La déficience en phosphore est l’un des facteurs limitant la production de légumineuses à graines, dont le haricot (Phaseolus vulgaris). La diversité génétique de cette espèce en Amérique latine a permis d'identifier avec le CIAT, des lignées recombinantes du croisement de DOR364 et BAT477 à fort pouvoir fixateur d'azote (FSN) et à forte efficacité d'utilisation de phosphore (EUP). Les résultats obtenus au cours de ce travail nous ont permis de répondre à notre objectif principal de thèse qui consistait à vérifier la pertinence des critères d'adaptation de la fixation symbiotique de l'azote chez le haricot à la disponibilité de P que nous avons déterminé. Nous avons démontré, d'une part, en milieu hydroaéroponique, que les racines nodulées du haricot, excrètent dans leur rhizosphère une quantité de H+ qui est corrélée à la perméabilité nodulaire. Ce qui laisse penser qu'une part d'H+ excrétée par les racines nodulées est liée à la fixation symbiotique de N2. D'autre part, nous avons démontré que sous déficience en P les lignées tolérantes acidifient plus leur rhizosphère que les sensibles. En multisites les facteurs les plus déterminants de la hiérarchisation des sites sont N-total et P-total. Deux groupes de lignées se distinguent, les tolérantes versus les sensibles à la faible fertilité des sols en P. La lignée locale se distingue des groupes précédents. Elle est capable de croître dans différents sols, notamment les contraignants avec une grande capacité à noduler, par conséquent, à compenser la déficience en N. Nous avons également pu démontrer que les biomasses nodulaire et aérienne sont corrélées au P Olsen du sol / Phosphorus deficiency is one of the limiting factor for grain legume production like bean (Phaseolus vulgaris). The results obtained during this work enabled us to confirm our main aim of this work which is checking the relevance of the criteria of adaptation of the symbiotic nitrogen fixation of bean to P availability. We showed, in controlled conditions that the nodulated roots of bean, release in their rhizosphere a quantity of H+ which is correlated with the nodul permeability. What lets think that a share of H+ released is related to the symbiotic N2 fixing. In addition, we showed that under P deficiency the tolerant lines acidify more their rhizosphere that the sensitive ones. Into multisite trial the most determining factors of the hierarchisation of the sites are total-N and total-P. Two groups of lines are distinguished: tolerant versus sensitive to P deficiency. The local farmer line is distinguished from the studied lines. It is able to grow in various soil fertility, in particular the constraining soils. ones with a great capacity with noduler, consequently, to compensate for deficiency in NR. We also could show that shoot and nodule biomass are correlated with the Olsen-P Olsen of the soil

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-02-21T14:31:39Z No. of bitstreams: 1 Penellope Teles Viveiros da Silva.pdf: 2225516 bytes, checksum: 71cbc5ed73dc00ce129b5c93f8fe83c3 (MD5) / Made available in DSpace on 2017-02-21T14:31:39Z (GMT). No. of bitstreams: 1 Penellope Teles Viveiros da Silva.pdf: 2225516 bytes, checksum: 71cbc5ed73dc00ce129b5c93f8fe83c3 (MD5) Previous issue date: 2016-07-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The cultural characterization of bacterial isolates is usually the first step in the process of getting the isolated and evaluation of biodiversity. These evaluations are important and allow for a preliminary screening of isolated by separating them into groups genera, for example. This study aimed to characterize phenotypically bacterial strains derived from root nodules of forages: Desmanthus pernambucanus and Mimosa tenuiflora grown in fertilized soil or not. Samples of soil for cultivation of plants in the screened greenhouse were collected in the cities of Arcoverde, Ibimirim and Serra Talhada, Pernambuco. The isolates were characterized as the time of growth of colonies, determine the shape and diameter, high transparency, exopolysaccharides production and coloration of the colonies, change of pH. They determined the number and biomass of nodules, production indoles and phenotypic characterization of bacterial isolates. Cultural assessment, were seen as key features: rapid growth time, flat elevation, exopolysaccharides production of low to moderate color ranging from cream to white and smooth surface. Legumes had nodulation in the three soils in the absence or presence of manure. Manuring does not increase the number of nodules of Mimosa tenuiflora plants, but increases the number of nodules Desmantus pernambucanus. The bacterial strains are capable of producing indole compounds without the addition of tryptophan. / A caracterização cultural dos isolados bacterianos geralmente é a primeira etapa do processo de obtenção dos isolados e avaliação de sua biodiversidade. Estas avaliações