O objetivo com este estudo foi avaliar o efeito de teores crescentes de proteína no concentrado de vacas leiteiras no terço inicial da lactação mantidas em pastagens de capim elefante (Pennisetum purpureum), manejadas com altas taxas de lotação durante o período chuvoso, sobre as seguintes variáveis: 1) produção e composição de leite; 2) peso vivo e condição corporal dos animais; 3) consumo de matéria seca; 4) parâmetros sanguíneos; 5) parâmetros ruminais; 6) composição bromatológica e morfológica da pastagem; e 7) comportamento animal. Buscou-se também a caracterização da PB do capim e a adequação proteica da dieta, visando otimizar a eficiência de utilização do nitrogênio. Foram utilizadas 30 vacas (HPB × Jersey) distribuídas em 10 grupos homogêneos, cada um compondo um quadrado latino 3x3, de acordo com a composição racial, dias em lactação e produção de leite e receberam suplemento concentrado com três níveis de PB durante períodos de 21 dias. Os concentrados continham milho moído fino, minerais, vitaminas e os três níveis de proteína (T1: 7,8; T2: 15,4; T3: 20,5% PB) foram obtidos substituindo-se parte do milho por farelo de soja. O capim elefante foi adubado com 50 kg N ha-1 por pastejo e apresentou teor médio de PB de 15,5%. As produções de leite e de leite corrigido para 3,5% de gordura, os teores e produções de gordura, proteína e caseína no leite, o consumo de matéria seca (CMS) não foram influenciados (P>0,05) pelos teores de PB dos concentrados. A concentração de nitrogênio ureico no leite aumentou linearmente (P<0,01) com o aumento dos teores de PB dos concentrados. A excreção de N pela urina foi maior (P<0,01) para os animais recebendo os concentrados contendo farelo de soja (T2 e T3) em relação àqueles consumindo o concentrado contendo apenas milho (T1). Além disso, a concentração de N amoniacal no rúmen foi menor (P<0,05) para estes últimos animais, que apresentaram, ainda, perda de peso durante o período experimental. A excreção total de N no ambiente acompanhou o comportamento do teor de PB dos concentrados. O fornecimento de farelo de soja no concentrado de vacas em terço inicial de lactação, mantidas em pastagens tropicais manejadas intensivamente, não resulta em benefício produtivo, mas favorece a acréscimo do peso corporal dos animais. / The aim of this study was to evaluate the effects of increasing levels of protein in the concentrate for early lactation cows maintained in an elephant grass pasture (Pennisetum purpureum), which was managed under high stocking rate during the raining season. The following variables were evaluated: 1) milk yield and composition; 2) body weight and condition score; 3) dry matter intake; 4) blood parameters; 5) rumen parameters; 6) chemical and morphological composition of the pasture; and 7) animal behavior. Protein fractionation of the pasture, and diet protein balancing were also studied as a way to improve efficiency of nitrogen use. Thirty Holstein × Jersey cows, distributed in 10 homogenous groups according to breed composition, days on lactation, and milk production, were used. In each group a 3×3 Latin square experiment was designed. Treatments consisted of 3 levels of crude protein in the concentrate supplement (T1: 7.8; T2: 15.4; T3: 20.5% CP). Concentrates were composed of finely ground corn, and mineral and vitamin mixture. Protein levels were obtained by adding soybean meal. Elephant grass pasture was fertilized with 50 kg N ha-1 after every grazing period, and presented 15.5% CP in average. Milk yield, fat corrected milk (3.5% fat), milk fat (g day-1 and %), milk protein (g day-1 and %), milk casein (g day-1 and %), and dry matter intake were not affected (P>0.05) by the supplement protein level. Concentration of ureic nitrogen in the milk increased linearly (P<0.01) as protein supplementation was increased. Nitrogen excretion in the urine was higher (P<0.01) for cows receiving the concentrates with soybean meal (T2 and T3), in comparison with those fed supplements containing just corn (T1). Moreover, these later had lower N-NH3 concentration in the rumen (P<0.05), and a negative body weight gain. Total N excretion to the environment follows the increasing in the protein level supplementation. Feeding soybean meal to early lactation cows, maintained in an intensively managed tropical pasture, does not result in productive benefits; however, it promotes an increasing in body weight of the animals.

