Dissertations / Theses on the topic 'Metabolismo primario'

La crema prescrita consisteix en l’ús planificat del foc en condicions ambientals adequades per tal d’aconseguir objectius de gestió prèviament definits. S’executa amb l’objectiu de reduir el risc d’incendis tot i que també s’utilitza per a la gestió de pastures i la conservació de la biodiversitat. La crema sota arbrat pot reduir el risc d’incendi de capçades, però també pot afectar el metabolisme primari i secundari de l’arbre. Tanmateix, les cremes haurien de minimitzar els efectes negatius del foc als arbres i maximitzar els positius. Així doncs, és important comprendre de quina manera els components específics del règim de foc, com són la intensitat del foc, severitat i estació de crema, afecten el funcionament de l’arbre. L’objectiu d’aquesta tesi és comprendre la influència que els factors del règim de cremes prescrites tenen en el metabolisme primari i secundari de tres espècies de pi amb tolerància al foc contrastada: Pinus nigra ssp. salzmannii (Dunal) Franco, P. sylvestris L. i P. halepensis Mill., utilitzant una combinació de tècniques dendrocronològiques, isotòpiques i de quantificació terpènica. El creixement post-crema va dependre del temps transcorregut des de la crema, l’espècie, la resistència de l’arbre, la severitat del foc i del creixement de l’arbre abans de la crema. L’any de la crema, el creixement es va reduir en P. halepensis i no es va veure afectat en P. nigra i P. sylvestris. Amb el pas del temps, el creixement es va incrementar en P. nigra, es va recuperar en P. halepensis i es va reduir en P. sylvestris. La probabilitat de morir va ser menor en P. nigra que en P. sylvestris. L’estació de crema va emergir com un factor important per explicar la mortalitat inicial: la probabilitat que un pi mori és més alta a la primavera que a la tardor per a un cert nivell de capçada socarrimada. La mortalitat tardana va ser més alta a les cremes de tardor que a les de primavera degut als majors temps de combustió registrats a la base dels troncs. Una reducció rellevant de la competència arbrada va augmentar significativament el creixement mitjançant un efecte positiu en la fusta de tardor de P. nigra i P. sylvestris, a mesura que el dany causat pel foc a la tija va disminuir. A més, vam demostrar que una crema executada just després d’un any sec no redueix la resiliència dels pins en comparació amb la dels pins no cremats. En P. halepensis, una reducció significativa de la competència va resultar en majors creixements amb el pas del temps, especialment en pins amb un menor volum de capçada socarrimada. Aquest augment va coincidir amb un any sec i es va associar amb una major conductància estomàtica, el que suggereix que la disponibilitat d’aigua va millorar després de la crema. La crema també va afectar al metabolisme secundari, específicament a la quantitat i al tipus de terpens segons l’espècie de pi i la severitat. A major volum de capçada socarrimada, la concentració de terpens a l’acícula dels pins 24 h post-crema fou major, però 1 any després es va produir una notable disminució. Aquesta reducció va ser més pronunciada en pins beneficiats per l’augment de la disponibilitat de recursos després de la crema, el que suggereix que els productes assimilats pels pins es van assignar al creixement en lloc de a mecanismes de defensa. Aquesta tesi proporciona informació valuosa per ajudar en la millora de la planificació de les cremes als boscos de pi mediterranis, en termes d’intensitat, severitat i estació de crema, oferint una nova finestra d’oportunitat per a l’ús de la crema prescrita com a eina de gestió forestal.
Prescribed burning is the planned use of fire to meet clear management objectives under suitable environmental conditions. It is usually executed to reduce fire hazard, but also to manage range and conserve biodiversity. Prescribed burning applied under a forest canopy can reduce crown fire hazard; however, underburning might affect the primary and secondary metabolism of trees. Planning underburning to reduced fire hazard, while minimizing the negative effects and maximizing the positive effects on trees, requires understanding how specific components of the fire regime, such as fire intensity, severity and season, affect tree performance. The goal of this doctoral thesis is to understand the influence of prescribed burning regime factors and related fire impacts on the primary and secondary metabolisms of three pine species with contrasting fire tolerances: Pinus nigra ssp. salzmannii (Dunal) Franco, P. sylvestris L. and P. halepensis Mill., using a combination of dendrochronological, isotope and terpene quantification techniques. Post-burning growth variations depended on the time since burning, the pine species, tree resistance, fire severity and tree performance before burning. In the year of burning, growth was reduced in P. halepensis and unaffected in P. nigra and P. sylvestris. However, as time passed, growth increased in P. nigra, recovered in P. halepensis and decreased in P. sylvestris. P. nigra had a lower probability of dying than P. sylvestris. Burning season emerged as an important factor for explaining initial post-burning pine mortality, since for a certain level of crown injury the probability of a pine dying was higher in spring than in fall. In contrast, delayed pine mortality was higher in fall than in spring burns probably due to the longer combustion times recorded during the fall burns at the base of the trunk. A relevant release of tree competition increased growth through a positive effect on the latewood of P. nigra and P. sylvestris as stem injury decreased. Moreover, we showed that burning just after a dry year did not reduce the growth resilience of pines in comparison with unburned pines. In P. halepensis a relevant competition release, especially in pines with lower crown volume scorched, resulted in higher growth rates as time since burning increased. This growth response coincided with a dry year and was associated with higher stomatal conductance, suggesting that water availability was enhanced after burning. Burning also affected the secondary metabolism of pines, and specifically the amount and type of terpene production depending on the pine species and fire severity. Thus, as crown injury increased, needle terpene concentration 24h post-burning also augmented. However, a remarkable decrease occurred at one year post-burning. This reduction was more pronounced in pines benefited by the increase in resource availability after burning, suggesting that pines were allocating assimilates to growth rather than to defence. From a fuel management point of view, this thesis provides valuable information that can be used to better plan prescribed burning in Mediterranean Pinus forests, in terms of required fire intensity, severity and burning season, offering a new window of opportunity for the use of prescribed burning as a forest management tool.

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Determina la prevalencia y factores asociados con la dislipidemia en trabajadores de algunos centros de salud del nivel primario. La investigación fue transversal. Se incluyó a 163 trabajadores, de algunos centros de salud del nivel primario durante el mes de agosto del año 2014 la mediana de edad fue 49,0 años y fluctúo entre 22 y 67 años. Se realizó una entrevista mediante un cuestionario que fue previamente sometido a juicio de experto, mejorando la presentación de las preguntas, posteriormente se realizó la prueba de piloto. El estado nutricional se determinó mediante el índice de masa corporal (IMC), siguiendo las normas técnicas para la valoración nutricional antropométrica del Ministerio de Salud. Adicionalmente se realizó la extracción de muestra de sangre a través de la punción venosa central para determinar las dislipidemias y se consideraron colesterol total (CT) elevado > 200 mg/dl, LDL-C > 100 mg/dl, HDL-C bajo (varones HDL-C < 40 mg/dl y mujeres HDL-C < 50 mg/ dl), y triglicéridos ≥ 150 mg/d. Las muestras fueron procesadas en un laboratorio particular. El perímetro abdominal se evaluó mediante la cinta métrica, se midió al participante en posición erguida, con el torso descubierto, y con los brazos relajados y con los pies separados por una distancia de 25 a 30 cm, de acuerdo a la Guía Técnica de valoración nutricional del Ministerio de Salud. Se solicitó el consentimiento informado por escrito para la extracción de muestras sanguíneas. Encuentra que la prevalencia de hipercolesterolemia fue 30,1%, hipertriglicéridemia 40,5%, HDL-C bajo 69,3%, LDL-C elevado 55,2%, y la dislipidemia global fue 87,7%. Los factores asociados a dislipidemia por Hipertrigliceridemia son la edad entre 50-59 años (OR 4, IC 95% : 1.2-13.6), obesidad (OR 2.8, IC 95% : 1.2- 6.7), riesgo cardiovascular muy alto según perímetro abdominal (OR 2.5, IC 95% : 1.1-1,7), relacionado a Hipercolesterolemia es la edad entre 40-49 años (OR 12.1, IC 95% : 1.4-100,7), 50-59 años (OR 8,9 IC 95% : 1.1-72.8), 60-69 años (OR 10.7, IC 95% : 1.2-93.6), sobrepeso (OR 2.8, IC 95% : 1.1-7,2), obesidad (OR 5.1, IC 95% : 1.9-13.8), por su parte el factor asociado a HDL-C bajo , es el riesgo cardiovascular muy alto (OR: 2.5, IC 95% : 1.1-5-9), y el factor asociado a LDL- C elevado es la edad entre 30-39 años (OR 6.7, IC 95% : 1.8-24.7), 40-49 años (OR 7.1, IC 95% : 1.9-25.7), 50-59 años (OR 6, IC 95% : 1.7-20, 6), 60-69 años (OR 4.3, IC 95% : 1.1-16.7) y la obesidad (OR 2.5, IC 95% : 1.1 – 5.8) Concluye que la prevalencia de hipertrigliceridemia fue de 40,5%, hipercolesterolemia 30,1%, HDL-C bajo 69,3 %, LDL-C elevado 55,2% en trabajadores de algunos centros de salud del nivel primario y la prevalencia de dislipidemia global fue 87,7%. La edad entre 50-59 años, la obesidad y el riesgo cardiovascular muy alto están asociados a la hipertrigliceridemia y la edad entre 40-49 años, 50–59 años, 60–69 años, el sobrepeso y la obesidad están asociados a la Hipercolesterolemia. El riesgo cardiovascular muy alto está asociado al HDL –C bajo. La edad entre 30-39 años, 40-49 años, 50–59 años, 60–69 años y la obesidad están asociados al LDL – C alto en trabajadores de algunos centros de salud del nivel primario.
Tesis

Introducción: Los cultivos de osteoblastos humanos obtenidos de explantes óseos son un buen modelo de estudio de la funcionalidad osteoblástica. Existe una elevada correlación entre la actividad celular que presentan los osteoblastos in vitro e in vivo (biopsias óseas). El cultivo primario de osteoblastos derivados de hueso trabecular es el mejor modelo para el estudio de anomalías intrínsecas de las células de pacientes con enfermedades metabólicas óseas.
Las estatinas o inhibidores específicos del enzima HMG-CoA reductasa actúan inhibiendo la síntesis hepática de colesterol. Las estatinas son utilizadas para disminuir los niveles de colesterol y proporcionar una importante vía de abordaje en el tratamiento de la hiperlipidemia y arteriosclerosis. En los últimos años se ha visto que pueden tener efectos pleiotrópicos en el hueso.
El objetivo del trabajo fue analizar la implicación de la funcionalidad osteoblástica en el metabolismo del hueso mediante cultivo primario de osteoblastos humanos. De esta manera el trabajo se ha separado en cuatro subestudios diferentes que tienen como columna vertebral el cultivo primario para el estudio de la función osteoblástica.
Objetivos: Subestudio I: Análisis del cultivo primario de osteoblastos humanos y la línea tumoral MG-63 como modelo para el estudio de patologías metabólicas óseas y el efecto de factores reguladores y de fármacos en el hueso. Subestudio II: Estudio de la proliferación celular y de la expresión génica del sistema OPG/RANKL, los factores Cbfa1 y BMP-2, y el colágeno tipo 1 y la osteocalcina, en cultivo primario de osteoblastos de pacientes con osteoporosis posmenopáusica. Subestudio III: Análisis del efecto de la simvastatina y la atorvastatina sobre la proliferación celular y la expresión génica del BMP-2, el colágeno tipo 1 y la osteocalcina en osteoblastos humanos primarios normales y la línea celular MG-63. Subestudio IV: Estudio de la proliferación celular y de la expresión génica del colágeno tipo 1 y la osteocalcina en cultivo primario de osteoblastos de varones con osteoporosis idiopática.
Metodología: Para el cultivo primario de osteoblastos humanos se utilizaron muestras de rodilla, de cabeza de fémur y de cresta ilíaca. Se extrajo el hueso trabecular y se troceó en explantes de 1mm3. Los explantes fueron lavados mediante agitación mecánica con una solución salina y depositados sobre una placa de Petri con medio completo suplementado con suero. Pasadas aproximadamente cuatro semanas el cultivo llegaba a la confluencia y en este momento, previamente a realizar experimentos se procedía a la caracterización de los osteoblastos mediante la detección de la actividad fosfatasa alcalina y la expresión génica de la osteocalcina, usando en ambos casos fibroblastos humanos primarios como control negativo.
Para la cuantificación de la expresión de los genes analizados en cada subestudio se utilizó la técnica de la PCR a tiempo real. A partir de las células tratadas de forma específica en cada uno de los subestudios se realizó la extracción de RNA total utilizando el método del Trizol. Cada una de las muestras de RNA aislado se evaluó su integridad mediante un gel de agarosa y se cuantificó para la posterior reacción de retrotranscripción a partir de 1mg de RNA. Finalmente se cuantificaron los niveles de expresión génica mediante la PCR a tiempo real. Esta técnica consiste en monitorizar la reacción de PCR a medida que ésta tiene lugar. Se realizó mediante la tecnología TacMan que utiliza unas sondas fluorogénicas que permiten la detección de los productos de PCR específicos a medida que se van sintetizando. En esta técnica se marca un umbral de fluorescencia en un punto donde la reacción se encuentra en fase exponencial. El ciclo en el cual la reacción de amplificación atraviesa el umbral de fluorescencia se denomina Ct (Treshold Cycle) y depende de la cantidad de cDNApresente en la muestra.
Resultados: Subestudio I: Todas las líneas de osteoblastos primarios utilizadas mostraron fenotipo osteoblástico ya que todas ellas presentaron actividad fosfatasa alcalina y expresaron el gen de la osteocalcina. Subestudio II: Se obtuvieron un total de 21 muestras procedentes de mujeres menopáusicas sometidas a intervención quirúrgica por prótesis de rodilla. Se separaron en dos grupos en función de patología osteoporótica siguiendo los criterios densitométricos de la OMS de -2.5DS. Resultó un total de 9 muestras en el grupo de pacientes osteoporóticas y 12 muestras en el grupo de pacientes no osteoporóticas. No se observaron diferencias estadísticamente significativas en la proliferación celular entre los dos grupos. Se observó una disminución estadísticamente significativa en los niveles de expresión génica y COL1A1 en el grupo de pacientes osteoporóticas. Se observó una diferencia estadísticamente significativa en los niveles de expresión génica de OPG bajo el estímulo de vitamina D y 17?-estradiol. Subestudio III: Se observó una disminución estadísticamente significativa de la proliferación celular en los cultivos tratados con distintas concentraciones de simbastatina y atorvastatina en los osteoblastos primarios y en la línea celular MG-63. Se observó un aumento de la expresión génica de COL1A1, osteocalcina y BMP-2 en los osteoblastos primarios y en la línea celular MG-63. Subestudio IV: Se obtuvieron un total de 30 muestras procedentes de biopsia de cresta ilíaca de pacientes varones. Se separaron en dos grupos en función de patología osteoporótica siguiendo los criterios densitométricos de la OMS de -2.5DS. Resultó un total de 14 muestras en el grupo de pacientes osteoporóticas y 16 muestras en el grupo de pacientes no osteoporóticas. Se observó una disminución estadísticamente significativa de la proliferación celular en las muestras del grupo de pacientes osteoporóticos. Se observó una tendencia a la disminución de la expresión génica de COL1A1 en condiciones basales y con vitamina D en el medio. Se observó una disminución estadísticamente significativa de los niveles de expresión génica de osteocalcina en presencia de vitamina D en las muestras del grupo de pacientes con osteoporosis.
Conclusiones: Subestudio I: El cultivo primario de osteoblastos es un buen modelo para el estudio de patologías metabólicas óseas (como la osteoporosis) y del efecto de factores y fármacos en el hueso. Subestudio II: Los osteoblastos procedentes de pacientes con osteoporosis posmenopáusica tienen un defecto que afecta a la expresión de genes característicos de la función osteoblástica. Subestudio III: Las estatinas (simvastatina y atorvastatina) afectan a la función osteoblástica favoreciendo la diferenciación a través de la inhibición de la proliferación celular y el incremento de la expresión de genes característicos del osteoblasto diferenciado con elevada actividad secretora. Subestudio IV: Los osteoblastos procedentes de pacientes con osteoporosis masculina idiopática tienen un defecto que afecta a la proliferación celular y a la expresión de genes característicos de la función osteoblástica.
Introduction: Human osteoblast cultures obtained from bone explants are a good model for studying the osteoblastic function. A strong correlation exists between the osteoblast activity in vitro and in vivo (bone biopsies). The osteoblasts primary culture derived from trabecular bone is the best model for the study of cell intrinsic anomalies from patients with metabolic bone illnesses.
Statins or specific HMG-CoA reductase inhibitors operate inhibiting the hepatic synthesis of cholesterol. Statins are used to reduce cholesterol levels and are an important way of approaching the treatment of hyperlipidemia and arteriosclerosis. In the last years, it has been demonstrated that they can have pleiotropic effects in the bone.
The objectives of this work were to analyze the implications of the osteoblastic function in bone metabolism using human osteoblast primary cultures. The work has been separated in to four substudies that although combine different strategies all share the common backbone of using primary cultures to study the osteoblastic function.
Objectives: Substudy I: Analysis of primary human osteoblasts and the tumoral cell line MG-63 culture as a model for the study of bone metabolic pathologies and the effect of regulatory factors and drugs in the bone. Substudy II: Study of the cell proliferation and gene expression of OPG/RANKL, the factors Cbfa1 and BMP-2, collagen type 1 and osteocalcin in osteoblasts primary cultures in patients with postmenopausal osteoporosis. Substudy III: Analysis of the effect of simvastin and atorvastin in cell proliferation and the gene expression of BMP-2, collagen type 1 and osteocalcin in normal primary human osteoblasts and in the cell line MG-63. Substudy IV: Study of the cell proliferation and the gene expression of collagen type 1 and osteocalcin in primary cultures of osteoblasts of men with idiopathic osteoporosis.
Patients and methods: For the primary culture of human osteoblasts, knee, femur and iliac crest samples were used. The trabecular bone was extracted, and cut into explants of 1mm3.
The explants were washed with a saline solution by mechanic agitation and then placed on a Petri dish with complete medium complemented with serum. After approximately four weeks the culture reached confluence, and before any experiments, the osteoblasts were characterized measuring alkaline phosphatase activity and the gene expression of osteocalcin, using, in both cases, primary human fibroblasts as a negative control.
For the quantification of the expression of the analyzed genes in every substudy, real time PCR was used. The different cells evaluated in every substudy were treated by the Trizol method to extract the total RNA. The integrity and the quantity of every one of the RNA samples isolated were evaluated by agarose gel electrophoresis. 1?g of RNA was used for every retrotranscription. Finally, gene expression levels were quantified by real time PCR. This technique consists in monitorizing the PCR reaction at the same time as it takes place. It was carried out using the TacMan technology that use fluorescent probes that permit the detection of the specific PCR products the moment they have been synthesized. In this technique, a fluorescent threshold is determined at a point where the reaction is in its exponential phase. The cycle in which the amplification reaction reaches the fluorescent threshold is called Ct (Threshold cycle) and depends on the quantity of cDNA in the sample.
Results: Substudy I: All the osteoblast primary cell lines used displayed osteoblastic phenotypes as all of them presented alkaline phosphatase activity and expressed the osteocalcin gene. Substudy II: 21 samples were obtained from menopausal women submitted to chirurgic intervention for knee prosthesis. They were separated into two groups depending on the osteoporotic pathology following densitrometric criteria. As a result, 9 samples of osteoporotic women and 12 samples of non osteoporotic women were obtained. No statistically relevant differences were observed between the cell proliferation of the two groups. Statistically significant decreases were observed in gene expression levels and COL1A1 in the osteoporotic group. Furthermore, a statistically significant difference was observed in the OPG gene expression post-stimulation with vitamin D and 17?estradiol. Substudy III: A statistically significant decrease was also observed in cell proliferation in the cultures treated with different concentrations of simbastatin and atorvastatin in primary osteoblasts and in the MG-63 cell line. An increase in COL1A1, osteocalcin and BMP-2 gene expression was observed in primary osteoblasts and in the MG-63 cell line before the addition of statins. Substudy IV: A total of 30 samples from iliac crest biopsies of male patients were obtained. They were separated in to two groups depending on the osteoporotic pathology following densitrometric criteria. As a result, a total of fourteen samples in the osteoporotic patient group and sixteen in the non osteoporotic group were obtained. A statistically significant decrease in cell proliferation in the samples of the ostheoporotic group was observed. A tendency to decrease was observed in COL1A1 gene expression in basal conditions and with vitamin D in the medium. A statistically significant decrease in the osteocalcin gene expression levels in the presence of vitamin D was observed in the samples of the group of osteoporotic patients.
Conclusions: Substudy I: The primary culture of osteoclasts is a good model for the study of bone metabolic illnesses (like osteoporosis) and the effect of factors and drugs in the bone. SubstudyII: The osteoblasts coming from patients with postmenopausal osteoporosis have a defect that affects the expression of characteristic genes of the osteoblastic function. Substudy III: Statins (simvastin and atorvastin) affect the osteoblastic function favouring differentiation through the inhibition of cellular proliferation and the increase of the expression of genes characteristic of the differentiated osteoblast with elevated secretive activity. Substudy IV: Osteoblasts coming from patients with masculine idiopathic osteoporosis have a defect that affects cell proliferation and characteristic genes of the osteoblastic function.