são importantes e permitem a seleção preliminar de isolados, separando-os em grupos de gêneros, por exemplo. A realização deste estudo teve como objetivo caracterizar fenotipicamente estirpes bacterianas oriundas de nódulos radiculares das espécies forrageiras: Desmanthus pernambucanus e Mimosa tenuiflora cultivadas em solos adubados ou não. As amostras de solos para cultivo das plantas em estufa telada foram coletadas nos municípios de Arcoverde, Ibimirim e Serra Talhada, Pernambuco. Os isolados foram caracterizados quanto ao tempo de crescimento de colônias, determinação da forma e diâmetro, elevação, transparência, produção de exopolissacarídeos e coloração das colônias, alteração do pH do meio. Foram determinados o número e a biomassa de nódulos, produção de compostos indólicos e caracterização fenotípica dos isolados bacterianos. Na avaliação cultural, foram observadas como principais características: tempo de crescimento rápido, elevação plana, produção de exopolissacarídeos de pouca a moderada, coloração variando de creme a branca e superfície lisa. As leguminosas tiveram nodulação nos três solos na ausência ou presença de esterco. Adubação com esterco não aumenta o número dos nódulos das plantas Mimosa tenuiflora, porém aumenta o número de nódulos das Desmantus pernambucanus. Os isolados bacterianos são capazes de produzir compostos indólicos sem adição de triptofano.

Nódulos de tireoide são achados clínicos comuns e, atualmente, o método diagnóstico de escolha para diferenciar lesões benignas de lesões malignas é a análise citopatológica dos nódulos através de punção aspirativa por agulha fina (PAAF). Estudos prévios já indicaram que os níveis séricos de TSH podem estar associados ao risco de malignidade nodular. O objetivo deste estudo foi avaliar se o TSH sérico é um preditor de malignidade em nódulos de tireoide em pacientes submetidos à PAAF. A amostra contemplou 100 indivíduos puncionados consecutivamente no Centro de Pronto Diagnóstico Ambulatorial, CPDA, HCPA e que apresentavam níveis de TSH dentro da normalidade. Todos os pacientes foram submetidos à PAAF da tireoide com controle ultrassonográfico e tiveram, posteriormente, a análise citopatológica da PAAF e a avaliação histopatológica do bloco celular. A análise estatística baseou-se em dados de frequências e testes não-paramétricos foram utilizados para correlacionar as variáveis. A população de estudo foi composta por 100 pacientes, sendo 89 mulheres e 11 homens. A média de idade foi de 54,1 ± 14,2 anos e o tamanho médio dos nódulos foi de 2.53 ± 1.36 centímetros. Vinte e seis % destes pacientes apresentavam algum tipo de doença tireoidiana prévia. A média do nível de TSH sérico entre os 100 indivíduos foi de 1.81 ± 1.08 uUI/mL. De acordo com o diagnóstico citopatológico da PAAF complementado pelos achados do bloco celular foram classificados como malignos 8% dos nódulos, 70% benignos, 11% suspeitos/ indeterminados, 8% insuficientes e 3% lesões foliculares. A média de TSH para os grupos maligno, benigno, suspeito/indeterminado, insuficiente e lesão folicular foi de, respectivamente, 2.48, 1.59, 2.21, 2.35 e 2.20 uUI/ml (p>0.05). Não houve diferença estatística significante entre os grupos diagnósticos avaliados, apesar de haver uma variação entre os níveis de TSH entre os grupos refletindo, provavelmente, o pequeno tamanho da amostra. / Thyroid nodules are common and currently the first choice of investigation in distinguishing benign from malignant disease is the cytological analysis of fine needle aspiration biopsy (FNAB). Previous studies have indicated that serum TSH levels might be associated with the likelihood of malignancy. The aim of this study was to evaluate whether serum TSH is a predictor of malignancy of thyroid nodules in patients undergoing FNAB. One hundred consecutive patients, who underwent FNAB as part of clinical investigation of thyroid nodule in a multidisciplinary setting tertiary hospital, underwent ultrasonography followed by FNAB, cytology and cell block analysis. Independent-Samples Kruskal-Wallis test was used to compare the groups. The study population comprised of 89 female and 11 male patients. The mean age was 54.1 ± 14.2 years. 26% had previous thyroid disease. Mean TSH levels was 1.81 ± 1.08 uUI/mL and the mean nodule size was 2.53 ± 1.36cm. Final cytology/cell block diagnosis classified 8% as malignant, 70% as benign, 11% suspicious/indeterminate, 8% insufficient and 3% follicular lesion. The mean TSH values for malignant, benign, suspect, insufficient and follicular lesion group were as follows: 2.48, 1.59, 2.21, 2.35 and 2.20 uUI/ml, respectively. No statistical significance was detected between TSH levels and final cytology/cell block diagnosis, possibly reflecting the small sample size (P>0.05). We observed a variation between TSH levels among the groups covered in this study, but there was no statistically significant difference among them.