Disputats. Universität Hohenheim, 2002. / Haves kun i elektronisk udg.

L’atmosphère terrestre est très riche en azote (N2). Mais cet azote diatomique est sous une forme très stable, inutilisable par la majorité des êtres vivants malgré qu’il soit indispensable pour la synthèse de matériels organiques. Seuls les procaryotes diazotrophiques sont capables de vivre avec le N2 comme source d’azote. La fixation d’azote est un processus qui permet de produire des substances aminées à partir de l’azote gazeux présent dans l’atmosphère (78%). Cependant, ce processus est très complexe et nécessite la biosynthèse d’une vingtaine de protéines et la consommation de beaucoup d’énergie (16 molécules d’ATP par mole de N2 fixé). C’est la raison pour laquelle ce phénomène est rigoureusement régulé. Les bactéries photosynthétiques pourpres non-sulfureuses sont connues pour leur capacité de faire la fixation de l’azote. Les études faites à la lumière, dans le mode de croissance préféré de ces bactéries (photosynthèse anaérobie), ont montré que la nitrogénase (enzyme responsable de la fixation du diazote) est sujet d’une régulation à trois niveaux: une régulation transcriptionnelle de NifA (protéine activatrice de la transcription des gènes nif), une régulation post-traductionnelle de l’activité de NifA envers l’activation de la transcription des autres gènes nif, et la régulation post-traductionnelle de l’activité de la nitrogénase quand les cellules sont soumises à un choc d’ammoniaque. Le système de régulation déjà décrit fait intervenir essentiellement une protéine membranaire, AmtB, et les deux protéines PII, GlnB et GlnK. Il est connu depuis long temps que la nitrogénase est aussi régulée quand une culture photosynthétique est exposée à la noirceur, mais jusqu’aujourd’hui, on ignore encore la nature des systèmes intervenants dans cette régulation. Ainsi, parmi les questions qui peuvent se poser: quelles sont les protéines qui interviennent dans l’inactivation de la nitrogénase lorsqu’une culture anaérobie est placée à la noirceur? Une analyse de plusieurs souches mutantes, amtB- , glnK- , glnB- et amtY- poussées dans différentes conditions de limitation en azote, serait une façon pour répondre à ces interrogations. Alors, avec le suivi de l’activité de la nitrogénase et le Western Blot, on a montré que le choc de noirceur provoquerait un "Switch-off" de l’activité de la nitrogénase dû à une ADP-ribosylation de la protéine Fe. On a réussit aussi à montrer que ii tout le système déjà impliqué dans la réponse à un choc d’ammoniaque, est également nécessaire pour une réponse à un manque de lumière ou d’énergie (les protéines AmtB, GlnK, GlnB, DraG, DraT et AmtY). Or, Rhodobacter capsulatus est capable de fixer l’azote et de croitre aussi bien dans la micro-aérobie à la noirceur que dans des conditions de photosynthèse anaérobies, mais jusqu'à maintenant sa régulation dans l’obscurité est peu étudiée. L’étude de la fixation d’azote à la noirceur nous a permis de montrer que le complexe membranaire Rnf n’est pas nécessaire à la croissance de R. capsulatus dans de telles conditions. Dans le but de développer une façon d’étudier la régulation de la croissance dans ce mode, on a tout d’abord essayé d’identifier les conditions opératoires (O2, [NH4 + ]) permettant à R. capsulatus de fixer l’azote en microaérobie. L’optimisation de cette croissance a montré que la concentration optimale d’oxygène nécessaire est de 10% mélangé avec de l’azote. / The atmosphere of the Earth is very rich in nitrogen (N2). However, diatomic nitrogen is very stable and therefore unusable by the majority of life forms even though it is necessary for the synthesis of a variety of organic compounds. Only diazotrophic procaryotes are capable of using N2 as nitrogen source. Their nitrogen fixation allows the production of aminated compounds from atmospheric nitrogen (78 %). However, this process is very complex and requires the biosynthesis of about twenty proteins and the consumption of a lot of energy (16 molecules of ATP per molecule of N2 fixed), thus necessitating its tight regulation. The purple non-sulfur photosynthetic bacteria are known for their ability to carry out nitrogen fixation. Studies conducted in the light, the preferred mode of growth of these bacteria (anaerobic photosynthetic), have shown that nitrogenase (the enzyme responsible for dinitrogen fixation) is subject to regulation at three levels: transcriptional regulation of NifA (activator protein for the transcription of nif genes), posttranslational regulation of the activity of NifA to activate nif gene transcription, and posttranslational regulation of nitrogenase activity when cells are subjected to an ammonium shock. The control system already described involves essentially a membrane