Les Streptomyces sont des bactéries filamenteuses du sol à Gram +. Elles sont connues pour leur capacité à produire des métabolites secondaires utiles en médecine et en agriculture. S. coelicolor et S. lividans sont des souches modèles phylogénétiquement proches. Elles ont cependant des capacités contrastées à accumuler des lipides de réserve de la famille des triacylglycérol (TAG) et produire des métabolites secondaires alors qu’elles possèdent des voies de biosynthèse similaires pour ces deux types de molécules. En présence de glucose, S. coelicolor produit des niveaux élevés de métabolites secondaires spécifiques et son contenu en TAG est faible alors que c'est le contraire chez S. lividans. En revanche, en présence de glycérol, les deux souches accumulent une quantité de TAG similaire mais S. coelicolor produit aussi des métabolites secondaires. Le but de la présente thèse était de déterminer les caractéristiques métaboliques différentielles qui sous-tendent les différentes capacités biosynthétiques de ces deux souches modèles. Pour ce faire, une analyse protéomique comparative sans marquage des souches cultivées en milieu R2YE liquide ou solide en présence de glucose ou de glycérol comme principales sources de carbone a été réalisée en utilisant la technique de chromatographie liquide couplée à de la Spectrométrie de Masse en tandem (LC-MS / MS). Au total, 2024 et 4372 protéines ont été identifiées à partir des cultures liquides et solides, représentant 24% et 50% du protéome théorique. Les études en liquide ont révélé que le métabolisme de S. lividans était principalement glycolytique alors que le métabolisme de S. coelicolor était principalement oxydatif. Elles ont également indiqué que ces caractéristiques pourraient être liées au catabolisme préférentiel des acides aminés par rapport au glucose chez S. coelicolor par rapport à S. lividans. De plus, cette thèse constitue la première analyse protéomique du métabolisme de ces deux souches modèles en présence de glycérol
Streptomyces are filamentous Gram+ soil bacteria well known for their ability to produce secondary metabolites useful in medicine and agriculture. S. coelicolor and S. lividans are phylogenetically closely-related model strains but they have contrasted abilities to accumulate storage lipids of the TriAcylGlycerol (TAG) family and to produce secondary metabolites whereas they possess similar pathways for the biosynthesis of these molecules. In the presence of glucose, S. coelicolor produces high levels of specific secondary metabolites and its TAG content is low whereas it is the opposite for S. lividans. In contrast, in the presence of glycerol, the two strains accumulated similar amount of TAG but S. coelicolor still produces secondary metabolites. The aim of the present thesis was to determine the differential metabolic features supporting such different biosynthetic abilities. To do so, a comparative label-free shotgun proteomic analysis of the strains grown in liquid or solid R2YE media with glucose or glycerol as main carbon sources was carried out using Liquid chromatography−tandem mass spectrometry (LC−MS/MS). A total of 2024 and 4372 proteins were identified in liquid and solid cultures, representing 24% and 50% of the theoretical proteome, respectively. These studies revealed that S. lividans metabolism was mainly glycolytic whereas S. coelicolor metabolism was mainly oxidative. They also suggested that these features might be related to the preferential catabolism of amino acids over glucose of S. coelicolor compared to S. lividans. Furthermore, this thesis constituted the first proteomic analysis of the metabolism of these two model strains in the presence of glycerol
Streptomyces es un género de bacterias filamentosas Gram+ provenientes del suelo que son conocidas por su capacidad para producir metabolitos secundarios útiles en la medicina y agricultura. S. coelicolor y S. lividans son cepas modelo filogenéticamente próximas que presentan capacidades opuestas para acumular lípidos de reserva de la familia de los triglicéridos (TAG) y para producir metabolitos secundarios en tanto que ambas cepas poseen rutas metabólicas idénticas para la biosíntesis de éstas moléculas. En presencia de glucosa, S. coelicolor produce altos niveles de metabolitos secundarios específicos y su contenido de TAG es bajo mientras que en S. lividans el comportamiento es opuesto. Sin embargo, en presencia de glicerol, ambas cepas acumulan cantidades similares de TAG y S. coelicolor produce metabolitos secundarios. El objetivo de ésta tesis fue de determinar las características metabólicas que distinguen las diferentes capacidades biosintéticas mencionadas previamente. Por esto, un análisis protéomico comparativo sin marcaje de tipo “shotgun” fue realizado con las dos cepas cultivadas en medio R2YE líquido y sólido usando glucosa o glicerol como fuentes principales de carbono mediante Cromatografía Líquida en “tándem” acoplada a Espectrometría de Masas (LC-MS/MS). Un total de 2024 y 4372 proteínas fueron identificadas en cultivos en medio líquido y sólido, representando 24% y 50% del proteoma teórico, respectivamente. El presente estudio demostró que el metabolismo de S. lividans fue principalmente glicolítico mientras que el metabolismo de S. coelicolor fue principalmente oxidativo. También se sugiere que éstas características pueden estar relacionadas con la preferencia catabólica de aminoácidos sobre el catabolismo de glucosa de S. coelicolor comparada con S. lividans. Además, la presente tesis constituye el primer análisis proteómico del metabolismo de éstas dos cepas modelo en presencia de glicerol

Le marché mondial des fruits représente des centaines de milliards d’euro par an et l’amélioration de la qualité organoleptique et nutritionnelle des fruits est l’un des principaux objectifs de ces dernières années. Le métabolisme primaire est une cible toute trouvée pour tenter de répondre à ces exigences. En effet c’est lui qui va fournir les briques nécessaires à la croissance et au développement, mais également les composés qui confèrent les valeurs gustatives tels que les sucres et les acides organiques. C’est pourquoi la compréhension de son fonctionnement au cours du développement des fruits est nécessaire. Pour cela le métabolisme primaire a été étudié chez huit espèces de fruits charnus qui diffèrent en termes de durée de développement, de taille de fruit, de famille botanique, de qualité gustative (sucrosité, acidité…), et sujettes ou non à une crise respiratoire au début de la maturation. Des données physiologiques et biochimiques ont été collectées tout au long du développement du fruit, de l’anthèse à la maturité physiologique. La modélisation de la croissance des fruits a permis de standardiser les stades de développement et ainsi d’améliorer la comparaison entre espèces. La composition de la biomasse a ensuite été caractérisée qualitativement et quantitativement par des approches analytiques ciblées et non ciblées mettant en évidence les similitudes et les différences de composition et d’évolution au cours du développement. Des modèles linéaires généralisés combinant la composition et les données de croissance ont été utilisés pour comparer différentes phases de développement du fruit et une analyse discriminante par régression des moindres carrés partiels (PLS-DA) a permis de séparer les fruits climactériques des non climactériques. Dans les deux cas, les composés des parois cellulaires, les protéines et les lipides interviennent dans la différentiation des groupes. Enfin, une étude détaillée du métabolome et de l’activome du fruit a été réalisée chez trois espèces de Solanacées. Elle montre qu’au sein d’une même famille botanique la régulation diffère au cours du développement, notamment au niveau du métabolisme des sucres et de la glycolyse. Ces travaux revisitent le caractère climactérique des fruits, le positionnant bien en amont du déclenchement de la crise respiratoire, et, plus généralement, permettent de mieux comprendre le métabolisme primaire au cours du développement du fruit
The world fruit market represents hundreds of billions of euros per year and improving the organoleptic and nutritional quality of fruit has been one of the main objectives in recent years. Primary metabolism is a target that can be used to try and meet these requirements. Indeed, it provides the bricks necessary for growth and development but also the compounds that contribute to taste such as sugars and organic acids. Therefore, it is necessary to understand how it operates during fruit development. For this purpose, primary metabolism has been studied in eight species of fleshy fruits that differ in terms of development duration, fruit size, botanical family, taste quality (sweetness, acidity, etc.), and are subject or not to a respiratory crisis at the initiation of ripening. Physiological and biochemical data have been collected throughout the fruit development from anthesis to physiological maturity. Fruit growth modelling allowed standardizing the stages of development and thus improved comparison between species. The composition of biomass was then characterized qualitatively and quantitatively by targeted and non-targeted analytical approaches highlighting similarities and differences in composition and changes during development. Generalized linear models combining composition and growth data were used to compare different phases of fruit development and a discriminant partial least square regression analysis (PLS-DA) was used to separate climacteric and non-climacteric fruits. In both cases, cell wall compounds, proteins and lipids were involved in group differentiation. Finally, a detailed study of the fruit metabolome and activome was performed in three species of Solanaceae. It revealed that within the same botanical family, regulation differs during development, particularly for sugar metabolism and glycolysis. This work revisits the climacteric character of fruits, positioning it long before the onset of the respiratory crisis, and, more generally, provides a better understanding of primary metabolism during fruit development

Orientador: Carmen Silvia Fernandes Boaro
Resumo: Espécies vegetais são capazes de produzir diversidade de substâncias, que desempenham funções importantes para sua sobrevivência e adaptação ao ecossistema. O metabolismo primário, é essencial para o crescimento, desenvolvimento, maturação e reprodução de qualquer espécie. O metabolismo especializado, dependente do primário, é responsável por originar o óleo essencial, que são misturas de metabólitos especializados voláteis, representados principalmente por monoterpenos e sesquiterpenos. Cada espécie vegetal produz um óleo essencial de composição característica específica, podendo ser influenciado por fatores bióticos e abióticos. A fenologia pode influenciar processos bioquímicos e rotas metabólicas capazes de modificar a formação de substâncias biologicamente ativas, alterando diretamente o conteúdo e a qualidade dos óleos essenciais. Sendo assim, o objetivo deste trabalho foi avaliar se as fases fenológicas, vegetativa e reprodutiva modificam o desempenho fotossintético e o perfil do óleo essencial de Xylopia aromatica (Lam.) Mart., influenciando sua atividade biológica na defesa antioxidante e ação antifúngica. As variáveis, fluorescência da clorofila a, trocas gasosas, carboidratos, atividade enzimática e peroxidação lipídica, potencial água, conteúdo relativo de água das folhas, extração, rendimento, caracterização química e atividade antifúngica do óleo essencial de Xylopia aromatica foram avaliadas em 24 plantas, 12 no estádio vegetativo e 12 no reprodutivo, coletadas... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Research aimed at the knowledge of plant species allows the elaboration of projects that aim at the understanding of development, conservation of biodiversity and sustainable exploitation of natural resources. The primary metabolism, represented by photosynthesis and the specialized one, that synthesizes the essential oil, can be influenced by the environmental and phenological conditions, which can influence the chemical profile of the essential oil and the biological activity in the vegetal defense, including against fungi, bacteria and virus. Compounds from the specialized metabolism present biological activity and potential for the production of bactericides and fungicides. Therefore, it is necessary to know the stage of development of plant species in which the substances of interest, with economic potential, are more concentrated, thus orienting, if appropriate, the collection period, aiming at the conservation and sustainable use. There are scientific studies that reveal biological activity of essential oils, as observed for the genus Xylopia, but none of them relates the primary and specialized metabolism to the stage of development in which the species is found. In this way, the objective of this research was to evaluate if the phenological, vegetative and reproductive phases of Xylopia aromatica (Lam.) Mart. modify the photosynthetic performance and the profile of the essential oil, which may influence its biological activity in the antioxidant defense and antifunga... (Complete abstract click electronic access below)
Mestre