Lung cancer is the leading cause of cancer death in the United States. While low-dose computed tomography (CT) screening reduces lung cancer mortality by 20%, 97% of suspicious lesions are found to be benign upon further investigation. Computer-aided diagnosis (CAD) tools can improve the accuracy of CT screening, however, current CAD tools which focus on imaging characteristics of the nodule alone are challenged by the limited data captured in small, early identified nodules. We hypothesize a CAD tool that incorporates quantitative CT features from the surrounding lung parenchyma will improve the ability of a CAD tool to determine the malignancy of a pulmonary nodule over a CAD tool that relies solely on nodule features. Using a higher resolution research cohort and a retrospective clinical cohort, two CAD tools were developed with different intentions. The research-driven CAD tool incorporated nodule, surrounding parenchyma, and global lung measurements. Performance was improved with the inclusion of parenchyma and global features to 95.6%, compared to 90.2% when only nodule features were used. The clinically-oriented CAD tool incorporated nodule and parenchyma features and clinical risk factors and identified several features robust to CT variability, resulting in an accuracy of 71%. This study supports our hypothesis that the inclusion of parenchymal features in the developed CAD tools resulted in improved performance compared to the CAD tool constructed solely with nodule features. Additionally, we identified the optimal amount of lung parenchyma for feature extraction and explored the potential of the CAD tools in a clinical setting.

Somatostatinrezeptoren (SSTRs) hemmen die Hormonsekretion und Proliferation in einer Vielzahl von neuroendokrinen Geweben. Eine erhöhte Dichte dieser Rezeptoren konnte im Zusammenhang mit verschiedenen Schilddrüsenpathologien nachgewiesen werden. Mittels Gallium-68 (Ga-68) DOTA-Phe(1)-Tyr(3)-Octreotid (DOTATOC) Positronen-Emissions-Tomographie (PET), einem nuklearmedizinischen Untersuchungsverfahren, ist die funktionelle Darstellung der SSTR-Expression in vivo möglich. Unser Studienziel war es, den Ga-68-DOTATOC Uptake als Korrelat für die SSTR-Dichte in gesundem und pathologisch verändertem Schilddrüsengewebe zu quantifizieren. Die Ga-68-DOTATOC PET Bilder von insgesamt 165 Patienten wurden mittels (ROI)- Technik ausgewertet und die Studienteilnehmer verschiedenen Schilddrüsenpathologiegruppen zugeordnet. Ergänzend erfolgte eine schilddrüsenspezifische Anamnese, eine Ultraschalluntersuchung der Schilddrüse, sowie die Bestimmung der Laborparameter TSH and Anti-TPO-Antikörper für jeden Studienteilnehmer. Normale Schilddrüsen, ohne eine erkennbare Pathologie, zeigten eine klar nachweisbare SSTR-Expression mit einer großen Spannweite innerhalb der ermittelten TBR-Werte. In acht Fällen war es möglich, Folgeuntersuchungen von Patienten mit normaler Schilddrüse, aber erhöhten Uptake-Werten in der Gallium-68-DOTATOC PET Untersuchung, auszuwerten (TBR>4). Der Abstand der durchgeführten Kontrollen lag im Mittel bei 11,4 Monaten mit einer Spannweite von sechs bis vierzehn Monaten. In keiner der durchgeführten Kontrolluntersuchungen konnte eine neu aufgetretene Schilddrüsenpathologie nachgewiesen werden. Eine erhöhte SSTR-Dichte (TBR>3.4) zeigte sich zudem im Fall von autonomen Adenomen, disseminierten Schilddrüsenautonomien, sowie bei den meisten Patienten (fünf von acht) mit aktiven Hashimoto-Thyreoiditiden. Vor allem die gesunden Schilddrüsen von männlichen Studienteilnehmern fielen mit erhöhten Radiotraceruptake-Werten in der durchgeführten Untersuchung auf. Diese unterschieden sich signifikant von der Gruppe weiblicher Studienteilnehmer mit normaler Schilddrüse. Patienten ohne eine nachweisbare Schilddrüsenpathologie, aber mit erhöhten Uptake-Werten (TBR>4.0), zeigten in den späteren Kontrolluntersuchungen keinen Hinweis auf eine sich entwickelnde Pathologie, speziell keine Hinweise auf eine sich entwickelnde Hashimoto-Thyreoidititis. Alle Patienten mit verschiedenen Formen der Schilddrüsenautonomie zeigen einen erhöhten DOTATOC Uptake in unserer Studie.