protein, AmtB and both PII proteins, GlnK and GlnB. It has long been known that nitrogenase is regulated when light is suddenly removed from a culture, but until now it is unclear whether these systems are also involved in the regulation of nitrogen fixation in dark. Thus, one outstanding question is what are the proteins involved in the inactivation of nitrogenase when a light-grown culture is placed in the dark? An analysis of several mutant strains; amtB-, glnK-, glnB-, and amtY- under different conditions of nitrogen deficiency was used to address this question. Using measurements of nitrogenase activity and Fe protein modification by Western blotting, we were able to show that darkness causes a "switch-off” of nitrogenase due to ADP- ribosylation of Fe protein. Thus, the system that has already been described as involved in the response to a lack of ammonia, is also required for a response to a lack of light or energy (AmtB, GlnK, GlnB, DraG, and DraT, and AmtY). However, Rhodobacter capsulatus is also able to fix nitrogen and grow micro-aerobically in the dark as well as photosynthetically under anaerobic conditions, but so far its regulation in the dark has been little studied. The study of nitrogen fixation in the dark allowed us to show that the Rnf membrane complex is not required for growth of R. capsulatus in such conditions. In order to develop a way to study its regulation during this growth mode, we have attempted to identify the operating conditions (O2, [NH4+]), allowing R. capsulatus to fix nitrogen micro-aerobically. The optimization of this conditions has shown that the optimal concentration of oxygen required is 10% mixed with nitrogen.

Notre étude a consisté d'abord, en un travail de caractérisation moléculaire du gène SBNRT1.1 codant pour un transporteur de nitrate et présent en 3 copies chez le sorgho. Nous avons donc analysé leur structure et leur expression dans différents organes de la plante. Une analyse de leurs séquences nucléotidiques et protéiques a été également conduite. Notre étude a montré que les 3 co-orthologues (SBNRT1.1A, B et C) sont exprimés aussi bien dans les feuilles que dans les tiges et les racines du sorgho. Par ailleurs, nous avons révélé, pour la première fois, l'existence de 5 transcrits, de taille variable pour le gène SBNRT1.1B. Dans un autre volet, l'analyse des paramètres de croissance et d'activités enzymatiques clés dans les métabolismes azoté et carboné chez deux écotypes de sorgho connus pour leur différence de croissance dans des conditions de cultures différentes en apport azote, a montré que ces plantes de sorgho se caractérisent par une capacité à croitre à des niveaux élevés d'apports en azote, avec une réponse différentielle des deux écotypes. Cette tolérance s'est manifestée par une accumulation de chlorophylle, d'acides aminés et de protéines. D'autre part, les activités enzymatiques d'enzymes clés des métabolismes azoté et carboné, semblent être liées a la capacité des plantes de sorgho à faire face au stress azoté. Nos résultats pourraient ainsi fournir un cadre initial pour l'identification de marqueurs biochimiques qui pourraient contribuer utilement à la sélection des génotypes utilisant l'azote plus efficacement et donnant un rendement en biomasse et /ou grain, plus élevé, même dans des conditions de stress azoté. / Our study consisted firstly, in the molecular characterization of SbNRT1.1 gene, coding for a nitrate transporter and present in three copies in sorghum plants. We report here their structure and expression patterns in different organs of sorghum. We have also conducted a comparison of their nucleotide and polypeptide sequences with orthologous sequences from other species. Our study showed that the 3 co-orthologous (SbNRT1.1A, B and C) are expressed in leaves, stems and roots of sorghum. Moreover, we have for the first time revealed the existence of 5 alternative transcripts for the SbNRT1.1B gene. In another component of our research program, biochemical and physiological traits of two sorghum ecotypes differing in sensitivity to nitrogen were investigated and have shown that these sorghum plants are able to grow at high levels of nitrogen inputs, with differential response to nitrogen sources and rates. This tolerance was manifested by accumulation of high accumulation of chlorophyll, amino acids and protein. On the other hand, the enzymatic activities of certain key enzymes of nitrogen and carbon metabolisms, seem to be related to the capacity of sorghum plants to deal with nitrogen stress. Hence, our findings could provide an initial framework for the identification of biochemical markers for the selection of genotypes using nitrogen more efficiently and giving high yield of biomass and/or grain even under nitrogen stress conditions.