Des de la pèrdua de la biodiversitat fins a la degradació de terra i la contaminació de les masses d'aigua, els nostres sistemes de suport vital estan en declivi. La nau espacial Terra té problemes. Nosaltres tenim problemes. La ciència de la sostenibilitat ha sorgit com a resposta, oferint models del nostre camí a la salvació. Això no obstant, l'esperit dels esforços de modelatge en la ciència de la sostenibilitat està dominat per les nocions de predicció i optimització. Mentre que la predicció i l'optimització han demostrat ser extremadament exitoses en altres camps, portant a la creació de coets i telèfons intel·ligents entre d'altres, aquests plantejaments no aconsegueixen comprendre les intangibilidades més essencials dels sistemes socio-ecològics. Aquestes nocions han colonitzat efectivament el futur, recolzant un règim de promeses tecnocientífiques i reconfortants raons ex-post. Aquesta dissertació explora un enfocament alternatiu de la ciència de la sostenibilitat. Un enfocament basat en estudis d'anticipació i en la idea que els sistemes socio-ecològics són sistemes adaptatius complexos. A partir de les teories de l'metabolisme social i la biologia relacional, es realitza una revisió exhaustiva de la base conceptual de la modelització de la sostenibilitat. Posteriorment, s'utilitza aquesta revisió per fonamentar la caracterització dels sistemes socioeconòmics com a sistemes de reparació metabòlica, és a dir, organismes. D'aquesta manera es llança nova llum sobre les megatendències mundials de la globalització i la urbanització, a través de les quals les societats estan perdent el control sobre les seves identitats. Posteriorment, els enfocaments de modelització proporcionades pel metabolisme social i la biologia relacional es discuteixen amb els de la filosofia de la ment i la filosofia de l'llenguatge per reconceptualitzar l'arquitectura dels espais de coneixement socio-ecològics, dins dels quals trobem els models. Amb especial deteniment és analitzat el paper de la justificació, l'explicació i els relats normatius en la creació dels límits de l'espai de coneixement i en la ruptura de les impredicatividades. Havent establert una sòlida base conceptual, es presenten dos estudis de cas. El primer, una narració quantitativa sobre el ràpid desplegament de fonts alternatives d'energia elèctrica per descarbonitzar l'economia, posa en relleu diverses deficiències en els actuals esforços de governança. S'afirma, per exemple, que la forma precipitada en què es considera l'emmagatzematge d'energia en els debats contemporanis sobre la transició energètica està portant a la societat a una greu situació de desajust estructural-funcional. El segon, una narració quantitativa sobre la reinternalització de l'agricultura, posa en relleu un conjunt de preocupacions en matèria de seguretat relacionades amb els nivells extrems d'externalització de l'agricultura observats en els sistemes socioeconòmics moderns. Cap de les narracions quantitatives presentades en aquesta dissertació fa cap intent de predir el futur. S'ofereixen com històries d'aprenentatge, ajudant a la societat a aclarir la seva visió d'un futur desitjable. Encara que són crítiques, cap de les tesis d'aquesta dissertació presenten arguments per eliminar els enfocaments convencionals de la modelització. Aquesta dissertació no és més que un esforç per trencar l'hegemonia de la predictivitat i la optimizabilidad, per complementar aquestes idees amb nocions de impredictibilitat. En última instància, es proposa un paradigma de sostenibilitat supercrítica, que és una manera de sostenibilitat en el qual l'auto-referencialitat dels sistemes complexos s'entén com un cicle virtuós, no viciós. La sostenibilitat supercrítica reobre el debat sobre el futur trencat, aportant idees sobre la creació deliberada de models socio-ecològics extensibles que donin suport vies de desenvolupament responsables.
Desde la pérdida de la biodiversidad hasta la degradación del suelo y la contaminación de las masas de agua, nuestros sistemas de soporte vital están en declive. La nave espacial Tierra tiene problemas. Nosotros tenemos problemas. La ciencia de la sostenibilidad ha surgido como respuesta, ofreciendo modelos de nuestro camino a la salvación. No obstante, el espíritu de los esfuerzos de modelado en la ciencia de la sostenibilidad está dominado por las nociones de predicción y optimización. Mientras que la predicción y la optimización han demostrado ser extremadamente exitosas en otros campos, llevando a la creación de cohetes y teléfonos inteligentes entre otros, estos planteamientos no logran comprender las intangibilidades más esenciales de los sistemas socio-ecológicos. Estas nociones han colonizado efectivamente el futuro, apoyando un régimen de promesas tecnocientíficas y reconfortantes razones ex-post. Esta disertación explora un enfoque alternativo de la ciencia de la sostenibilidad. Un enfoque basado en estudios de anticipación y en la idea de que los sistemas socio-ecológicos son sistemas adaptativos complejos. A partir de las teorías del metabolismo social y la biología relacional, se realiza una revisión exhaustiva de la base conceptual de la modelización de la sostenibilidad. Posteriormente, se utiliza esta revisión para fundamentar la caracterización de los sistemas socioeconómicos como sistemas de reparación metabólica, es decir, organismos. De este modo se arroja nueva luz sobre las megatendencias mundiales de la globalización y la urbanización, a través de las cuales las sociedades están perdiendo el control sobre sus identidades. Posteriormente, los enfoques de modelización proporcionadas por el metabolismo social y la biología relacional se discuten con los de la filosofía de la mente y la filosofía del lenguaje para reconceptualizar la arquitectura de los espacios de conocimiento socio-ecológicos, dentro de los cuales encontramos los modelos. Con especial detenimiento es analizado el papel de la justificación, la explicación y los relatos normativos en la creación de los límites del espacio de conocimiento y en la ruptura de las impredicatividades. Habiendo establecido una sólida base conceptual, se presentan dos estudios de caso. El primero, una narración cuantitativa sobre el rápido despliegue de fuentes alternativas de energía eléctrica para descarbonizar la economía, pone de relieve varias deficiencias en los actuales esfuerzos de gobernanza. Se afirma, por ejemplo, que la forma precipitada en que se considera el almacenamiento de energía en los debates contemporáneos sobre la transición energética está llevando a la sociedad a una grave situación de desajuste estructural-funcional. El segundo, una narración cuantitativa sobre la reinternalización de la agricultura, pone de relieve un conjunto de preocupaciones en materia de seguridad relacionadas con los niveles extremos de externalización de la agricultura observados en los sistemas socioeconómicos modernos. Ninguna de las narraciones cuantitativas presentadas en esta disertación hace ningún intento de predecir el futuro. Se ofrecen como historias de aprendizaje, ayudando a la sociedad a clarificar su visión de un futuro deseable. Aunque son críticas, ninguna de las tesis de esta disertación presentan argumentos para eliminar los enfoques convencionales de la modelización. Esta disertación no es más que un esfuerzo por romper la hegemonía de la predictividad y la optimizabilidad, para complementar esas ideas con nociones de impredictibilidad. En última instancia, se propone un paradigma de sostenibilidad supercrítica, que es un modo de sostenibilidad en el que la auto-referencialidad de los sistemas complejos se entiende como un ciclo virtuoso, no vicioso. La sostenibilidad supercrítica reabre el debate sobre el futuro roto, aportando ideas sobre la creación deliberada de modelos socio-ecológicos extensibles que apoyen vías de desarrollo responsables.
From biodiversity loss to soil degradation to pollution of water bodies, our life support systems are in decline. Spaceship Earth is in trouble. We are trouble. Sustainability science has emerged in response, offering to model our way to safety. The spirit of modeling efforts in the sustainability science is, however, dominated by notions of prediction and optimization. While prediction and optimization have proven extremely successful in other domains, leading to the creation of rockets and smartphones and so forth, they fail to grasp the essential intangibilities of social-ecological systems. They have effectively colonized the future, supporting a regime of techno-scientific promises and comforting ex-post motives. This dissertation explores an alternative approach to sustainability science, one based on anticipation studies and the idea of social-ecological systems as complex adaptive systems. A thorough revision of the conceptual basis of modeling for sustainability is made, based on insights from societal metabolism and relational biology. That revision is then used to inform the characterization of social-economic systems as metabolic-repair systems, meaning organisms. New light is thereby shed on global megatrends of globalization and urbanization, through which societies are losing control over their identities. Insights on modeling provided by societal metabolism and relational biology are then crossed with insights from philosophy of mind and philosophy of language to re-conceptualize the architecture of social-ecological knowledge spaces, within which models exist. An emphasis is made on the role of justification, explanation and normative narratives in creating knowledge space bounds and breaking impredicativities. Having established a robust conceptual basis, two case studies are presented. The first, a quantitative storytelling on the quick deployment of alternative sources of electrical energy to decarbonize the economy, highlights several shortcomings of current governance efforts. It is asserted, for example, that the hasty way energy storage is considered in contemporary energy transition discussions is leading society towards a grave situation of structural-functional mismatch. The second case study, a quantitative storytelling on agricultural re-internalization, highlights a set of security concerns associated with the extreme levels of agricultural externalization found in modern social-economic systems. Neither of the quantitative storytellings presented in this dissertation make any attempt to predict the future. Their offering is as learning-type storylines, helping society clarify its vision of a desirable future. Indeed, although critical of them, none of the insights in this dissertation are arguments for the elimination of conventional approaches to modeling. This dissertation is merely an effort to break the hegemony of predictivity and optimizability, to complement those ideas with notions of impredicativity. A paradigm of supercritical sustainability is ultimately proposed, being a mode of sustainability where the self-referentiality of complex systems is understood to be a virtuous cycle, not a vicious one. Supercritical sustainability re-opens discussion of the ruptured future, providing insights into the deliberative creation of extensible social-ecological models in support of responsible development pathways.
Universitat Autònoma de Barcelona. Programa de Doctorat en Ciència i Tecnologia Ambientals

In this study, the biosynthesis of albizziine has been elucidated, and a direct precursor relationship shown to exist between uracil and albizziine. This was confirmed by the demonstration that [2-¹⁴C]uracil specifically labels C-5 of albizziine. It is concluded that the biosynthetic sequence involves the hydroxylation of uracil to isobarbituric acid, then amination to 5-aminouracil, followed by hydrogenation and ring-opening, to yield albizziine. 2,3-Diaminopropanoic acid was shown to be formed from albizziine by the action of β-ureidopropionase. Thus, the formation of albizziine and 2,3-diaminopropanoic acid represents a further aspect of the interfacing of pyrimidine primary and secondary metabolism through uracil. Lathyrine was shown to be catabolyzed in Lathyrus tingitanus to yield the non-protein amino acid 4-hydroxyhomoarginine, and it was thus confirmed that 4-hydroxyhomoarginine is a catabolite rather than a precursor of lathyrine. 2-Amino-4-carboxypyrimidine, the immediate precursor of the lathyrine ring-system, was shown to be synthesized enzymically from uracil. The relative amount of exogenously supplied uracil diverted into production of the isomeric pyrimidinyl amino acids willardiine and isowillardiine in Pisum sativum, and also that diverted into the production of the pyrimidine amino acid lathyrine in Lathyrus tingitanus was determined. Uracil was shown to have a pronounced inhibitory effect on the germination and growth of Phaseolus aureus and Glycine max. As these plants do not produce pyrimidine-derived secondary products, this observation is consistent with the view that production of such compounds is a detoxification mechanism for bioactive pyrimidines.

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of host nutrient dependency, allowing comparisons that provide insight into parasite adaptation. A key aspect of this is the relationship between parasite metabolism and the metabolite profile of its host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor. First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts that had mutations in amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. This research highlights the importance of aspartate and asparagine to early post-attachment metabolism in both P. aegyptiaca and T. versicolor and through host deficiencies possibly associated with decreased growth in P. aegyptiaca. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.
Ph. D.
Parasitic weeds of the plant family Orobanchaceae attach to the roots of host plants via haustoria. Parasite haustoria embed into the host plant and are capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of the extent to which a parasitic plant may depend on its host for nutrients. This allows comparisons that provide insight into the ways in which parasites adapt. A key aspect of this is the relationship between the metabolite profile of the parasite and the metabolite profile of the host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate parasite Phelipanche aegyptiaca (which cannot perform photosynthesis) and the facultative parasite Triphysaria versicolor (which can perform photosynthesis). First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts. These hosts had mutations in enzymes related to amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.

The tricarboxylic acid (TCA) cycle is one of the central pathways in respiration and also plays an important role in a variety of metabolic processes including the synthesis of secondary metabolites and the provision of carbon skeletons for ammonium assimilation and amino acid biosynthesis. Effective regulation of these multiple demands on the TCA cycle is likely to be very important for plant fitness. One way that this regulation could be achieved is through metabolite channelling. This occurs when metabolites are transferred between enzyme active sites without diffusing into the bulk aqueous phase of the cell, and is known to be important in regulating demands in metabolic pathways. Although there is evidence that metabolite channelling exists in animals, there have been no attempts to investigate it in plant. The first aim of this thesis was therefore to investigate whether metabolite channelling exists in the plant TCA cycle. Isotope dilution experiments were developed to investigate metabolite channelling, and were able to show that metabolite channelling was present between certain enzymes of the TCA cycle in both S. tuberosum and A. thaliana mitochondria. The second aim of the thesis was investigate whether metabolite channelling is important in regulating the TCA cycle in plant mitochondria. The pattern of metabolite channelling did not change in mitochondria isolated from the light and the dark, or from mitochondria with increased or decreased TCA cycle rates, but it was not possible to say whether the metabolite channelling altered in a quantitative fashion. Overall the thesis provides the first direct evidence of channelling in the TCA cycle in plants, and further work should help to elucidate what role, if any, it plays.

