased as dietary RDP level increased; however, method of barley processing had no effect on N use. Dietary treatment had no effect on urea-N kinetics; however, endogenous production of urea-N (UER) exceeded N intake. For all diets, 0.669 to 0.742 of UER was recycled to the GIT; however, 0.636 to 0.756 of the GER was returned to the ornithine cycle. In Experiment 2, the objective was to delineate the effects of partial defaunation of the rumen on urea-N kinetics in lambs fed low or high N diets. Treatments were: 1) partial defaunation (PDFAUN) vs. faunation (FAUN); and 2) low (10%, LOW) vs. high (15%, HIGH) dietary CP. Linoleic acid-rich sunflower oil was fed as a partially-defaunating agent. Partial defaunation decreased ruminal NH3-N concentrations. The UER and urinary urea-N excretion (UUE) were lower, and the GER tended to be lower in PDFAUN as compared to FAUN lambs; however, as a proportion of UER, GER was higher and the proportion of recycled urea-N that was utilized for anabolism (i.e., UUA) tended to be higher in PDFAUN lambs. The UER, GER and UUE were higher in lambs fed diet HIGH; however, as a proportion of UER, GER and its anabolic use were higher in lambs fed diet LOW. In Experiment 3, the objective was to delineate how, at similar N intakes, interactions between ruminal partial defaunation and altering dietary RFC may alter urea-N kinetics and N metabolism in lambs. Treatments were: 1) PDFAUN vs. FAUN; and 2) DRB vs. PB. Urinary N excretion was lower and retained N was higher in PDFAUN compared to FAUN lambs. The UER was similar across treatments; however, the GER, expressed as absolute amounts or as a proportion of UER, UUA, and microbial N supply were higher in PDFAUN compared to FAUN lambs. As a proportion of UER, GER was higher, whereas UUE was lower in lambs fed PB compared to those fed DRB. In Experiment 4, the objective was to determine the effects of feeding oscillating dietary CP compared to static dietary CP concentration on N retention and in vitro urea flux across ruminal epithelia. Dietary treatments consisted of a medium CP diet (MEDIUM; 12.8% CP) or diets with oscillating CP content (OSC) fed in two different sequences i.e., 2 d of low CP (9.7% CP) followed by 2 d of high CP (16.1% CP; OSC-HIGH) or vice-versa (OSC-LOW). Ruminal epithelial tissues were collected and mounted in Ussing chambers under short-circuit conditions and the serosal-to-mucosal urea flux (Jsm-urea) was measured using 14C-urea. Although N intake was similar, retained N and microbial N supply were greater in lambs fed the OSC diets compared to those fed the MEDIUM diet. The total Jsm-urea was higher in lambs fed the OSC-LOW compared to those fed the OSC-HIGH diet. Across diets, the addition of phloretin (a known specific inhibitor of facilitative urea transporter-B; UT-B) reduced Jsm-urea; however, phloretin-insensitive Jsm-urea was the predominant route for transepithelial urea transfer. In summary, data presented in this thesis provide new insights that the improved N retention typically observed in defaunated ruminants and in ruminants fed oscillating dietary CP concentrations is partly mediated via increased urea-N recycling to the GIT and utilization of recycled urea-N for anabolic purposes.