Nódulos de tireoide são achados clínicos comuns e, atualmente, o método diagnóstico de escolha para diferenciar lesões benignas de lesões malignas é a análise citopatológica dos nódulos através de punção aspirativa por agulha fina (PAAF). Estudos prévios já indicaram que os níveis séricos de TSH podem estar associados ao risco de malignidade nodular. O objetivo deste estudo foi avaliar se o TSH sérico é um preditor de malignidade em nódulos de tireoide em pacientes submetidos à PAAF. A amostra contemplou 100 indivíduos puncionados consecutivamente no Centro de Pronto Diagnóstico Ambulatorial, CPDA, HCPA e que apresentavam níveis de TSH dentro da normalidade. Todos os pacientes foram submetidos à PAAF da tireoide com controle ultrassonográfico e tiveram, posteriormente, a análise citopatológica da PAAF e a avaliação histopatológica do bloco celular. A análise estatística baseou-se em dados de frequências e testes não-paramétricos foram utilizados para correlacionar as variáveis. A população de estudo foi composta por 100 pacientes, sendo 89 mulheres e 11 homens. A média de idade foi de 54,1 ± 14,2 anos e o tamanho médio dos nódulos foi de 2.53 ± 1.36 centímetros. Vinte e seis % destes pacientes apresentavam algum tipo de doença tireoidiana prévia. A média do nível de TSH sérico entre os 100 indivíduos foi de 1.81 ± 1.08 uUI/mL. De acordo com o diagnóstico citopatológico da PAAF complementado pelos achados do bloco celular foram classificados como malignos 8% dos nódulos, 70% benignos, 11% suspeitos/ indeterminados, 8% insuficientes e 3% lesões foliculares. A média de TSH para os grupos maligno, benigno, suspeito/indeterminado, insuficiente e lesão folicular foi de, respectivamente, 2.48, 1.59, 2.21, 2.35 e 2.20 uUI/ml (p>0.05). Não houve diferença estatística significante entre os grupos diagnósticos avaliados, apesar de haver uma variação entre os níveis de TSH entre os grupos refletindo, provavelmente, o pequeno tamanho da amostra. / Thyroid nodules are common and currently the first choice of investigation in distinguishing benign from malignant disease is the cytological analysis of fine needle aspiration biopsy (FNAB). Previous studies have indicated that serum TSH levels might be associated with the likelihood of malignancy. The aim of this study was to evaluate whether serum TSH is a predictor of malignancy of thyroid nodules in patients undergoing FNAB. One hundred consecutive patients, who underwent FNAB as part of clinical investigation of thyroid nodule in a multidisciplinary setting tertiary hospital, underwent ultrasonography followed by FNAB, cytology and cell block analysis. Independent-Samples Kruskal-Wallis test was used to compare the groups. The study population comprised of 89 female and 11 male patients. The mean age was 54.1 ± 14.2 years. 26% had previous thyroid disease. Mean TSH levels was 1.81 ± 1.08 uUI/mL and the mean nodule size was 2.53 ± 1.36cm. Final cytology/cell block diagnosis classified 8% as malignant, 70% as benign, 11% suspicious/indeterminate, 8% insufficient and 3% follicular lesion. The mean TSH values for malignant, benign, suspect, insufficient and follicular lesion group were as follows: 2.48, 1.59, 2.21, 2.35 and 2.20 uUI/ml, respectively. No statistical significance was detected between TSH levels and final cytology/cell block diagnosis, possibly reflecting the small sample size (P>0.05). We observed a variation between TSH levels among the groups covered in this study, but there was no statistically significant difference among them.