O uso da metabolômica ambiental tem sido usada para avaliar a interação dos organismos com o ambiente. Apesar do alto impacto que os metais têm no ambiente, essa abordagem analítica ainda está em seu início, em especial para as macroalgas. Como membro do primeiro nível trófico da cadeia alimentar marinha, fornecendo nutrientes e microelementos para os níveis superiores, as macroalgas são um alvo apropriado tanto para o desenvolvimento de ensaios toxicológicos quanto como bioindicador de degradação do ambiente marinho. Também por causa da sua posição na cadeia alimentar, essas macrófitas são consideradas o principal vetor para a magnificação desses elementos tóxicos. Os efeitos tóxicos dos metais sobre o ambiente aquático são documentados e bem conhecidos, mas relacionam principalmente ao desbalanço do potencial redox intracelular e, assim, o estresse oxidativo em organismos vivos. Com o objetivo de compreender a relação das macroalgas com o metal essencial Cu2+ e o metal não essencial Cd2+, a macroalga vermelha Gracilaria domingensis foi selecionada. Após 48h de exposição aos metais em águas do mar sintética e natural, as amostras foram extraídas e analisadas em cromatografia gasosa acoplada à espectrometria de massas. Em seguida, os dados foram pré-processados e pré-tratados para serem utilizados nas análises estatísticas multivariadas (AEM) de PCA e OPLS-DA. A G. domingensis exposta aos metais em água do mar sintética não foram significativamente influenciadas, como indicado pela MVA e pela análise de vias. Apesar disso, alterações significativas foram observadas na exposição aos metais em água do mar natural. Os principais resultados para o Cu2+ foram a interação do metabolismo de glicina, serina e treonina com o metabolismo de glioxilato e dicarboxilato. Foi sugerido que a macroalga poderia estar alterando o modo de adquirir carbono para uma via não fotossintetizante, uma vez que essa via está prejudicada na exposição ao metal. Também, o metabolismo de fenilalanina foi impactado por essa exposição, uma vez que é uma via fundamental para sintetizar compostos fenólicos antioxidantes. Por outro lado, apesar de oito vias terem sido identificadas como significativamente alteradas na exposição ao Cd2+, somente o metabolismo de arginina e prolina parece ter sido significativamente influenciado com o objetivo de produzir prolina, um aminoácido reconhecido por suas propriedades antioxidantes e protetoras em organismos estressados por metais. Em conclusão, os metais essenciais e não essenciais parecem ter mecanismos diferentes na tentativa de promover o combate aos danos gerados pela exposição aos metais.
The environmental metabolomics approach has been used to evaluate the interaction of organisms with their environment. Besides the high impact metals have on the environment, this method of analysis is still in its infancy, in special for macroalgae. As members of the first trophic level in the marine food chain, providing nutrients and microelements to upper levels, macroalgae are appropriate target organisms both for the development of toxicological assays and as a bioindicator of marine degradation. Also, because of their marine food chain position, these macrophytes are considered the main vectors to magnify these toxic elements. The toxic effects of metals on the aquatic environment are documented and well known, but regards mainly to the unbalance of intracellular redox potential and, therefore, oxidative stress in living organisms. In order to understand the relationship of macroalgae with the essential metal Cu2+ and the non-essential metal Cd2+, the red macroalga Gracilaria domingensis was chosen. After 48h of metal exposure in synthetic and natural seawater, the samples were extracted and analysed in gas chromatograph coupled to mass spectrometry. Afterward, the data were preprocessed and pretreated for the multivariative analysis (MVA) with PCA and OPLS-DA statistics. G. domingensis exposed to metals in synthetic seawater were not significantly affected, as indicated by MVA and pathway analysis. Though, significant changes were observed on exposure to metals in natural seawater. The main results for Cu2+ were the interlay of glycine, serine and threonine metabolism with glyoxylate and dicarboxylate metabolism. It was suggested that the macroalga could be shifting the metabolism to acquire carbon from a non-photosynthetic pathway, since it is injured on metal exposure. Also, phenylalanine metabolism was impacted by this metal exposure, since it is a pivotal source of phenolic antioxidant compounds. On the other hand, besides eigth pathways were identified as significantly changed on Cd2+ exposure, only arginine and proline metabolism seemed to be significantly affected, in order to produce proline, known for its antioxidative and protective properties in metal stressed organims. In conclusion, essential and non essential metals seem to have distinct mechanism to mitigate the damaged caused by metal exposure.

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FACEPE
Primary brain calcification (PBC), also known as idiopathic brain calcification or Fahr's disease, is a rare neurological condition that is characterized by calcium phosphate deposits in the basal ganglia and adjacent areas, movement disorders, headache and neuropsychiatric symptoms. It presents autosomic dominant inheritance and it is associated with two inorganic phosphate transporter coding genes: SLC20A2 and XPR1. Two other genes related to the blood-brain barrier maintenance and integrity are also linked to PBC, the platelet-derived growth factor-β and its receptor (PDGFB and PDGFRB), although their roles in the formation mechanism of the calcifications is not clear yet. For this study, besides the four genes above mentioned, other members of the platelet-derived grown factor family (PDGFA, PDGFRA, PDGFC and PDGFD) have also been selected as candidate genes, for which new primer pairs were designed. All genes above were screened for new variants by Sanger sequencing in fifteen Brazilian unrelated patients with brain calcifications. Sequence in silico analysis was performed using CLC Main Workbench 6.9 software and online tools available in NCBI and GOLDENPATH platforms, resulting in the identification of the first de novo SLC20A2 mutation in a patient diagnosed with PBC (NM_006749.4:c.1158C>G; NP_006740.1:p.Y386*). SLC20A2 is to-date the main gene associated with PBC, with affecting-variants observed in ~50% cases. In order to find SLC20A2 deletions and/or duplications not detected by sequencing, all Brazilian probands were screened by QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) and a duplication of the terminal exon was found in a patient with brain calcifications and hyperparatiroidism. Simultaneously, twenty-four French unrelated patients with PBC were also analyzed by QMPSF and partial SLC20A2 deletions were detected in four patients: two with deletion of the exon 2, where the start codon is located; one with deletion of the exon 4; and one with deletion of exons 4 and 5. These results reinforce SLC20A2 role as the main gene associated to PBC, as well as demonstrate that copy number variation analyses, even when revealing only partial deletions or duplications of a gene, are complementary to sequencing and work side by side in the search of genetic variations involved in this disease.
Introdução: A calcificação cerebral primária (CCP), também conhecida como calcificação idiopática dos núcleos da base ou doença de Fahr, é uma condição neurológica caracterizada por depósitos de fosfato de cálcio dos núcleos da base e região de entorno, parkinsonismo e sintomas neuropsiquiátricos. Apresenta herança autossômica dominante e é associada a dois genes codificantes de transportadores de fosfato inorgânico: o SLC20A2 e o XPR1. Dois outros genes relacionados à manutenção e à integridade da barreira hemato-encefálica, o fator de crescimento plaquetário B e seu receptor (PDGFB e PDGFRB), também foram associados à CCP, embora seus papeis no mecanismo de formação das calcificações ainda não estejam claros. Materiais e Métodos: Além dos quatro genes acima, foram selecionados como candidatos outros genes da família dos fatores de crescimento plaquetário (PDGFA, PDGFRA, PDGFC e PDGFD) e das protocaderinas (PCDH12), para os quais foram confeccionados pares de primers utilizados no seu sequenciamento e para análise de variação de número de cópia. Resultados e Discussão: Quinze famílias brasileiras com CCP foram triadas para novas variantes nos genes candidatos por sequenciamento. A análise in silico do sequenciamento foi feita através do software CLC Combined Workbench versão 6.9 e das ferramentas disponíveis nas plataformas online do NCBI e do GOLDENPATH. A partir dessa análise, foi identificada em um probando a primeira mutação de novo do SLC20A2, o principal gene associado a CCP (NM_006749.4:c.1158C>G; NP_006740.1:p.Y386*). A fim de encontrar deleções e/ou duplicações do SLC20A2 não detectadas por sequenciamento, todos os probandos brasileiros com calcificações cerebrais foram triados através da técnica de QMPSF (do inglês, Quantitative Multiplex PCR of Short fluorescent Fragments). Foi encontrada uma duplicação do exon terminal do mesmo gene em um paciente brasileiro com calcificações cerebrais e hiperparatireoidismo. Simultaneamente, foram identificadas deleções parciais no mesmo gene em quatro famílias francesas com CCP. Conclusões: Esses resultados reafirmam o SLC20A2 como o principal gene associado a CCP, bem como demonstram que análises de variação de número de cópia (CNV), ainda que parciais, são complementares ao sequenciamento na busca por variantes genéticas relacionadas a esta doença.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Most terrestrial plants make use of beneficial symbiotic associations to obtain nutrients (eg. nitrogen (N) and phosphorous (P)) from fungi in exchange for photoautotrophic carbon. However, plant parasitism (defined here as the ability of certain plants to parasitize other living material) has evolved in the plant kingdom and such plants obtain some, or all, of their nutritional needs from a host, which is severely negatively impacted by the parasite. While the physiological adaptations are well studied, the underlying molecular and biochemical mechanisms of plant parasitism remain largely unknown. As a first approach, a biochemical blueprint of primary metabolites present within parasitic plant species was constructed. The metabolomes of nineteen parasitic plants, ranging from hemi- and holoparasitism to mycoheterotrophism, were profiled via gas chromatography mass spectrometry (GC MS) based technology and targeted spectrophotometric assays. Based on these analyses, three important observations were made. First, parasitic plants were severely carbon deprived, despite being successful in colonizing and exploiting their hosts. Second, the levels of organic acids participating in mitochondrial respiration decreased and certain amino acids and soluble protein content increased. This suggests that parasitic plants utilize alternative respiratory substrates to compensate for a limitation in carbon supply. Third, although characterized by reduced carbohydrate pools, minor sugars normally not associated with plant metabolism, dominated the soluble sugar pool. The presence and significance of one of these sugars, namely turanose (α-D-glucopyranosyl-(1→3)-α-D-fructofuranose), was further investigated. Turanose biosynthetic reactions could be demonstrated in Orobanche minor extracts. Protein purification and mass spectrometry identification suggested that turanose biosynthesis occurred uniquely in parasitic plants. Future work will elucidate the functional significance of turanose metabolism in plant parasitism. Taken together, this study significantly contributes to our understanding of plant parasitism through development of metabolic signatures associated with distinct parasitic classes. These biochemical profiles highlighted several important strategies and alternative metabolic pathways that are either expressed or constitutively activated during parasitism. This knowledge broadens the scope of using parasitic plants in several biotechnological applications or as a novel research tool to address fundamental questions in plant science.
AFRIKAANSE OPSOMMING: Meeste landelike plante maak gebruik van voordelige simbiotiese assosiasies met swamme om voedinsgtowwe (bv. stikstof (N) en fosfor (P)) van hulle te verkry in ruil vir koolstof geproduseer deur die plant. Plant parasitisme (gedefinieer hier as die vermoë van sekere plante om ander lewende materiaal te parasiteer) het ontwikkel in die planteryk waar hulle sommige, of al hul voedings stowwe van 'n gasheer plant ontvang, wat erg negatief geraak word deur die parasiet. Terwyl die fisiologiese aanpassings goed gebestudeer is, is die onderliggende molekulêre en biochemiese meganismes van plant parasitisme steeds grootliks onbekend. As 'n eerste benadering, was hierdie projek geïnisieer om 'n biochemiese bloudruk op te bou van primêre metaboliete teenwoordig in parasitiese plante. Die metabolome van negentien parasitiese spesies, wat wissel van hemi - en holoparasiete tot mikoheterotrofiese plante, is ondersoek deur gas chromatografie – massa spektrometrie (GC MS) gebaseerde tegnologie en geteikende spektrofotometriese toetse. Gebaseer op hierdie ontledings was drie belangrike waarnemings gemaak. Eerstens, parasitiese plante was erg koolstof arm, ten spyte daarvan dat hulle suksesvol is in die aanhegting en ontginning van voedingstowwe vanaf gasheer plante. Tweedens, die vlakke van organiese sure wat deelneem aan mitochondriale respirasie het afgeneem, terwyl sekere aminosure en oplosbare proteïen inhoude toegeneem het. Dit dui daarop dat parasitiese plante gebruik maak van alternatiewe respiratoriese substrate om te vergoed vir 'n beperking in koolstof aanbod. Derde, alhoewel parasitiese plante gekenmerk word deur verminderde koolhidraat inhoude, het skaarse suikers wat normaalweg nie verband hou met plant metabolisme nie, hulle oplosbare suiker inhoud oorheers. Die teenwoordigheid en betekenis van een van hierdie suikers, naamlik turanose (α -D -glucopyranosyl-(1→3)-α-D-fructofuranose), was verder ondersoek. Die sintese reaksie van turanose kan gedemonstreer word in Orobanche hederae uittreksels. Proteïen suiwering en massa spektrometrie identifikasie het voorgestel dat turanose biosintese uniek plaasvind in parasitiese plante. Toekomstige werk sal aandui wat die betekenis is van turanose metabolisme in plant parasitisme. Saamgevat het hierdie studie aansienlik bygedra tot ons begrip van plant parasitisme deur ontwikkeling van metaboliese handtekeninge wat verband hou met onderskeie parasitiese klasse. Hierdie biochemiese profiele beklemtoon verskeie belangrike strategieë en alternatiewe metaboliese paaie wat óf uitgedruk of konstitutief geaktiveer word tydens parasitisme. Hierdie kennis verbreed die omvang van die gebruik van parasitiese plante in verskeie biotegnologiese toepassings of as 'n nuwe navorsings instrument om fundamentele vrae in plant wetenskap aan te spreek.

Dissertation presented to obtain the Ph.D degree in Biochemistry, Neuroscience
Brain energy metabolism results from a complex group of pathways and trafficking mechanisms between all cellular components in the brain, and importantly provides the energy for sustaining most brain functions. In recent decades, 13C nuclear magnetic resonance (NMR) spectroscopy and metabolic modelling tools allowed quantifying the main cerebral metabolic fluxes in vitro and in vivo. These investigations contributed significantly to elucidate neuro-glial metabolic interactions, cerebral metabolic compartmentation and the individual contribution of neurons and astrocytes to brain energetics. However, many issues in this field remain unclear and/or under debate.
To the financial support provided by Fundação para a Ciência a Tecnologia (SFRH/BD/29666/2006; PTDC/BIO/69407/2006) and to the Clinigene – NoE (LSHBCT2006- 010933). I further acknowledge the Norwegian Research Council for a fellowship that allowed me to perform part of my PhD work at NTNU, Norway.