O objetivo com este estudo foi avaliar o efeito de teores crescentes de proteína no concentrado de vacas leiteiras no terço inicial da lactação mantidas em pastagens de capim elefante (Pennisetum purpureum), manejadas com altas taxas de lotação durante o período chuvoso, sobre as seguintes variáveis: 1) produção e composição de leite; 2) peso vivo e condição corporal dos animais; 3) consumo de matéria seca; 4) parâmetros sanguíneos; 5) parâmetros ruminais; 6) composição bromatológica e morfológica da pastagem; e 7) comportamento animal. Buscou-se também a caracterização da PB do capim e a adequação proteica da dieta, visando otimizar a eficiência de utilização do nitrogênio. Foram utilizadas 30 vacas (HPB × Jersey) distribuídas em 10 grupos homogêneos, cada um compondo um quadrado latino 3x3, de acordo com a composição racial, dias em lactação e produção de leite e receberam suplemento concentrado com três níveis de PB durante períodos de 21 dias. Os concentrados continham milho moído fino, minerais, vitaminas e os três níveis de proteína (T1: 7,8; T2: 15,4; T3: 20,5% PB) foram obtidos substituindo-se parte do milho por farelo de soja. O capim elefante foi adubado com 50 kg N ha-1 por pastejo e apresentou teor médio de PB de 15,5%. As produções de leite e de leite corrigido para 3,5% de gordura, os teores e produções de gordura, proteína e caseína no leite, o consumo de matéria seca (CMS) não foram influenciados (P>0,05) pelos teores de PB dos concentrados. A concentração de nitrogênio ureico no leite aumentou linearmente (P<0,01) com o aumento dos teores de PB dos concentrados. A excreção de N pela urina foi maior (P<0,01) para os animais recebendo os concentrados contendo farelo de soja (T2 e T3) em relação àqueles consumindo o concentrado contendo apenas milho (T1). Além disso, a concentração de N amoniacal no rúmen foi menor (P<0,05) para estes últimos animais, que apresentaram, ainda, perda de peso durante o período experimental. A excreção total de N no ambiente acompanhou o comportamento do teor de PB dos concentrados. O fornecimento de farelo de soja no concentrado de vacas em terço inicial de lactação, mantidas em pastagens tropicais manejadas intensivamente, não resulta em benefício produtivo, mas favorece a acréscimo do peso corporal dos animais. / The aim of this study was to evaluate the effects of increasing levels of protein in the concentrate for early lactation cows maintained in an elephant grass pasture (Pennisetum purpureum), which was managed under high stocking rate during the raining season. The following variables were evaluated: 1) milk yield and composition; 2) body weight and condition score; 3) dry matter intake; 4) blood parameters; 5) rumen parameters; 6) chemical and morphological composition of the pasture; and 7) animal behavior. Protein fractionation of the pasture, and diet protein balancing were also studied as a way to improve efficiency of nitrogen use. Thirty Holstein × Jersey cows, distributed in 10 homogenous groups according to breed composition, days on lactation, and milk production, were used. In each group a 3×3 Latin square experiment was designed. Treatments consisted of 3 levels of crude protein in the concentrate supplement (T1: 7.8; T2: 15.4; T3: 20.5% CP). Concentrates were composed of finely ground corn, and mineral and vitamin mixture. Protein levels were obtained by adding soybean meal. Elephant grass pasture was fertilized with 50 kg N ha-1 after every grazing period, and presented 15.5% CP in average. Milk yield, fat corrected milk (3.5% fat), milk fat (g day-1 and %), milk protein (g day-1 and %), milk casein (g day-1 and %), and dry matter intake were not affected (P>0.05) by the supplement protein level. Concentration of ureic nitrogen in the milk increased linearly (P<0.01) as protein supplementation was increased. Nitrogen excretion in the urine was higher (P<0.01) for cows receiving the concentrates with soybean meal (T2 and T3), in comparison with those fed supplements containing just corn (T1). Moreover, these later had lower N-NH3 concentration in the rumen (P<0.05), and a negative body weight gain. Total N excretion to the environment follows the increasing in the protein level supplementation. Feeding soybean meal to early lactation cows, maintained in an intensively managed tropical pasture, does not result in productive benefits; however, it promotes an increasing in body weight of the animals.