Nódulos de tireoide são achados clínicos comuns e, atualmente, o método diagnóstico de escolha para diferenciar lesões benignas de lesões malignas é a análise citopatológica dos nódulos através de punção aspirativa por agulha fina (PAAF). Estudos prévios já indicaram que os níveis séricos de TSH podem estar associados ao risco de malignidade nodular. O objetivo deste estudo foi avaliar se o TSH sérico é um preditor de malignidade em nódulos de tireoide em pacientes submetidos à PAAF. A amostra contemplou 100 indivíduos puncionados consecutivamente no Centro de Pronto Diagnóstico Ambulatorial, CPDA, HCPA e que apresentavam níveis de TSH dentro da normalidade. Todos os pacientes foram submetidos à PAAF da tireoide com controle ultrassonográfico e tiveram, posteriormente, a análise citopatológica da PAAF e a avaliação histopatológica do bloco celular. A análise estatística baseou-se em dados de frequências e testes não-paramétricos foram utilizados para correlacionar as variáveis. A população de estudo foi composta por 100 pacientes, sendo 89 mulheres e 11 homens. A média de idade foi de 54,1 ± 14,2 anos e o tamanho médio dos nódulos foi de 2.53 ± 1.36 centímetros. Vinte e seis % destes pacientes apresentavam algum tipo de doença tireoidiana prévia. A média do nível de TSH sérico entre os 100 indivíduos foi de 1.81 ± 1.08 uUI/mL. De acordo com o diagnóstico citopatológico da PAAF complementado pelos achados do bloco celular foram classificados como malignos 8% dos nódulos, 70% benignos, 11% suspeitos/ indeterminados, 8% insuficientes e 3% lesões foliculares. A média de TSH para os grupos maligno, benigno, suspeito/indeterminado, insuficiente e lesão folicular foi de, respectivamente, 2.48, 1.59, 2.21, 2.35 e 2.20 uUI/ml (p>0.05). Não houve diferença estatística significante entre os grupos diagnósticos avaliados, apesar de haver uma variação entre os níveis de TSH entre os grupos refletindo, provavelmente, o pequeno tamanho da amostra. / Thyroid nodules are common and currently the first choice of investigation in distinguishing benign from malignant disease is the cytological analysis of fine needle aspiration biopsy (FNAB). Previous studies have indicated that serum TSH levels might be associated with the likelihood of malignancy. The aim of this study was to evaluate whether serum TSH is a predictor of malignancy of thyroid nodules in patients undergoing FNAB. One hundred consecutive patients, who underwent FNAB as part of clinical investigation of thyroid nodule in a multidisciplinary setting tertiary hospital, underwent ultrasonography followed by FNAB, cytology and cell block analysis. Independent-Samples Kruskal-Wallis test was used to compare the groups. The study population comprised of 89 female and 11 male patients. The mean age was 54.1 ± 14.2 years. 26% had previous thyroid disease. Mean TSH levels was 1.81 ± 1.08 uUI/mL and the mean nodule size was 2.53 ± 1.36cm. Final cytology/cell block diagnosis classified 8% as malignant, 70% as benign, 11% suspicious/indeterminate, 8% insufficient and 3% follicular lesion. The mean TSH values for malignant, benign, suspect, insufficient and follicular lesion group were as follows: 2.48, 1.59, 2.21, 2.35 and 2.20 uUI/ml, respectively. No statistical significance was detected between TSH levels and final cytology/cell block diagnosis, possibly reflecting the small sample size (P>0.05). We observed a variation between TSH levels among the groups covered in this study, but there was no statistically significant difference among them.