L'ammonium (NH4+) est une source d'azote d'un grand intérêt dans le cadre d'une agriculture durable. Son application en champs avec des inhibiteurs de nitrification s’est montré efficace pour limiter les pertes de N par rapport à l'utilisation de nitrate (NO3-). NH4+ est un intermédiaire commun impliqué dans de nombreuses voies métaboliques. Cependant, des concentrations élevées peuvent conduire à une situation de stress chez la plante provoquant un « syndrome ammoniacal », caractérisé par une croissance réduite. Ces symptômes sont causés par la combinaison, entre autres, d'une reprogrammation métabolique, d'une perturbation de la photosynthèse, d'une dérégulation du pH et d'un déséquilibre ionique. De nombreuses études ont décrit la façon dont la plante s’adapte à la nutrition ammoniacale. Cependant, le stade de développement des organes a été souvent négligé.Pour combler cette lacune, dans le premier chapitre nous étudions comment le métabolisme s’adapte en fonction de la position des feuilles sur l'axe vertical de plants de tomates (Solanum lycopersicum) cultivées en présence de NH4+, NO3- ou NO3NH4. Nous avons disséqué la composition de la biomasse foliaire et le métabolisme grâce à une analyse complète des métabolites, ions et activités enzymatiques. Nos résultats montrent que l'ajustement métabolique du C et du N en fonction de la source d'azote était plus intense chez les feuilles âgées par rapport au plus jeunes. Surtout, nous révélons un compromis entre l'accumulation de NH4+ et l'assimilation afin de préserver les jeunes feuilles du stress ammoniacal. Par ailleurs, les plantes alimentées en NH4+ présentaient un réarrangement des squelettes carbonés impliquant un coût énergétique élevé. Nous expliquons une telle réallocation par l'action du pH-stat biochimique, pour compenser la production différentielle de protons dépendante de la forme azotée fournie.La nutrition ammoniacale peut limiter l'expansion cellulaire. Entre autres, la croissance cellulaire dépend largement de la pression interne exercée par la vacuole sur la paroi cellulaire. Cependant, l’impact du stress ammoniacal sur la vacuole a été rarement abordé. Dans le second chapitre, nous évaluons l'effet de la nutrition ammoniacale sur le développement des feuilles en se focalisant sur l'expansion et le métabolisme vacuolaire. Pour cela, nous avons suivi le développement d’une feuille depuis son apparition jusqu'à son expansion complète avec du NH4+ ou NO3- comme seule source d'azote. Nous avons d’abord mis en évidence que la réduction de l’expansion cellulaire en nutrition ammoniacal était associée à des vacuole plus petite et aussi plus acide que celles recevant du NO3-. De plus, un modèle a été construit pour prédire l'équilibre thermodynamique de différentes espèces solubles de part et d’autre du tonoplaste. Le modèle intègre les volumes subcellulaires, les gradients électrochimiques et la formation de complexe ionique dans la vacuole afin de prédire les concentrations subcellulaires des ions, acides organiques et sucres mesurée dans la feuille. De plus, ces prédictions ont été validées avec des données obtenus par fractionnement non aqueux. Finalement, l’estimation des flux de soluté dans la vacuole nous a permis de démontrer que la déficience en malate dans les cellules des feuilles nourries avec NH4+ est central dans la limitation de l'expansion vacuolaire. De plus, nous concluons que le coût énergétique du transport de soluté dans la vacuole est plus élevé sous nutrition ammoniacale en raison du gradient électrochimique plus élevé généré de part et d’autre du tonoplaste. Ce travail souligne l'importance de considérer l'état phénologique des feuilles lors de l'étude du métabolisme de l'azote. De plus, notre approche place le contrôle du pH cytosolique et l'expansion des vacuoles au centre de l'adaptation des feuilles de tomate à ce stress et ouvre la voie à de futures études dans le domaine de la nutrition ammoniacal
Ammonium (NH4+) is a nitrogen source of great interest in the context of sustainable agriculture. Its application in the field together with nitrification inhibitors has been extensively proven efficient to limit detrimental N losses compared to the use of nitrate (NO3-). NH4+ is a common intermediate involved in numerous metabolic routes. However, high NH4+ concentrations may lead to a stress situation provoking a set of symptoms collectively known as “ammonium syndrome” mainly characterized by growth retardation. Those symptoms are caused by a combination of, among others, a profound metabolic reprogramming, disruption of photosynthesis, pH deregulation and ion imbalance. Numerous studies have described the way plant copes to ammonium nutrition. However, the organ developmental stage has been generally neglected.To fill in this gap, in the first chapter we first aimed studying how the metabolism is adapted in function of the leaf position in the vertical axis of the tomato plants (Solanum lycopersicum) grown with NH4+, NO3- or NO3NH4 supply. To do so, we dissected leaf biomass composition and metabolism through a complete analysis of metabolites, ions and enzyme activities. The results showed that C and N metabolic adjustment in function of the nitrogen source was more intense in older leaves compared to younger ones. Importantly, we propose a trade-off between NH4+ accumulation and assimilation to preserve young leaves from ammonium stress. Besides, NH4+-fed plants exhibited a rearrangement of carbon skeletons with a higher energy cost respect to plants supplied with NO3-. We explain such reallocation by the action of the biochemical pH-stat, to compensate the differential proton production that depends on the nitrogen form provided.Ammonium nutrition may limit cell expansion, suggesting that the cellular processes involved would be altered. Among others, cell growth is largely dependent of the internal pressure exerted on the cell wall by the vacuole. However, the role of the vacuole in ammonium stress has been rarely addressed. In the second chapter, we evaluated the effect of ammonium stress on leaf development with a special focus on vacuole expansion and metabolism. To carry out this aim, we monitored the leaf development from its appearance until its complete expansion in plants grown under NH4+ or NO3- as unique nitrogen source. Cytological analysis evidenced that the reduced cell expansion under ammonium nutrition was associated with smaller vacuole size. Besides, we reported an acidification of the vacuole of NH4+-fed plants compared to nitrate nutrition. Moreover, a model was built to predict the thermodynamic equilibrium of different soluble species across the tonoplast. The model was set up through an extensive reviewing of vacuolar transporters and integrated subcellular volumes, vacuolar electrochemical gradients and the formation of ionic complex in the vacuole to fit the subcellular concentration of ions, organic acids and sugars measured in the leaf. Further, predictions obtained with the model were cross validated with data from non-aqueous fractionation. Firstly, the entrance of solutes was higher in vacuoles of NO3--fed leaves but was not associated with higher vacuolar osmolarity likely because of the adjustment of the vacuolar volume. In this sense, we proposed that the lack of malate in cells of ammonium-fed leaves was central in the limitation of vacuolar expansion. Secondly, we conclude that the energy cost of solute transport into the vacuole is higher under NH4+ nutrition because of the higher electrochemical gradient generated by the proton pumps across tonoplast.This work highlights the importance of considering leaf phenological state when studying nitrogen metabolism. In addition, our integrated approach place cytosolic pH control and vacuole expansion in the center of tomato leaf adaptation to ammonium stress and pave the way for future studies in the field of ammonium nutrition

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Despite numerous attempts involving a variety of target genes, the successful transgenic manipulation of sucrose accumulation in sugarcane remains elusive. It is becoming increasingly apparent that enhancing sucrose storage in the culm by molecular means may depend on the modification of the activity of a novel gene target. One possible approach to identify target genes playing crucial coarse regulatory roles in sucrose accumulation is to assess gene expression during the developmental transition of the culm from active growth to maturation. This study has resulted in the successful optimisation of a mRNA hybridisation technique to characterise the expression of 90 carbohydrate metabolism-related genes in three developmentally distinct regions of sugarcane culm. A further goal of this work was to extend the limited knowledge of the regulation of sucrose metabolism in sugarcane, as well as to complement existing data from physiological and biochemical studies. Three mRNA populations derived from the different culm regions were assayed and their hybridisation intensities to the immobilised gene sequences statistically evaluated. The relative mRNA transcript abundance of 74 genes from three differing regions of culm maturity was documented. Genes exhibiting high relative expression in the culm included aldolase, hexokinase, cellulase, alcohol dehydrogenase and soluble acid invertase. Several genes (15) were demonstrated to have significantly different expression levels in the culm regions assessed. These included UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which were down-regulated between immature and mature internodes. Conversely, sucrose phosphate synthase, sucrose synthase and neutral invertase exhibited up-regulation in maturing internodal tissue. A variety of sugar transporters were also found to be up-regulated in mature culm, indicating a possible control point of flux into mature stem sink tissues. Combined with knowledge of the levels of key metabolites and metabolic intermediates this gene expression data will contribute to identifying key control points of sucrose accumulation in sugarcane and assist in the identification of gene targets for future manipulation by transgenic approaches.
AFRIKAANSE OPSOMMING: Ondanks verskeie pogings, waartydens verskeie gene geteiken is, is daar nog weinig sukses behaal om sukrose-akkumulering te verhoog. Toenemend wil dit voorkom asof suksesvolle genetiese manipulering van sukroseberging in die stingel van die verandering van ‘n nuwe geen afhanklik sal wees. Een van die moontlike benaderings wat gevolg kan word om potensiële teiken gene wat ‘n belangrike rol in die beheer van sukrose-opberging speel te identifiseer, is om geen uitdrukkingspatrone in die stingel tydens die omskakeling van aktiewe groei tot volwassenheid te karakteriseer. In hierdie studie is ‘n metode gebaseer op die hibridisering van mRNA geoptimiseer en suksesvol aangewend om die uitdrukkingspatrone van 90 verskillende geselekteerde gene, wat vir sleutelensieme in die beheer van koolhidraatmetabolisme kodeer, te bestudeer. Die doel met die ondersoek was om die beperkte kennis oor die regulering van koolhidraatmetabolisme uit te brei en om die bestaande inligting afgelei van fisiologiese en biochemiese-studies aan te vul. Drie verskillende mRNApopulasies, verkry uit verskillende dele van die stingel, is ontleed deur verskillende peilers te gebruik. Die gegewens is statisties ontleed en dit het afleidings oor die verandering in uitdrukking van hierdie gene moontlik gemaak. Die relatiewe konsentrasies van 74 verskillende gene is gedokumenteer. Gene wat sterk uitgedruk word het aldolase, heksokinase, sellulase, alkoholdehidrogenase en ongebonde suurinvertase ingesluit. Die uitdrukkingspatrone van 15 gene het tussen die verkillende weefsels gevarieer. Gene waarvan die uitdrukking tydens die oorgang na volwassenheid verlaag sluit in UDP-glukose pirofosforilase en UDP-glukose dehidrogenase en waarvan die uitdrukking verhoog sukrosefosfaatsintase, sukrosesintase en neutrale invertase in. Die uitdrukking van verskeie suikertransporter gene verhoog tydens volwassewording. Hierdie inligting te same met die huidige kennis oor heersende metabolietvlakke sal bydrae tot die identifisering van geenteikens vir toekomstige genetiese manupulering.

Dissertation presented to obtain a Ph. D. degree in Biochemistry by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica.
The human brain is the product of 600 million years of evolution and it is by far the most complex structure in the known universe. The vertebrates’ brain is composed of several different cell types, which perform the different functions required. The two most abundant cell types are neurons, which are the brain function unit, and glial cells, which are responsible for a myriad of housekeeping, homeostatic and structural functions. Glial metabolism is so far interconnected with the neuronal metabolism, that it is difficult to state where one finishes and the other starts. Trafficking between the two compartments include metabolites of almost all metabolic pathways (glycolysis, TCA, amino acids, ketone bodies, etc) and also ions.(...)
Fundação para a Ciência e a Tecnologia – PRAXIS XXI, for the financial support that made this doctoral work possible (PRAXIS XXI/BD/21532/99).

Metabolismus in Pflanzenzellen ist stark kompartimentiert. Viele Stoffwechselwege haben Reaktionen in mehr als einem Kompartiment. Zum Beispiel wird während der Photosynthese in pflanzlichen Mesophyllzellen Kohlenstoff in Form von Stärke in den Chloroplasten synthetisiert, während es im Zytosol in Form von Sacharose gebildet und in der Vakuole gespeichert wird. Diese Reaktionen sind strikt reguliert um ein Gleichgewicht der Kohlenstoffpools der verschiedenen Kompartimente aufrecht zu erhalten und die Energieversorgung aller Teile der Zelle für anabolische Reaktionen sicher zu stellen. Ich wende eine Methode an, bei der die Zellen unter nicht-wässrigen Bedingungen fraktioniert werden und daher der metabolische Status der während der Ernte herrschte über den ganzen Zeitraum der Auftrennung beibehalten wird. Durch die Kombination von nichtwässriger Fraktionierung und verschiedener Massenspektrometrietechniken (Flüssigchromotagraphie- und Gaschromotagraphie basierende Massenspekrometrie) ist es möglich die intrazelluläre Verteilung der meisten Intermediate des photosynthetischen Kohlenstoffstoffwechsels und der Produkte der nachgelagerten metabolischen Reaktionen zu bestimmen. Das Wissen über die in vivo Konzentrationen dieser Metabolite wurde genutzt um die Änderung der freien Gibbs Energie in vivo zu bestimmen. Mit Hilfe dessen kann bestimmt werden, welche Reaktion sich in einem Gleichgewichtszustand befinden und welche davon entfernt sind. Die Konzentration der Enzyme und der Km Werte wurden mit den Konzentrationen der Metabolite in vivo verglichen, um festzustellen, welche Enzyme substratlimitiert sind und somit sensitiv gegenüber Änderungen der Substratkonzentration sind. Verschiedene Intermediate des Calvin-Benson Zyklus sind gleichzeitig Substrate für andere Stoffwechselwege, als da wären Dihyroxyaceton-phosphat (DHAP, Saccharosesynthese), Fructose 6-phosphat (Fru6P, Stärkesynthese), Erythrose 4-phosphat (E4P, Shikimat Stoffwechselweg) und Ribose 5-phosphat (R5P, Nukleotidbiosynthese). Die Enzyme, die diese Intermediate verstoffwechseln, liegen an den Abzweigungspunkten zu diesen Stoffwechselwegen. Diese sind Trisose phosphat isomerase (DHAP), Transketolase (E4P), Sedoheptulose-1,7 biphosphat aldolase (E4P) und Ribose-5-phosphat isomerase (R5P), welche nicht mit ihren Substraten gesättigt sind, da die jeweilige Substratkonzentration geringer als der zugehörige Km Wert ist. Für metabolische Kontrolle bedeutet dies, dass diese Schritte am sensitivsten gegenüber Änderungen der Substratkonzentrationen sind. Im Gegensatz dazu sind die regulierten irreversiblen Schritte von Fructose-1,6.biphosphatase und Sedoheptulose-1,7-biphosphatase relativ insensitiv gegenüber Änderungen der Substratkonzentration. Für den Stoffwechselweg der Saccharosesynthese konnte gezeigt werden, dass die zytosolische Aldolase eine geringer Bindeseitenkonzentration als Substratkonzentration (DHAP) aufweist, und dass die Konzentration von Saccharose-6-phosphat geringer als der Km Wert des synthetisierenden Enzyms Saccharose-phosphatase ist. Sowohl die Saccharose-phosphat-synthase, also auch die Saccharose-phosphatase sind in vivo weit von einem Gleichgewichtszustand entfernt. In Wildtyp Arabidopsis thaliana Columbia-0 Blättern wurde der gesamte Pool von ADPGlc im Chloroplasten gefunden. Das Enzyme ADPGlc pyrophosphorylase ist im Chloroplasten lokalisiert und synthetisiert ADPGlc aus ATP und Glc1P. Dieses Verteilungsmuster spricht eindeutig gegen die Hypothese von Pozueta-Romero und Kollegen, dass ADPGlc im Zytosol durch ADP vermittelte Spaltung von Saccharose durch die Saccharose Synthase erzeugt wird. Basierend auf dieser Beobachtung und anderen veröffentlichten Ergebnissen wurde geschlußfolgert, dass der generell akzeptierte Stoffwechselweg der Stärkesynthese durch ADPGlc Produktion via ADPGlc pyrophosphorylase in den Chloroplasten korrekt ist, und die Hypothese des alternativen Stoffwechselweges unhaltbar ist. Innerhalb des Stoffwechselweges der Saccharosesynthsese wurde festgestellt, dass die Konzentration von ADPGlc geringer als der Km Wert des Stärkesynthase ist, was darauf hindeutet, dass das Enzym substratlimitiert ist. Eine generelle Beobachtung ist, dass viele Enzmye des Calvin-Benson Zyklus ähnliche Bindeseitenkonzentrationen wie Metabolitkonzentrationen aufweisen, wohingegen in den Synthesewegen von Saccharose und Stärke die Bindeseitenkonzentrationen der Enzyme viel geringer als die Metabolitkonzentrationen sind.
Metabolism in plant cells is highly compartmented, with many pathways involving reactions in more than one compartment. For example, during photosynthesis in leaf mesophyll cells, primary carbon fixation and starch synthesis take place in the chloroplast, whereas sucrose is synthesized in the cytosol and stored in the vacuole. These reactions are tightly regulated to keep a fine balance between the carbon pools of the different compartments and to fulfil the energy needs of the organelles. I applied a technique which fractionates the cells under non-aqueous conditions, whereby the metabolic state is frozen at the time of harvest and held in stasis throughout the fractionation procedure. With the combination of non-aqueous fractionation and mass spectrometry based metabolite measurements (LC-MS/MS, GC-MS) it was possible to investigate the intracellular distributions of the intermediates of photosynthetic carbon metabolism and its products in subsequent metabolic reactions. With the knowledge about the in vivo concentrations of these metabolites under steady state photosynthesis conditions it was possible to calculate the mass action ratio and change in Gibbs free energy in vivo for each reaction in the pathway, to determine which reactions are near equilibrium and which are far removed from equilibrium. The Km value and concentration of each enzyme were compared with the concentrations of its substrates in vivo to assess which reactions are substrate limited and so sensitive to changes in substrate concentration. Several intermediates of the Calvin-Benson cycle are substrates for other pathways, including dihydroxyacetone-phosphate (DHAP,sucrose synthesis), fructose 6-phosphate (Fru6P, starch synthesis), erythrose 4-phosphate (E4P,shikimate pathway) and ribose 5-phosphate (R5P, nucleotide synthesis). Several of the enzymes that metabolise these intermediates, and so lie at branch points in the pathway, are triose-phosphate isomerase (DHAP), transketolase (E4P, Fru6P), sedoheptulose-1,7-bisphosphate aldolase (E4P) and ribose-5-phosphate isomerase (R5P) are not saturated with their respective substrate as the metabolite concentration is lower than the respective Km value. In terms of metabolic control these are the steps that are most sensitive to changes in substrate availability, while the regulated irreversible reactions of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase are relatively insensitive to changes in the concentrations of their substrates. In the pathway of sucrose synthesis it was shown that the concentration of the catalytic binding site of the cytosolic aldolase is lower than the substrate concentration of DHAP, and that the concentration of Suc6P is lower than the Km of sucrose-phosphatase for this substrate. Both the sucrose-phosphate synthase and sucrose-phosphatase reactions are far removed from equilibrium in vivo. In wild type A. thaliana Columbia-0 leaves, all of the ADPGlc was found to be localised in the chloroplasts. ADPglucose pyrophosphorylase is localised to the chloroplast and synthesises ADPGlc from ATP and Glc1P. This distribution argues strongly against the hypothesis proposed by Pozueta-Romero and colleagues that ADPGlc for starch synthesis is produced in the cytosol via ADP-mediated cleavage of sucrose by sucrose synthase. Based on this observation and other published data it was concluded that the generally accepted pathway of starch synthesis from ADPGlc produced by ADPglucose pyrophosphorylase in the chloroplasts is correct, and that the alternative pathway is untenable. Within the pathway of starch synthesis the concentration of ADPGlc was found to be well below the Km value of starch synthase for ADPGlc, indicating that the enzyme is substrate limited. A general finding in the comparison of the Calvin-Benson cycle with the synthesis pathways of sucrose and starch is that many enzymes in the Calvin Benson cycle have active binding site concentrations that are close to the metabolite concentrations, while for nearly all enzymes in the synthesis pathways the active binding site concentrations are much lower than the metabolite concentrations.