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-07-05T14:36:45Z No. of bitstreams: 1 Artenisa Cerqueira Rodrigues.pdf: 1487841 bytes, checksum: e529e19a4038e9e52c84b90e95264477 (MD5) / Made available in DSpace on 2016-07-05T14:36:45Z (GMT). No. of bitstreams: 1 Artenisa Cerqueira Rodrigues.pdf: 1487841 bytes, checksum: e529e19a4038e9e52c84b90e95264477 (MD5) Previous issue date: 2012-06-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / It has been a recurrent the use of plant growth-promoting bacteria (PGPB) in association with the legume-rhizobia symbiotic system to increase the biological nitrogen fixation (BNF). Thus, the search for using alternative vehicles that increase quality and efficiency of inoculum leading to reduced costs and possible environmental impacts have been investigated. The BNF change in response to biological and environmental factors and their effectiveness can be measured by the concentrations of compounds involved in this process, being an important point to evaluate biochemical indicators related to the metabolisms of nitrogen, carbon and antioxidant. In this context, this study aimed at verifying the survival of cowpea to bacterial colonization and evaluating the interrelationship of Bradyrhizobium sp. and PGPB providing better symbiotic performance and cowpea development; optimizing the BNF and cowpea development, inoculated and co-inoculated with Bradyrhizobium sp. and PGPB, as well as evaluating the activities of enzymes as indicators of efficiency, senescence and stress/oxidative protection during and after the establishment of symbiosis; and determining the efficiency of inoculation and co-inoculation of cowpea with Bradyrhizobium sp. and PGPB using variables of growth and production and, jointly, characterizing the exopolysaccharide used as a vehicle for seed inoculation. Three experiments were conducted in greenhouse of the Agronomy Institute of Pernambuco (IPA). The legume used was cowpea cv. "IPA 206" inoculated with Bradyrhizobium sp. (BR 3267) and co-inoculated with different strains of PGPB. In the first experiment, the experimental design was randomized blocks with 24 treatments one inoculated (BR 3267), and 22 co-inoculated (BR 3267 + PGPB); and an absolute control (AC), with three replications. In the experiment II, the experimental design was randomized blocks with 2x4 factorial arrangement, two harvest periods (FP, flowering point; BS, beginning of senescence) and four treatments (inoculated and co-inoculated) + AC, with four repetitions. In the experiment III, the experimental design was a randomized block with 2x6 factorial arrangement, two harvest periods (FP, flowering point; GF, grain filling) and six treatments, one inoculated (BR 3267), and three co-inoculated (BR 3267 + PGPB), and two controls (AC and NC), with four replications. In the results was found synergism between BR 3267 and PGPB in cowpea especially in combination of BR 3267 with strains Paenibacillus graminis (MC 04.21) and P. durus (C 04.50), which exhibited better symbiotic response. Pre-selected strains of PGPB were combined with BR 3267, forming two symbiotic pairs (BR 3267 + MC 04.21; BR 3267 + C 04.50) and a tripartite (BR 3267 + MC 4.21 + C 04.50) where the results showed significant differences for growth variables as well as the biochemical indicators related to the metabolisms of nitrogen, carbon and antioxidative. There was increased concentration of total soluble proteins in nodules of plants co-inoculated compared to those inoculated in isolation with BR 3267. There was intense proteolytic activity and a decline in enzyme activity in the BS, but there was delayed of the deleterious effects of aging in the tripartite co-inoculation, allowing better symbiotic performance in cowpea plants. It can be suggested the use of inoculating compounds with bacteria studied aiming at improving the BNF and delaying senescence of nodules ensuring the availability of nitrogen for a longer period of time. This response emphasizes the importance of the combination and compatibility of microorganisms introduced, in combination, to promote a better symbiotic efficiency of cowpea, especially for tripartite co-inoculation, with respect to Rhizobium native to soil, and this synergistic response resulted in improved variables of production and efficiency of N2 fixation. With respect to chemical and rheological characteristics, the EPS used as vehicle for inoculation presents as a polyanionic heteropolysaccharide with low-viscosity fluid pseudoplastic, revealing an endothremal peak by the technique of