Background: Reported results for thyroid nodule fine-needle aspiration (FNA) cytology mainly originate from tertiary centers. However, thyroid nodule FNA cytology is mainly performed in primary care settings for which the distribution of FNA Bethesda categories and their respective malignancy rates are largely unknown. Therefore, this study investigated FNA cytology malignancy rates of a large primary care setting to determine to what extent current evidence-based strategies for the malignancy risk stratification of thyroid nodules are applied and applicable in such primary care settings. Methods: In a primary care setting, 9460 FNAs of thyroid nodules were retrospectively analyzed from 8380 patients evaluated by one cytologist (I.R.) during a period of two years. The 8380 FNA cytologies were performed by 64 physicians in different private practices throughout Germany in primary care settings. Results: The cytopathologic results were classified according to theBethesda Systemas non-diagnostic in 19%, cyst/ cystic nodule in 21%, benign (including thyroiditis) in 48%, atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) in 6%, follicular neoplasms/suspicious for follicular neoplasm (FN/SFN) in 4%, suspicious for malignancy (SFM) in 1%, and malignant in 1%. The proportion of patients proceeding to surgery or with a follow-up of at least one year and the observed risks of malignancy were 22%/8% for AUS/FLUS, 69%/ 17% for FN/SFN, 78%/86% for SFM, and 71%/98% for malignant. For 112 cytologically suspicious and malignant FNAs, there were 102 true positives and 10 false positives, considering histology as gold standard. Conclusion: At variance with other data mostly originating from tertiary centers, these data demonstrate low percentages for malignant, SFM, FN/SFN, and AUS/FLUS, and high percentages for cysts/cystic nodules in this primary care setting in Germany. The risks of malignancy for malignant, SFM, AUS/FLUS, and FN/SFN FNA cytologies are according to Bethesda recommendations.:1 ABKÜRZUNGSVERZEICHNIS ............................................................................................................... 1 2 EINFÜHRUNG .......................................................................................................................................... 2 2.1 DAS ORGAN SCHILDDRÜSE ................................................................................................................................ 3 2.1.1 Anatomie der Schilddrüse ......................................................................................................................... 3 2.1.2 Funktion der Schilddrüse .......................................................................................................................... 3 2.2 SCHILDDRÜSENKNOTEN ..................................................................................................................................... 4 2.2.1 Epidemiologie der Schilddrüsenknoten .............................................................................................. 4 2.2.2 Ätiologie der Schilddrüsenknoten ......................................................................................................... 4 2.2.3 Jodversorgung in Deutschland ................................................................................................................ 5 2.2.4 Symptomatik der Schilddrüsenknoten ................................................................................................ 5 2.2.5 Diagnostik von Schilddrüsenknoten ..................................................................................................... 6 2.2.6 Die Feinnadelaspirationszytologie (FNA) ......................................................................................... 9 2.2.7 Therapeutisches Management ............................................................................................................ 11 2.3 DIE ABLEITUNG DER RATIONALE .................................................................................................................. 13 3 PUBLIKATIONSMANUSKRIPT ........................................................................................................ 14 4 ZUSAMMENFASSUNG ......................................................................................................................... 22 4.1 SCHLUSSFOLGERUNG ....................................................................................................................................... 26 5 LITERATURVERZEICHNIS ................................................................................................................ 28 6 ANLAGEN ............................................................................................................................................... 33 6.1 TABELLE 1: MALIGNITÄTSRISIKO NACH SONOGRAPHISCHEM ERSCHEINUNGSBILD UND FNA INDIKATION FÜR SCHILDDRÜSENKNOTEN (36) ........................................................................................................... 33 6.2 TABELLE 2: DAS BETHESDA SYSTEM FOR REPORTING THYROID CYTHOPATHOLOGY. DIAGNOSTISCHE KATEGORIEN UND MALIGNITÄTSRISIKEN (36) ........................................................................................................... 34 6.3 ABBILDUNG 1: ALGORITHMUS FÜR PATIENTEN MIT SCHILDDRÜSENKNOTEN NACH DER AMERICAN THYROID ASSOCIATION (36) .......................................................................................................................................... 35 6.4 DARSTELLUNG DES EIGENEN BEITRAGS AN DER PUBLIKATION ............................................................... 36 6.5 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT .......................................................... 54 6.6 LEBENSLAUF ..................................................................................................................................................... 55 6.7 VERZEICHNIS WISSENSCHAFTLICHER VERÖFFENTLICHUNGEN ................................................................ 56 6.8 DANKSAGUNG ................................................................................................................................................... 57

No description available.