Although it has been known for almost 90 years that cancer cells metabolize glucose differently than normal cells, the reason for this difference has been poorly understood and controversial. Unlike healthy cells, which completely oxidize most of their glucose to carbon dioxide, cancer cells metabolize most of their glucose to lactate even in the presence of oxygen. Paradoxically, despite cancer proliferation needing more energy, this metabolism generates less ATP per glucose. This phenomenon, also known as the Warburg Effect, is influenced by the differential isoform expression of pyruvate kinase (PK). PK catalyzes the final step of glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. While many differentiated cells express the M1 isoform (PKM1), cancer cells and other rapidly dividing tissues express the M2 isoform (PKM2). PKM2 has regulatable and overall less activity than PKM1, but it remains unclear how PKM2 benefits rapid proliferation. To study the importance of PKM2 in rapidly dividing cells, we examined the changes to proliferation and metabolism that occur following deletion of PKM2 in primary cells derived from mice harboring a PKM2 conditional allele. We find that acute deletion of PKM2 results in expression of PKM1 and a concomitant arrest in proliferation that is independent of senescence induction and cell death. Co-expression of PKM1 and PKM2 mimics the effect of expressing PKM1 alone, suggesting that gain of PKM1, not loss of PKM2, is responsible for the arrest. Slowed proliferation after exposure of cells to small molecule activators of PKM2 further argues that gain of pyruvate kinase activity is responsible for proliferation arrest. Metabolic flux analysis using U-13C-glucose and mass spectrometry reveals that PKM1 expression reduces flux to specific biosynthetic pathways and increases flux to the TCA cycle. Consistently, U-14C-glucose labeling of CO2 reveals increased glucose oxidation in PKM1-expressing MEFs. Exogenously supplied deoxyribonucleosides were able to rescue the proliferation arrest of PKM1-expressing MEFs. These data suggest that in comparison to PKM1, PKM2 expression promotes anabolism in proliferating cells by favoring metabolic flux that supports nucleotide biosynthesis.

Historically, whole stream open channel metabolism has been measured over short periods in conjunction with nutrient injections to assess nutrient dynamics within streams. The purpose of my study was to understand the seasonal changes in metabolism within and among streams as well as the impacts of different land use. This was addressed by monitoring nine different watersheds in the Little Tennessee River watershed in southwestern North Carolina.  The nine study watersheds were selected to represent a gradient of forested, agricultural, and developed land use / land cover types. Data loggers were deployed to collect continuous oxygen, temperature, conductivity, and stage height data from 2010-2011. I used these data to estimate gross primary production (GPP) and ecosystem respiration (ER). GPP and ER were compared to stream chemistry, light, land cover, and storms. I found that there is greater influence of local riparian land cover than watershed land cover on GPP and ER. Streams had varying annual GPP, but generally the peak in GPP occurred in late winter- early spring with lows in fall. GPP was most strongly influenced by the amount of available light, which is directly related to the amount of canopy cover. ER was much more variable than GPP within and among streams but generally peaked in summer and was lowest in the winter. ER was most strongly related to the proportion of agricultural land cover in the local riparian area. My results suggest that local riparian vegetation may have a greater impact on metabolism than mountainside development.
Master of Science

L'acide myristique (c14 : 0) est present en faible quantite dans les tissus animaux (1% des acides gras) mais relativement abondant dans la matiere grasse laitiere (7-12%). Le metabolisme et les fonctions de l'acide myristique sont mal connus. Ce travail a consiste en une etude metabolique et fonctionnelle de l'acide myristique dans l'hepatocyte de rat en culture primaire. Nos resultats montrent que l'acide myristique d'origine alimentaire est la source majeure de cet acide gras dans le foie : la biosynthese hepatique d'acide myristique a partir d'acetyl-coa et par retroconversion d'acide palmitique est faible. L'etude du metabolisme des acides myristique, palmitique et laurique exogenes indique que ces acides gras n'ont pas le meme devenir dans la cellule : leur captage, leur incorporation dans les lipides, leur niveau de -oxydation, leurs vitesses d'elongation et de desaturation sont differents. L'acide myristique possede un avenir court dans la cellule : il est a la fois rapidement -oxyde et rapidement allonge en acide palmitique. De plus, un apport en acide myristique n'induit pas la production de cholesterol. Ces donnees ne vont pas dans le sens d'un effet negatif de l'acide myristique alimentaire, par rapport aux autres acides gras satures, sur les aspects etudies. Ce travail amene egalement des informations originales sur les fonctions de l'acide myristique. Dans l'hepatocyte, l'acide myristique se lie de facon covalente a plusieurs proteines. Nous montrons la myristoylation n-terminale (liaison amide) de la sous-unite d'une proteine g heterotrimerique, et l'acylation laterale (liaison thioester ou ester) de proteines du cytosquelette (actine, cytokeratine) et de petites proteines g. L'acide myristique exogene pourrait aussi etre le precurseur de l'acide palmitique qui est utilise pour l'acylation de ces proteines. L'acide myristique aurait donc un role essentiel dans l'activation de certaines proteines et dans la regulation de plusieurs fonctions cellulaires.

Objetivo: Determinar las variables clínicas asociadas al deterioro del metabolismo glucémico en población nomoglucémica con factores de riesgo para la diabetes mellitus tipo 2 (DM2) o prediabéticos. Material y métodos: Se diseñan 3 cohortes de seguimiento, con pacientes de las consultas de atención primaria: normoglucémicos con factores de riesgo de DM2, prediabéticos, y pacientes con DM2 de reciente diagnóstico, llamados diabéticos. Se analizan los antecedentes personales y antropométricos, antecedentes familiares, hábitos tóxicos, actividad física, síntomas y complicaciones relacionadas con la diabetes y datos analíticos. Resultados: 545 pacientes, evaluados al inicio, a los 2 y 5 años de seguimiento. Edad media 60±12 años, IMC 30,1±5,1, HTA 48,6%, HbA1c media normoglucémicos, 4,8%(4,8-5), prediabéticos 5,2%(4,9-5,3%) diabéticos 6,5% (5,6-8,5%). A los 5 años el 17,1% de los normoglucémicos son prediabéticos y el 22,6% de los prediabéticos son diabéticos. En los normoglucémicos se ha relacionado el consumo fármacos diabetogénicos, especialmente los diuréticos, con una incidencia aumentada de prediabetes a los 5 años (OR 24,6 95%; 6,25-96,8). Los niveles elevados de colesterol a los 5 años también se ha relacionado entre los normoglucémicos con la incidencia de prediabetes (OR 4,0 IC95%; 1,13-14,2). En los prediabéticos, la incidencia de diabetes, se relaciona a los 2 años con la elevación de los triglicéridos (OR 7,6 IC95%;1,1-62) y la edad ≥ 60 años, (OR 4,6 IC 95%; 1,47-14,7), y a los 5 años, con la elevación de colesterol total (OR 4,6 IC95%; 1,3-16,1) y de triglicéridos (OR 3,7 IC95%; 1,23-11,2). Conclusiones: La incidencia de prediabetes entre los normoglucémicos fue del 17%, de DM2 entre los prediabéticos del 22,6% a los 5 años del seguimiento. Los factores identificados relacionados con la progresión a DM2 han sido la edad ≥60 años, colesterol y triglicéridos elevados. El consumo de fármacos, sobre todo diuréticos, también se ha relacionado con la evolución a DM2.
Objective: To determine the clinical variables associated with a deterioration in glycemic metabolism in normoglycemic sample with risk factors for diabetes mellitus type 2 (DM2) or who are prediabetic. Methods: Three follow-up cohorts with patients in primary care consultations were designed: normoglycemic patients with risk factors for DM2, prediabetic patients and newly diagnosed DM2 patients. Personal and anthropometric history, family history, toxic habits, physical activity, symptoms and complications related to diabetes and laboratory data were analyzed. Results: 545 patients, assessed at baseline, and 2 and 5 years follow-up. Mean age 60 ± 12 years, BMI 30.1 ± 5.1, 48.6 % hypertension , average in HbA1c normoglycemic patients, 4.8 % ( 4.8 to 5 ), in prediabetic patients 5.2% ( from 4.9 to 5, 3%) and in diabetic patients 6.5% ( 5.6 to 8.5 %). At 5 years follow-up, 17.1% of normoglycemic patients became prediabetics and 22.6 % of prediabetic patients became diabetic. In normoglycemic patients, the consumption of diabetogenic drugs, especially diuretics , was linked with an increased incidence of prediabetes at 5 years (OR 24.6 , 95% CI , 6.25 to 96.8). At 5 years, elevated levels of cholesterol was among normoglycemic related to the incidence of pre-diabetes (OR 4.0 95% CI, 1.13 to 14.2 ) . In prediabetic patients, the incidence of diabetes at 2 years of follow-up was association with elevated triglycerides levels (OR 7.6 95% CI, 1.1 to 52 ) and age ≥ 60 years (OR 4.6 95% CI: 1.47 to 14.7 ), and at 5 yearswas association with elevated total cholesterol levels (OR 4.6 95% CI: 1.3 to 16.1 ) and hight triglycerides levels (OR 3.7 95% CI ; 1, 23 to 11.2 ) . Conclusions: At 5 years follow up, the incidence of prediabetes among normoglycemic patients was 17% and the incidence of DM2 among prediabetic was 22.6%. The identified factors associated with progression to DM2 were age > 60 years, elevated cholesterol and triglycerides. The consumption of drugs, especially diuretics, has also been associated with progression to DM2 .

The products of the five-membered PRS gene family in S. cerevisiae exist as three entities, Prs1/Prs3, Prs2/Prs5 and Prs4/Prs5, each capable of supporting cell viability. The signal generated from an integrated GFP-Prs1 construct is dependent on the presence of Prs3. Prs1 not only interacts with Prs3 but also with Slt2, the MAPK of the Cell Wall Integrity (CWI) pathway as shown by co-immunoprecipitation and confirming previous Y2H studies that the central region of Prs1, NHR1-1, is essential for the interaction with the CWI pathway. Point mutations in PRS1 corresponding to missense mutations associated with human neuropathies or in the divalent cation- and/or PRPP-binding sites interfere with CWI signalling resulting in temperature and/or caffeine sensitivity. The synthetic lethality of a strain lacking PRS1 and PRS5 can only be rescued by versions of Prs1 containing NHR1-1, implying that impaired CWI contributes to this loss of viability. Prs5 is unusual in that it contains two NHRs, one, NHR5-2, contains three phosphorylation sites. Mutation of these amino acid residues impinges on the expression of the transcription factor, Rlm1, an endpoint of the CWI pathway and reduces the temperature-dependent transcription of FKS2, the gene encoding the stress-induced catalytic subunit of 1,3-β-glucan synthase essential for maintaining CWI. This finding emphasizes the functionality of the three phosphorylation sites located in NHR5-2 of Prs5. The data presented support the hypothesis that the PRS gene family has, as a result of gene duplication and acquisition of NHRs, evolved to link primary metabolism with CWI.

Les plantes sont des organismes sessiles. Elles doivent réagir rapidement et efficacement aux stress biotiques et abiotiques qu’elles subissent. Pour cela, elles utilisent plusieurs niveaux de régulation. L’un d’eux, rapide et réversible, consiste a effectuer des modifications post-traductionnelles (PTMs) sur ses enzymes. La PTM la plus répandue est la phosphorylation protéique, qui intervient dans diverses voies du métabolisme primaire. La glycolyse permet la production d’énergie (ATP) et de pouvoir réducteur à partir de glucose. La régulation de la phosphoglycérate mutase d’Arabidopsis thaliana (AtiPGAM) a été étudiée grâce à une analyse d’un site de phosphorylation dans le but d’élucider le mécanisme réactionnel. La photorespiration est un processus essentiel pour les organismes photosynthétiques. Ce cycle, initié par l’activité oxygénase de la ribulose-1,5-biphosphate carboxylase / oxygénase (RuBisCO), produit notamment une molécule de 2-phosphoglycolate (2-PG), toxique pour la plante. Le recyclage, couteux, du 2-PG par le cycle photorespiratoire se déroule dans quatre compartiments (chloroplaste, peroxysome, mitochondrie et cytosol). Sept des huit enzymes du cycle photorespiratoire sont phosphorylables. La phosphoglycolate phosphatase (AtPGLP1), première enzyme du cycle, est associée à quatre phosphosites. Des approches in vitro et in planta développées chez A. thaliana ont permis d’acquérir de nouvelles données sur la régulation post-traductionnelle de cette protéine, à la fois par phosphorylation et par oxydo-réduction
As sessile organisms, plants need to rapidly and effectively react to environmental abiotic and biotic stresses. To do so, various regulatory mechanisms exist that include post-translational modifications (PTMs) of proteins. One of the most prevalent PTM is protein phosphorylation that has been shown to occur in many metabolic pathways. Glycolysis allows the production of energy (as ATP) and reducing power from glucose. In this context, the regulation of Arabidopsis thaliana phosphoglycerate mutase (AtiPGAM) was studied by analysing a phosphorylation site potentially involved in the reaction mechanism of this glycolytic enzyme. The photorespiratory cycle is a major metabolic pathway occurring in all photosynthetic organisms. It is initiated by the oxygenase activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and leads to the production of toxic 2-phosphoglycolate (2-PG) molecules. The costly recycling of 2-PG by the photorespiratory cycle takes place in four different compartments (chloroplast, peroxisome, mitochondrion and cytosol). Seven of the eight core photorespiratory enzymes appear to be phosphorylated. Phosphoglycolate phosphatase (AtPGLP1), the first enzyme of the cycle that metabolizes 2-PG to glycolate, is associated with four phosphosites. In vivo and in vitro approaches using Arabidopsis thaliana have allowed us to obtain further insights into the post-translational regulation of this protein by protein phosphorylation and by oxidation-reduction