differential scanning calorimetry that represents a favorable feature considering its use on industrial scale. / Para o incremento da fixação biológica do nitrogênio (FBN) tem sido recorrente o uso de bactérias promotoras de crescimento em plantas (BPCP’s) em associação com o sistema simbiótico leguminosa-rizóbio. Desta forma, a busca da utilização de veículos alternativos que aumentem a qualidade e eficiência do inoculante levando a redução de custos e de possíveis impactos ambientais tem sido investigados. A FBN varia em resposta a fatores biológicos e ambientais e a sua efetividade pode ser mensurada através das concentrações dos compostos envolvidos neste processo, sendo um ponto relevante avaliar indicadores bioquímicos relacionados aos metabolismos do nitrogênio, do carbono e antioxidativo. Neste contexto, este trabalho teve como objetivos verificar a sobrevivência do caupi à colonização bacteriana, assim como avaliar a interrelação Bradyrhizobium sp. e BPCP’s visando uma melhor performance simbiótica e desenvolvimento do caupi; otimizar a FBN e o desenvolvimento do caupi, inoculados e co-inoculados com Bradyrhizobium sp. e BPCP’s, assim como avaliar as atividades de enzimas como indicadores de eficiência, senescência e de estresse/proteção oxidativo durante a após o estabelecimento da simbiose; e determinar a eficiência da inoculação e co-inoculação do caupi com Bradyrhizobium sp. e BPCP’s utilizando variáveis de crescimento e produção e, juntantemente, caracterizar o exopolissacarídeo utilizado como veículo de inoculação das sementes. Foram conduzidos três experimentos em casa de vegetação do Instituto Agronômico de Pernambuco (IPA). A leguminosa utilizada foi caupi cv. “IPA 206” inoculada com Bradyrhizobium sp. (BR 3267) e co-inoculadas com diferentes estirpes de BPCP’s. No experimento I, o delineamento experimental utilizado foi em blocos ao acaso com 24 tratamentos, sendo um com inoculação (BR 3267); 22 combinações (BR 3267 + BPCP’s); uma testemunha absoluta (TA), com três repetições. No experimento II, o delineamento experimental adotado foi em blocos ao acaso com arranjo fatorial 2x4, dois períodos de coleta (PF, ponto de florescimento; IS, início de senescência) e quatro tratamentos (inoculados e co-inoculados) + TA, com quatro repetições. No experimento III, o delineamento experimental utilizado foi em blocos ao acaso com arranjo fatorial 2x6, dois períodos de colheita (PF, ponto de florescimento; e EG, enchimento de grãos) e seis tratamentos, sendo um com inoculação (BR 3267), e três com co-inoculações (BR 3267 + BPCP’s); e duas testemunhas (TA e TN), com quatro repetições. Nos resultados constatou-se sinergismo entre BR 3267 e BPCP’s em caupi principalmente na combinação de BR 3267 com Paenibacillus graminis (MC 04.21) e P. durus (C 04.50), que exibiram melhor resposta simbiótica. As estirpes de BPCP’s pré-selecionadas foram combinadas com BR 3267, formando dois pares simbióticos (BR 3267 + MC 04.21; BR 3267 + C 04.50) e uma tripartite (BR 3267+ MC 04.21 + C 04.50) onde os resultados mostraram diferenças significativas para as variáveis de crescimento bem como para os indicadores bioquímicos relacionados aos metabolismos do nitrogênio, carbono e antioxidativo. Houve aumento da concentração de proteínas solúveis totais nos nódulos das plantas co-inoculadas em relação àquelas inoculadas isoladamente com a BR 3267. Ocorreu intensa atividade proteolítica e declínio na atividade das enzimas no IS, porém na co-inoculação tripartite houve retardo dos efeitos deletérios da senescência, o que permitiu uma melhor performance simbiótica na plantas de caupi. Pode-se sugerir o uso de inoculantes compostos com as bactérias estudadas visando incrementar a FBN e retardar a senescência dos nódulos assegurando a disponibilidade de nitrogênio por um maior período de tempo. Esta resposta ressalta a importância da combinação e compatibilidade de microrganismos introduzidos, em mistura, para promover uma melhor eficiência simbiótica do caupi, em especial para a co-inoculação em tripartite, com relação ao rizóbio nativo do solo, e esta resposta sinérgica resultou em melhoria nas variáveis de produção e eficiência da fixação de N2. Em relação às características químicas e reológicas do EPS, utilizado como veículo para inoculação, este se apresenta como um heteropolissacarídeo polianiônico com fluido pseudoplástico pouco viscoso, revelando-se pela técnica de calorimetria exploratória diferencial um pico endotérmico que representa uma característica favorável tendo em vista a possibilidade de seu uso em escala industrial.