<p>C₄-dicarboxylic acids appear to be metabolized via the TCA cycle in N₂-fixing bacteria (bacteroids) within legume nodules. In <em>Sinorhizobium meliloti</em> bacteroids from alfalfa, NAD⁺-malic enzyme (DME) is required for symbiotic N₂-fixation and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont <em>Rhizobium leguminosarum</em> pyruvate synthesis can occur via either the DME pathway or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK), pyruvate kinase (PYK), and pyruvate dehydrogenase. Here we report that <em>dme</em> mutants of <em>Sin</em>or<em>hizobium sp</em>. NGR234 formed root nodules on a broad range of plants and that the level of N₂-fixation varied from 90% to 20% of wild type depending on the host plants inoculated. NGR234 bacteroids had significant PCK activity and while single <em>pckA</em> and single <em>dme</em> mutants fixed N₂ on <em>Macroptilium atropurpureum</em> and <em>Leucaena leucocephala</em> (albeit at a reduced rate), a <em>pckA</em> <em>dme</em> double mutant had no N₂-fixing activity (Fix^-). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix^- phenotype of <em>S. meliloti dme </em>mutants may be specific to the alfalfa-<em>S. meliloti </em>symbiosis. We therefore examined the ME-like genes <em>azc3656 </em>and <em>azc0119 </em>from <em>Azorhizobium caulinodans</em>, as <em>azc3656 </em>mutants were previously shown to form Fix^- nodules on the tropical legume <em>Sesbania rostrata</em>. We found that purified AZC3656 protein is an NAD (P)⁺-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N₂ fixation in <em>A. caulinodans </em>and <em>S. meliloti</em>, in other rhizobia this activity can be bypassed via another pathway(s).</p> <p>In <em>S. meliloti</em> both malic enzymes DME and TME share similar apparent <em>K_m</em>s for substrate and cofactors, but differ in their responses to TCA cycle intermediates, with DME activity inhibited by acetyl-CoA and induced by succinate and fumarate. Previous results in our laboratory indicated that DME is essential for symbiotic N₂ fixation, while TME fails to functionally replace DME. One possible reason for it is that a high ratio of NADPH/NADP⁺in<em> S. meliloti </em>bacteroids prevents TME from functioning in nodules. We sought to lower the<em> </em>NADPH/NADP⁺ratio by overexpressing a soluble pyridine nucleotide transhydrogenase (STH). However, metabolite measurements indicated that overproducing STH failed to lower the ratio of NADPH/NADP⁺ in<em> S. meliloti</em>.</p> <p>Previous studies assumed that DME and TME might play different roles in central carbon metabolism. To gain insight of their physiological functions, genome-wide microarray analysis was conducted in <em>S. meliloti</em> single<em> dme and</em> <em>tme</em> mutants grown on glucose or succinate. The most striking changes of gene expression were observed in <em>S. meliloti</em> <em>dme</em> mutants grown on succinate. The functions of upregulated genes suggested that DME might play an important role in regulating TCA cycle intermediates, important for the maintenance of metabolic flux through TCA cycle during C₄-dicarboxylate oxidation. However, changes of gene expression found in <em>tme </em>mutants were not significant enough to predict the physiological functions of TME protein in central carbon metabolism.</p> / Doctor of Philosophy (PhD)