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Ecologia
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Lagos costeiros são ambientes que apresentam características particulares quanto a sua dinâmica, estado trófico, morfologia e interação das comunidades biológicas. Em geral, são ambientes rasos submetidos à intensa ação dos ventos. A lagoa do Peri localizada na ilha de Santa Catarina (27°44#S e 48°31#W) é um sistema costeiro subtropical de água doce com 5,7 km2 de área e 4,2 m de profundidade média. De acordo com a literatura, apresenta homogeneidade horizontal e vertical para clorofila a e nutrientes, característica oligotrófica para a concentração de nutrientes e presença de cianobactérias. Com o objetivo de verificar a ocorrência de autotrofia e heterotrofia na lagoa do Peri, determinaram-se as taxas de produção primária (bruta e líquida) e de respiração fitoplanctônica através de incubações em garrafas claras e escuras durante 24 horas no outono, inverno, primavera e verão. Incubações em garrafas claras e escuras, também foram conduzidas bimensalmente, em quatro profundidades em uma estação central da lagoa, para avaliar a variação vertical das taxas de produção primária (bruta e líquida) e de respiração bem como sua relação com a comunidade fitoplanctônica. Condições de heterotrofia foram encontradas no outono, quando as maiores taxas de respiração ocorreram devido ao aumento da disponibilidade de matéria orgânica e fósforo total. Condições de autotrofia ocorreram no verão devido às maiores taxas de produção primária líquida, favorecida pelas maiores temperaturas e luz. As taxas de produção primária, respiração e a densidade dos grupos fitoplanctônicos, em geral, não apresentaram variação vertical, em função da homogeneização promovida pelos ventos incidentes na lagoa do Peri. Condições limitantes de luz e nutrientes, para o crescimento fitoplanctônico, foram os fatores atribuídos as menores taxas de produção primária encontradas neste ambiente em relação a outros ambientes tropicais e subtropicais. A densidade fitoplanctônica e condições de temperatura e luz favoreceram a produção primária na primavera e no verão. Cyanobacteria e Chlorophyta foram os grupos mais importantes da comunidade fitoplanctônica, em termos de densidade e diversidade, com destaque para Cylindrospermopsis raciborskii que dominou na maior parte do período de estudo. Assim, a lagoa do Peri apresentou alternância entre períodos autotróficos e heterotróficos em função das diferentes condições ambientais e as taxas de produção primária foram menores que em outros ambientes, variando em função das variáveis bióticas e abióticas.
Coastal lakes are environments with distinct characteristics when it comes to dynamics, trophic state, morphology, and the interaction of biological communities. They are generally shallow environments subjected to intense winds. Peri lagoon located on the island of Santa Catarina (27°44'S and 48°31'W) is a subtropical coastal system of freshwater with an area of 5.7 km2 and average depth of 4.2 m. According to literature, it is horizontally and vertically homogenous for chlorophyll a and nutrients, oligotrophic characteristics for nutrient concentration and presence of Cyanobacteria. In order to verify the occurrence of autotrophy and heterotrophy in the Peri lagoon, primary production (gross and net) and respiration rates were determined by incubating in light and dark bottles over a period of 24 hours in the fall, winter, spring and summer. Incubations in light and dark bottles were also conducted every two months, at four depths in the central part of the lagoon, in order to evaluate the vertical variation in primary production rates (gross and net) and respiration and the relationship to the phytoplankton community. Heterotrophic conditions were found in autumn, where the highest rates of respiration were found due to an increased availability of organic matter and total phosphorus. Autotrophic conditions occurred in the summer due to higher rates of net primary production, favored by higher temperatures and light availability. In general, the primary production rates, respiration and phytoplankton group density, did not show vertical variation due to the homogenization promoted by winds in the Peri lagoon. Light and nutrients limitation were attributed to the lower rates of primary production found in this environment than other ones. However, phytoplankton density, temperature and light conditions favored the primary production rates in spring and summer. Cyanobacteria and Chlorophyta were the most important groups of the phytoplankton community in terms of density and diversity, especially Cylindrospermopsis raciborskii, which was dominant during the majority of the study period. Thus, the Peri lagoon had alternating autotrophic and heterotrophic periods due to different environmental conditions and the primary production rates were lower than other tropical environments, depending on biotic and abiotic variables.

Plants employ diverse defense mechanisms to combat attack by harmful organisms. For instance, plants produce constitutive physical barriers or use chemical compounds such as specialized secondary metabolites to resist herbivore or pathogen invasion. Considering the cost-efficiency and energy balance between defense, growth and reproduction, defense reactions in plants have to be regulated temporally and spatially. As more cost-efficient strategies, plants may induce their defense response only in the presence of the attacker or restrict constitutive defenses to specific tissues or cells. In this study, we investigated aspects of the spatial regulation and induced changes of primary and secondary metabolism in roots of Arabidopsis thaliana. Roots represent important organs for anchoring plants in the soil and taking up water and nutrients. Hence, it is assumed that roots are as well protected as aerial tissues by different defense mechanisms. The first part of this work is focused on the cell-type-specific biosynthesis of volatile terpenes in Arabidopsis roots. Terpenes are the most abundant specialized metabolites in plants and play an important role in plant defense against pathogens or herbivores. Terpene biosynthetic enzyme activities are often coordinated in specific tissues and cellular compartments. Fine-scale transcriptome maps of Arabidopsis roots have shown that terpene biosynthesis is restricted to particular cell types. However, the reasons and significance of this cell-type specificity are not well understood. We hypothesized that the formation of terpene metabolites is not restricted to specific cells but can be supported by different cell types. We, therefore, probed the plasticity of the cell-specific formation of terpenes by swapping the expression of the terpene synthase (TPS) genes, TPS08, TPS13 and TPS25, between different root cell types in the respective mutant background. To investigate the ectopic expression of TPSs at different levels, quantitative real-time PCR (qRT-PCR), confocal microscopy, and gas chromatography-mass spectrometry (GC-MS) were performed. We found that terpene synthase TPS08, which produces the diterpene rhizathalene and is normally expressed in the root vascular tissue, is functionally active when expressed in the epidermis or cortex, although at substantially lower levels compared to the wild type. We did not find an obvious correlation between the volatile emission level and gene transcript level of TPS08, which may be attributed to a reduced activity of the expressed TPS08-yellow fluorescent protein (YFP) fusion protein. When expression of TPS13 (producing the sesquiterpene (Z)-"-bisabolene) was directed from the cortex to the epidermis or stele, TPS13 gene expression and (Z)-"-bisabolene formation was supported by these cell types although to varying levels in comparison to wild type. TPS13-YFP fluorescent signal driven by the epidermal WER and GL3 promoters was primarily detected at the root tip. Terpene production was also observed for the (E)-"-farnesene sesquiterpene synthase TPS25 when its expression was switched from the endodermis and non-hair producing epidermal cells to hair producing epidermal cells although only a weak fluorescent signal was detected from the expressed TPS25-mGFP protein. Together, the results provide preliminary evidence for a relaxed cell specificity of terpene biosynthesis in Arabidopsis roots and suggest that tissue-specific terpene metabolite patterns could change depending on different selective pressures in rhizosphere. In the second part of this study, we performed global gene transcript profiling and primary metabolite analysis of Arabidopsis roots upon feeding by the generalist root herbivore, Bradysia (fungus gnat). In a microarray analysis, we identified 451 of 22,810 genes that were up-regulated more than 2-fold. Gene ontology (GO) analysis showed that 26% of those genes with predicted or known functions play a role in primary or secondary metabolism, while 24% are involved in cell signaling or in responses to stimulating factors, such as jasmonic acid (JA), ethylene, wounding, and oxidative stress. At the metabolite level, we observed only marginal changes of amino acid, sugar and carboxylic acid relative levels over a time course of 4 days of Bradysia feeding. There was a trend for increased levels of amino acids and the relative levels of sucrose were increased significantly ("=0.05) at the fourth day of feeding. In conclusion, the study provided evidence for the induction of genes related to primary and secondary metabolism and stress responses in Arabidopsis roots, but showed only marginal changes at the primary metabolite level. In addition, the work indicated that the formation of terpene-specialized metabolites in Arabidopsis roots is not restricted to specific cells, but can be supported by different cell types.
Master of Science

Lors de lesions nerveuses par retrait de co::(2), l'etude du metabolisme des lipides a montre qu'un traitement a la cdp-choline ne modifie pas la distribution et la synthese des phospholipides et des acides gras mais provoque par contre une diminution de la liberation d'acides gras. La cdp-choline diminue l'activite phospholiposique a1 au niveau des membranes et protege donc celles-ci de la degradation

Spatial heterogeneity in ecosystem metabolism may play a critical role in determining ecosystem functions. Variation in ecosystem metabolism between macrophyte patches in shallow wetlands at the extremes of freshwater habitats has not been investigated. We estimated ecosystem metabolism in mesocosms containing different macrophytes using 24-hour oxygen curves to test our hypotheses: (1) net aquatic production (NAP) during spring and summer would be similar among algal patches (metaphyton and Chara), (2) NAP in algal patches would be greater than patches dominated by the vascular plant Potamogeton foliosus, (3) heterotrophy and anaerobiosis would be greatest in patches dominated by Lemna, and (4) the pond would be autotrophic in the spring and fall but heterotrophic in the summer. We found that different patches generated differences in NAP but not always as we predicted. NAP was different among algal patches in the spring and summer, and only metaphyton was more heterotrophic than P. foliosus. In the summer Chara and Lemna patches were heterotrophic and metaphyton became autotrophic. As predicted, the pond was net autotrophic in the spring and heterotrophic in the summer with an absence of patchiness in fall attributed to the dominance of Lemna. This research suggests the importance of macrophyte patchiness in wetlands in determining patterns of ecosystem metabolism despite challenges in measuring 24 hour oxygen curves (e.g. oxygen supersaturation). Consequently, macrophyte traits may be important in determining spatial heterogeneity of ecosystem metabolism in shallow ponds.

Orientador: Ariovaldo Pereira da Cruz Neto
Banca: Denis Otavio Vieira de Andrade
Banca: Luiz Antonio Martinelli
Resumo: Nós estimamos a contribuição relativa de frutos e insetos como fonte de proteína na dieta de duas espécies de morcegos frugívoros (Artibeus lituratus e Carollia perspicillata) mediante o uso de isótopos estáveis de carbono (13C) e nitrogênio (15N). A contribuição de insetos como fonte de proteína na dieta foi predominante em ambas às espécies. Em A. lituratus, a contribuição de insetos foi de 95% na área fragmentada, e 75% na área contínua. No entanto, em C. perspicilllata, a contribuição de insetos foi de 85% na área fragmentada e 98% na área contínua. O nível trófico foi significativamente diferente entre A. lituratus e C. perspicilllata. A pesar da diferencia entre as duas espécies, os mesmos são muito semelhantes entre si, indicando que ambas as espécies se localizam na mesma posição trófica ao longo da cadeia alimentar. Para cada espécie estudada foram testados quinze modelos matemáticos concorrentes para determinar se a taxa metabólica basal (TMB) é explicada pela massa do corpo, nível trófico, produtividade primária liquida e temperatura do ambiente. Em A. lituratus, quatro modelos foram matematicamente plausíveis em explicar a taxa metabólica basal, dois desses modelos explicaram TMB em termos de produtividade primária liquida, e nível trófico. Em C. perspicillata, dois modelos explicaram a TMB, um desses modelos explicaram TMB em termos de massa do corpo, produtividade primária liquida, e temperatura ambiental. Nossos resultados parecem suportar a hipóteses do habito alimentar em A. lituratus e C. perspicillata
Abstract: We estimated the relative contribution of fruits and insects as protein source in the diet of two species of frugivorous bats (Artibeus lituratus and Carollia perspicillata) through the use of stable isotopes of carbon (13C) and nitrogen (15N). The contribution of insects as protein source in the diet was prevalent in both species. In A. lituratus, the contribution of insects was 95 percent in fragmented area, and 75 percent in continuous area. However, in C. perspicilllata, the contribution of insects was 85 percent in fragmented area, and 98 percent in continuous area. In spite of differences in the trophic level between A. lituratus and C. perspicilllata, they are very similar to each other, indicating that both species are found in the same trophic position along the food chain. For each species have been tested 15 mathematical models to determine whether basal metabolic rate (BMR) is explained by body mass, trophic level, net primary productivity and temperature. In A. lituratus, four models were mathematically plausible in explaining the basal metabolic rate, two of these models explained TMB in terms of net primary productivity, and trophic level. In C. perspicillata, two models explained the TMB, one of these models explained TMB in terms of body mass, net primary productivity, and ambient temperature. Our result seems to support the food habits hypotheses in A. lituratus and C. perspicilllata
Mestre

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Nós estimamos a contribuição relativa de frutos e insetos como fonte de proteína na dieta de duas espécies de morcegos frugívoros (Artibeus lituratus e Carollia perspicillata) mediante o uso de isótopos estáveis de carbono (13C) e nitrogênio (15N). A contribuição de insetos como fonte de proteína na dieta foi predominante em ambas às espécies. Em A. lituratus, a contribuição de insetos foi de 95% na área fragmentada, e 75% na área contínua. No entanto, em C. perspicilllata, a contribuição de insetos foi de 85% na área fragmentada e 98% na área contínua. O nível trófico foi significativamente diferente entre A. lituratus e C. perspicilllata. A pesar da diferencia entre as duas espécies, os mesmos são muito semelhantes entre si, indicando que ambas as espécies se localizam na mesma posição trófica ao longo da cadeia alimentar. Para cada espécie estudada foram testados quinze modelos matemáticos concorrentes para determinar se a taxa metabólica basal (TMB) é explicada pela massa do corpo, nível trófico, produtividade primária liquida e temperatura do ambiente. Em A. lituratus, quatro modelos foram matematicamente plausíveis em explicar a taxa metabólica basal, dois desses modelos explicaram TMB em termos de produtividade primária liquida, e nível trófico. Em C. perspicillata, dois modelos explicaram a TMB, um desses modelos explicaram TMB em termos de massa do corpo, produtividade primária liquida, e temperatura ambiental. Nossos resultados parecem suportar a hipóteses do habito alimentar em A. lituratus e C. perspicillata
We estimated the relative contribution of fruits and insects as protein source in the diet of two species of frugivorous bats (Artibeus lituratus and Carollia perspicillata) through the use of stable isotopes of carbon (13C) and nitrogen (15N). The contribution of insects as protein source in the diet was prevalent in both species. In A. lituratus, the contribution of insects was 95 percent in fragmented area, and 75 percent in continuous area. However, in C. perspicilllata, the contribution of insects was 85 percent in fragmented area, and 98 percent in continuous area. In spite of differences in the trophic level between A. lituratus and C. perspicilllata, they are very similar to each other, indicating that both species are found in the same trophic position along the food chain. For each species have been tested 15 mathematical models to determine whether basal metabolic rate (BMR) is explained by body mass, trophic level, net primary productivity and temperature. In A. lituratus, four models were mathematically plausible in explaining the basal metabolic rate, two of these models explained TMB in terms of net primary productivity, and trophic level. In C. perspicillata, two models explained the TMB, one of these models explained TMB in terms of body mass, net primary productivity, and ambient temperature. Our result seems to support the food habits hypotheses in A. lituratus and C. perspicilllata
FAPESP: 11/08959-0
CNPq: 130941/2011-5

Primary hyperparathyroidism (pHPT), characterized by elevated serum levels of calcium and parathyroid hormone (PTH), is associated with a number of metabolic derangements causing secondary manifestations. These include osteoporosis and increased risk of fractures, but also risk factors for cardiovascular morbidity and mortality. These risk factors include impaired glucose tolerance (IGT), dyslipidemia, increased body mass index and hypertension. While the skeletal abnormalities are mainly due to elevated PTH, the latter disturbances are still unexplained. Non-insulin dependent diabetes mellitus (NIDDM), IGT, dyslipidemia and hypertension are all included in the metabolic syndrome, also associated with morbidity and mortality in cardiovascular diseases.

In this thesis, decreased bone mineral density (BMD) and variables of the metabolic syndrome are explored in patients with mild and normocalcemic pHPT before and after parathyroidectomy. To further investigate the relationship between insulin sensitivity and calcium, a community-based cohort was investigated.