SUMMARY<p><p>Gap1, the general amino acid permease of Saccharomyces cerevisiae, is a plasma membrane protein which is synthesized and most active under conditions of poor nitrogen supply. Under these conditions, the role of Gap1 is to scavenge external amino acids to be used as nitrogen sources or directly as building blocks for protein synthesis. Gap1 is a member of the Yeast Amino Acid Transporter (YAT) family, a family of amino acid transporters highly conserved in bacteria and fungi. <p>The intracellular trafficking of Gap1 has been the subject of intense investigation as well as the role of lipids (in particular sphingolipids) in its activity and folding. These studies have all been carried out in the cellular context using versatile yeast genetics as exploratory tools. While such in vivo investigations allow to identify physiologically relevant features, they do not provide the details to understand the molecular basis of these phenomena. In order to decipher the molecular features responsible for biological functions, physiological analysis must be combined with biochemical, biophysical and structural studies which typically will be performed on isolated and purified proteins.<p>Our work during this study fits in this trans-disciplinary approach that aims at understanding different properties of the protein: (I) how sphingolipids modulate the activity of Gap1, (II) what part of the protein, and more specifically, what residues, are implicated in its regulation and (III) what are the molecular determinants of the multi-specificity of Gap1.<p>At the beginning of this work, Gap1 had not been previously produced and purified. During this thesis we have identified suitable expression and purification strategies for Gap1 and initiated first characterization studies. In the process, we have developed a new expression system in S. cerevisiae based on the nitrogen catabolite repression. <p>The capacity of this system to express other proteins was successfully tested for two other yeast transporters (Mep2 and Uga4) and for two human proteins: MD-2, a soluble protein and Vglut1, a vesicular transporter.<p>Therefore, we propose our expression design as a viable alternative to existing systems of production in yeast and as a valuable tool to be tested when starting the expression of a new target. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Manure production and nutrient management in pasture-based dairy production systems by Walter Svinurai The effect of dietary treatments on nutrient excretion was determined and an attempt to improving the retention of excreted nitrogen in stored manure using Aloe ferox leaf gel (AFLG) and pine bark was conducted at a pasture-based dairy farm. The animal mass-balance method was used to determine nutrient excretion by cows fed on three dietary treatments, Lolium perrene-based treatment (LP), Lolium multiforium-based treatment (LM) and Lolium perenne- Trifolium repens -based treatment (LTF). In separate experiments, slurry from dairy cows fed LM was amended with AFLG and pine bark at concentrations of 25, 50, 75 and 100 g/l and stored under anaerobic conditions for 16 days. The highest and lowest excretions of N, P and K were observed in LM and LP treatments, respectively. Increasing dietary N improved K and N retention in milk and, consequently increased milk urea nitrogen (MUN) and N excretion (P < 0.05). The concentrations of AFLG affected N and P retention in manure (P < 0.05). The highest retention in total Kjeldahl nitrogen (TKN) of the initial was 42% AFLG at 25g/l inclusion rate. Ammonium nitrogen (AMN) concentration increased significantly due to the interactive effect of AFLG inclusion rate and time. Pine bark powder significantly improved N and P retention in manure at all concentrations. The retention in TKN was higher (P < 0.05) at 25g/l pine bark powder than other concentrations. The interaction effect of inclusion rate and time increased iii AMN (P < 0.05). Dietary treatments significantly affected nutrient excretion, and AFLG and pine bark considerably improved N retention in stored slurry. Findings from the filed trial suggest the need for more attention on managing dietary nutrients in the post-rainy and cool-dry season when growth of pasture influenced choice of dietary treatments that led to high nutrient excretion. Field simulation of the additives to determine their efficacy and environmental hazards was recommended.