In two different patient cohorts of pHPT, lipoprotein alterations with decreased levels of HDL-cholesterol and elevated triglycerides were found in association with a high frequency of IGT, NIDDM and decreased insulin sensitivity. Parathyroidectomy had effects on the dyslipidemia and in part on the glucose metabolism. The disturbed glucose metabolism in pHPT was substantiated by results from the general population by a negative association between insulin sensitivity, measured by hyperinsulinemic clamp, and serum calcium.

In conclusion, normocalcemic, mild and overt pHPT are associated with a range of risk factors for cardiovascular diseases, development of NIDDM and decreased BMD in cortical as well as trabecular bone. These findings explain, at least in part, the elevated morbidity and mortality from cardiovascular disease as well as fractures, reported in pHPT patients. Moreover, in the general population, serum calcium is associated with decreased insulin sensitivity. Parathyroidectomy has positive effects on several, but not all, of the investigated metabolic parameters.

Dans le cadre de la promotion d'une viticulture durable, le développement d'alternatives écologiques à l’utilisation de pesticides de synthèse pour le contrôle des maladies de la vigne (Vitis vinifera) gagne en importance. Une des méthodes de bio-contrôle proposée est l'induction de l'immunité des plantes par l’application de substances biodégradables, non toxiques pour la santé et l'environnement, appelées éliciteurs ou stimulateurs de défense des plantes (SDP). La résistance conférée contre divers agents pathogènes peut être obtenue grâce à l’emploi de molécules utilisant le plus fréquemment les voies de signalisation de l'acide jasmonique (JA), de l'acide salicylique (SA) et/ou de l'éthylène (ET). De telles voies peuvent déclencher l'induction de gènes liés à la défense tels ceux codant des enzymes responsables de la biosynthèse des stilbènes, des métabolites phénoliques antimicrobiens parmi les plus importants des Vitacées. Au vignoble, pour contrôler les maladies, les éliciteurs peuvent être appliqués en complément des pesticides de synthèse et non en remplacement total car leur efficacité est souvent variable selon les agents pathogènes et les conditions environnementales. Afin de développer leur utilisation, des études supplémentaires qui pourraient notamment élucider leur mécanisme d'action sont nécessaires. L’objectif de cette thèse était d'étudier les réponses de la vigne à des éliciteurs de différents modes d'action, comme le jasmonate de méthyle (MeJA), impliqué dans la voie de signalisation du JA, l’acide S-méthyl ester 2,1,3-benzothiadiazole-7-carbothioïque (BTH), un analogue synthétique du SA, des phosphonates (PHOS), molécules à double action stimulateur-fongicide. Le profil des stéroïdes et des triterpénoïdes pentacycliques a été caractérisé par des analyses en chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS). Dans un premier temps, l'effet éliciteur du MeJA a été évalué dans des suspensions cellulaires (in vitro) de plusieurs cultivars de V. vinifera. Une surproduction de triterpénoïdes pentacycliques a été observée avec des différences selon la variété considérée : induction de l’accumulation de la bétuline et de l'acide oléanolique ou des phytostérols pour le Petit Verdot, le Gamay Teinturier) et le Cabernet Sauvignon, respectivement. Puis les élicitations ont été effectuées au niveau des feuilles de boutures de serre de V. vinifera cv. Cabernet Sauvignon. Un effet stimulant sur les triterpénoïdes pentacycliques liés à la défense a été démontré au détriment de la biosynthèse des stérols, composants structurels essentiels des membranes cellulaires. Par l’utilisation de puces NeoVigen et la chromatographie liquide à ultra haute performance couplée à la spectrométrie de masse (UHPLC-MS), l’induction de l’expression de gènes liés à la défense et l'accumulation de polyphénols (stilbènes, flavanols et flavonols) ont été notées suite aux trois traitements éliciteurs. La protection de la vigne conférée par les éliciteurs a été confirmée par des biotests sur disques foliaires inoculés par l’oomycète biotrophe Plasmopara viticola, l’agent responsable du mildiou. Par ailleurs, il est important d’avoir connaissance de l’impact des éliciteurs sur le métabolisme général afin d'obtenir l’effet optimal entre croissance, rendement et défense. Ainsi, une approche métabolomique utilisant la spectroscopie de résonance magnétique nucléaire du proton (RMN 1H) a été menée. Une reprogrammation similaire et/ou spécifique selon l'éliciteur considéré a été notée en particulier au niveau des glucides, des acides aminés et de certains intermédiaires du cycle de Krebs. Les recherches présentées dans cette thèse, démontrent que la compréhension approfondie de l'interaction entre l'éliciteur, les réponses moléculaires et métaboliques de la plante et le pathogène, est cruciale pour le développement de stratégies de protection efficaces basées sur l'utilisation des SDP pour contrôler les maladies de la vigne
In the frame of promoting sustainable vitiviniculture, the development of eco-friendly alternatives to synthetic chemical products for phytosanitary treatments against grapevine (Vitis vinifera) pests is gaining importance. One of the bio-control methods that can be proposed is the induction of plant immunity by using elicitors, also called plant defense stimulators (PDS), as these substances are biodegradable and, non-toxic to health and environment. A conferred resistance against various pathogens can be obtained with natural molecules acting most frequently through jasmonic acid (JA), salicylic acid (SA), and/or ethylene (ET) signaling pathways. These pathways are involved in the induction of defense-related genes such as those encoding enzymes responsible for the biosynthesis of stilbenes, which are the most important polyphenolic antimicrobial metabolites (phytoalexins) in Vitaceae. For vineyard protection, PDS can be applied as a complement for pesticides and not as a full replacement since their effectiveness is often variable according to pathogens and environmental conditions. In order to develop the strategies based on PDS use, more studies which could elucidate their mechanism of action are needed. The aim of this thesis was to examine the responses of grapevine to elicitors of different mode of action, as methyl jasmonate (MeJA), implicated in JA signaling pathway, 2,1,3-benzothiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic analogue of SA, and phosphonates (PHOS), molecules of a double stimulator-fungicide action. Due to scarce information about steroids and pentacyclic triterpenoids in grapevine, their profile after PDS treatment were characterized in different grapevine experimental models using gas chromatography-mass spectrometry (GC-MS) analyses. Firstly, the effect of elicitation with MeJA was evaluated in cell suspension cultures (in vitro) of V. vinifera. An overproduction of bioactive pentacyclic triterpenoids occurred with differences according to the cultivar studied, i.e., acumulation of betulin and oleanolic acid or phytosterols was noted in respectively Petit Verdot, Gamay Teinturier and Cabernet Sauvignon cell suspension cultures. Then, elicitations were effectuated on the leaves of V. vinifera cv. Cabernet Sauvignon greenhouse cuttings. A stimulatory effect on the potentially defense-related pentacyclic triterpenoids at the expense of the biosynthesis of sterols, which are essential structural components of cell membranes, was shown. By the use of NeoVigen microarrays, and ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS), the accumulation of defense-related transcripts and polyphenols (stilbenes, flavanols and flavonols) were noted after the three elicitors treatments. Grapevine protection conferred by these elicitors was confirmed on foliar discs against the biotrophic oomycete Plasmopara viticola, the causal agent of downy mildew. Furthermore, the impact of PDS on primary metabolism should be evaluated in order to ensure, in the longer term, the best trade-off between growth, yield and defense. Thus, a thorough metabolomic approach using proton nuclear magnetic resonance spectroscopy (1H-NMR) was performed. A reprogramming similar and/or specific to the elicitor applied was noted, particularly within carbohydrates, amino acids, and some of the Krebs cycle intermediates. The research presented in the current dissertation revealed that the thorough comprehension of the interaction between elicitor, plant molecular and metabolic responses and pathogen, is crucial for the development of effective protection strategies based on the use of PDS for grapevine diseases control

The trophic state of lakes is commonly defined by the concentration of nutrients in the water column. High nutrient concentrations generate high phytoplankton production, and lakes with low nutrient concentrations are considered low-productive. This simplified view of lake productivity ignores the fact that benthic primary producers and heterotrophic bacteria can be important basal producers in lake ecosystems. In this thesis I have studied clear-water and brown-water lakes with respect to primary production, respiration and bacterial production based on allochthonous organic carbon. These processes were quantified in pelagic and benthic habitats on temporal and spatial scales. I also calculated the net ecosystem production of the lakes, defined as the difference between gross primary production (GPP) and respiration (R). The net ecosystem production indicates whether a lake is net heterotrophic (GPP < R), net autotrophic (GPP > R) or in metabolic balance (GPP = R). Net heterotrophic lakes are sources of carbon dioxide (CO2) to the atmosphere since respiration in these lakes, by definition, is subsidized by an external organic carbon source. External organic carbon is transported to lakes from the terrestrial environment via inlets, and can serve as a carbon source for bacteria but it also limits light availability for primary producers by absorbing light. On a seasonal scale, four of the clear-water lakes studied in this thesis were dominated by primary production in the soft-bottom benthic habitat and by respiration in the pelagic habitat. Concentrations of dissolved organic carbon (DOC) were low in the lakes, but still high enough to cause the lakes to be net heterotrophic. However, the lakes were not low-productive due to the high production in the benthic habitat. One of the clear-water lakes was studied also during the winter and much of the respiration under ice was supported by the benthic primary production from the previous summer. This is in contrast to brown-water lakes where winter respiration is suggested to be supported by allochthonous organic carbon. By studying lakes in a DOC gradient (i.e. from clear-water to brown-water lakes) I could draw two major conclusions. The lakes became less productive since benthic primary production decreased with increasing light extinction, and the lakes became larger sources of CO2 to the atmosphere since pelagic respiration was subsidized by allochthonous organic carbon. Thus, lake carbon metabolism can have an important role in the global carbon cycle due to their processing of terrestrial organic carbon and to their possible feedback effects on the climate system.

O equilíbrio ecológico dos ecossistemas aquáticos é fundamental para a conservação da biodiversidade e para que tais ambientes possam desempenhar seus serviços ambientais. O metabolismo dos riachos pode ser utilizado como um indicador funcional do nível de perturbação do ambiente, devido à influência das condições da bacia hidrográfica sobre as características e estrutura dos ecossistemas aquáticos. A presente pesquisa avaliou a variação espaço-temporal das taxas de produção primária bruta (PPB) e respiração (R) em riachos localizados no Cerrado, como subsídio para o entendimento da influência de algumas características da água, do substrato e da vegetação ripária sobre o seu metabolismo. Para isso, foram estudados seis riachos em ambientes subtropicais (São Carlos e Brotas, SP, Brasil) com diferentes condições ambientais. O metabolismo foi modelado pelas mudanças de curto prazo nas concentrações de oxigênio dissolvido (OD) e da radiação solar fotossinteticamente ativa, a cada 10 min em períodos de 24h. Além disso, a modelagem considerou o coeficiente de reaeração em cada riacho, que foi estimado por meio de traçador gasoso (SF6). Foram determinadas variáveis de qualidade da água, hidrológicas, do substrato e porcentagem de cobertura vegetal. Modelos de regressão foram utilizados, entre outras análises estatísticas, para avaliar as principais variáveis preditoras das taxas metabólicas. Os riachos apresentaram vazões sempre inferiores a 100 L.s-1, e a porcentagem de cobertura vegetal variou entre 39 e 86%. As concentrações médias de nitrogênio e fósforo totais variaram entre 0,8-1,3 mg.L-1 e 9,4-42,4 μg.L-1, respectivamente, sendo os riachos classificados entre oligo e mesotróficos. A amplitude média das variações diárias de OD foi de 0,2 a 1,2 mg.L-1. As taxas de PPB apresentaram variação de 0,01 a 0,68 gO2 m-2.dia-1. Já as taxas de R variaram entre 0,61-42,08 gO2 m-2.dia-1. A partir do balanço entre a PPB e a R, os seis riachos apresentaram condições heterotróficas (respiração excedeu a produção primária bruta, com a produção primária líquida negativa), o que torna esses ambientes mais vulneráveis ao eventual aporte de cargas orgânicas que gerem demanda por oxigênio. Embora as concentrações de fósforo tenham sido correlacionadas com as taxas metabólicas, as principais variáveis preditoras, pelos modelos de regressão, foram vazão e a porcentagem de cobertura vegetal. A cobertura vegetal provavelmente foi responsável pela redução da incidência de radiação solar e, consequentemente, o principal limitante da disponibilidade de luz subaquática para algas bentônicas. A vazão apresentou possível efeito na redução da biomassa de algas pela abrasão decorrente da velocidade da água. Espera-se que, além de gerarem um conjunto de dados sobre ambientes aquáticos de um bioma ainda relativamente pouco estudado, o Cerrado, as informações apresentadas sobre os principais fatores intervenientes nos processos metabólicos dos riachos possam oferecer direcionamentos a projetos para sua conservação e para a manutenção de seus serviços ambientais.
The ecological balance of aquatic ecosystems is fundamental for biodiversity conservation and for to perform the environmental services. Stream metabolism can be used as a functional indicator of the level of disturbance of the environment due to the influence of watershed conditions on the characteristics and structure of aquatic ecosystems. The present study evaluated the spatial-temporal variation of gross primary production (GPP) and ecosystem respiration (R) rates, in streams located in the Cerrado, as to understanding the influence of water and the substrate characteristics and vegetation in metabolism. For this, six subtropical streams (São Carlos and Brotas, SP, Brazil) were studied with different environmental conditions. The metabolism was modeled by the short-term changes in the concentrations of dissolved oxygen (DO) and the photosynthetically active solar radiation, every 10 min in periods of 24h. In addition, the modeling considers the reaeration rates in each stream, which was estimated by gas tracer (SF6). Variables of water quality, substrate, hydrological characteristics, and percentage of canopy cover were determined. Regression models were used, among other statistical analyzes, to evaluate the main predictors of metabolic rates. The streams had discharge rates lower than 100 L.s-1, and the percentage of canopy cover varied between 39 and 86%. The mean concentrations of nitrogen and phosphorus varied between 0.8-1.3 mg.L-1 and 9.4-42.4 μg.L-1, respectively, and the streams were classified as oligo and mesotrophic. The mean amplitude of daily DO variations was 0.2 to 1.2 mg.L-1. Rates of GPP ranged from 0.01 to 0.68 gO2 m-2.day-1. R rates varied from 0.61 to 42.08 gO2 m-2.day-1. From the balance between PPB and R, the six streams presented heterotrophic conditions (respiration exceeded gross primary production, with negative net primary production), which makes these streams more vulnerable to the eventual contribution of organic loads that generate demand for oxygen. Although phosphorus concentrations were correlated with metabolic rates, the main predictors of metabolic rates, by regression models, were discharge and canopy cover percentage. The canopy cover was probably responsible for reducing the incidence of solar radiation and, consequently, the main limitation of the availability of underwater light for benthic algae. The discharge indicated a possible effect on the algae biomass reduction due to the abrasion caused by the water velocity. It is expected that, in addition to the generation of data set on aquatic environments of the relatively little studied biome, the Cerrado, the information presented about the main drivers in the metabolic processes of the streams can offer directions to projects for its conservation and for the support of their environmental services.

Diel dissolved oxygen (DO) data were used to characterize seasonal, inter-annual, and longitudinal variation in production and respiration for the James River Estuary. Two computational methods (Bayesian and bookkeeping) were applied to these data to determine whether inferences regarding DO metabolism are sensitive to methodology. Net metabolism was sensitive to methodology as Bayesian results indicated net heterotrophy (production < respiration) while bookkeeping results indicated net autotrophy (production > respiration). Differences in net metabolism among the methods was due to low seasonal variation in respiration using the Bayesian method, whereas bookkeeping results showed a strong correlation between production and respiration. Bayesian results suggest a dependence on allochthonous organic matter (OM) whereas bookkeeping results suggest that metabolism is dependent on autochthonous OM. This study highlights the importance in considering the method used to derive metabolic estimates as it can impact the assessment of trophic status and sources of OM supporting an estuary.