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 &ldquo;atmospheric epiphytic bromeliad&rdquo; 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 &ldquo;young epiphytic bromeliad without tank&rdquo; was suggested to refer the bromeliad V. gigantea in the juvenile phase

Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosolische NADP-ICDH) zeigen. Die transgene Pflanzen wiesen mehrheitlich keine erkennbare Veränderung eines Wachstumphänotyps auf. Obwohl die photosyntetische Leistung keine Änderungen gezeigt hatte, war die mitochondriale Respiration gestiegen, begleitet von einem reduzierten Kohlenstoff-fluss durch den Citratzyklus. Darüber hinaus waren die CS Pflanzen charakterisiert durch wesentliche Änderungen im Blattmetabolismus, einschließlich eines eingeschränkten Niveaus des photosynthetischen Pigments und Zwischenprodukten des Citratzyklus zusammen mit einer Akkumulation von Nitraten, verschiedenen Aminosäuren und Stärken. Zusammengefasst deuten diese Ergebnisse auf eine Einschränkung der Nitrat-Aufnahme hin. Das mit Hilfe von TOM1 Mikroarrays und quantitativer RT-PCR durchgeführte Transcript-profiling hat gezeigt, dass die fehlende Aktivität der mitochondrialen CS teilweise von einer gestiegenen, peroxisomalen CS Isoform ausgeglichen wird. Die metabolische Verschiebung ergab eine Verstärkung der photorespiratorischen Leistung, die vermutlich eine ausgleichende Rolle in der Produktion organischer Säuren und der Wiederherstellung der Redox-Balance spielt. Interessantenweise war die metabolische Antwort von Blättern auf Stickstoffmangel in NADP-ICDH Pflanzen dramatischer als in NAD-IDH Pflanzen, was darauf hindeutet, dass die cytosolische Isoform der Hauptlieferant von 2-Oxoglutarat im Tomatenmetabolismus sein könnte. / Although the TCA cycle is a respiratory metabolic pathway of central importance for all living organisms, relatively few molecular physiological studies of plants were performed to date. Here, I report the generation and functional analysis of tomato plants (Solanum lycopersicum) independently displaying mildly limited activity of mitochondrial citrate synthase (CS) and two isocitrate dehydrogenases, namely mitochondrial NAD-IDH and cytosolic NADP-ICDH. The transgenic plants revealed minor phenotypic alterations. Although the leaf photosynthetic performance was largely unaltered, the changes in mitochondrial respiration and carbon flux through the TCA cycle were observed. Moreover, the plants were characterized by significant modifications in the leaf metabolic content and in maximal catalytic activities of several enzymes involved in primary C and N metabolism. These results hint towards limitations in nitrate assimilation pathway. The transcript profiling performed by utilizing TOM1 microarrays and quantitative RT-PCR approach revealed that the deficiency in mitochondrial CS activity was partially compensated by up-regulation of peroxisomal CS isoform. The limitations in the activities of isocitrate dehydrogenases resulted in up-regulation of the photorespiratory pathway, which presumably played a compensatory role in supporting organic acid production and re-establishing redox balance in the transgenic leaves. Interestingly, the leaf metabolic response towards nitrogen starvation conditions was far more dramatic in NADP-ICDH transgenic plants than NAD-IDH plants, hinting that the cytosolic isoform may be the major 2-oxoglutarate supplier in tomato metabolism.