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Das Enzym Glutaminsynthetase (GS) wird in Organen mit niedriger enzymatischer Aktivität in zumeist allen Zellen exprimiert. Auf der anderen Seite ist die Expression in Geweben mit hoher Aktivität auf spezialisierte Zellen beschränkt. So findet man in der Säugerleber Expression der GS nur in Hepatozyten, die in ein bis drei Zellreihen um die Zentralvenen lokalisiert sind. In der vorliegenden Arbeit wurde die Frage gestellt, ob der zwischen verschiedenen Spezies hoch konservierte 3’-Bereich der nicht-translatierten Region des GS-Gens an der Regulation der Expression und der Zonierung beteiligt ist. Hierzu wurden Reportergenstudien, transiente Transfektionen sowie Northern-Blot-Experimente unter Verwendung von primären Hepatozyten aus dem periportalen und perizentralen Bereich der Rattenleber durchgeführt. Die Ergebnisse der Arbeit lassen eine über das 3’-Ende vermittelte selektive Destabilisierung der GS-mRNA in periportalen (GS-negativen) Hepatozyten vermuten. Zudem zeigte sich, dass die Wechselwirkung des 3’-UTRs mit Bereichen des 5’-UTRs, bzw. dem GS-Promotor für die eigentliche Regulation verantwortlich ist. Es lässt sich vermuten, dass eine posttranskriptionale Regulation neben den in den letzten Jahren aufgeklärten Mechanismen der Regulation der Transkription mit zur Feinsteuerung der Expression der GS beiträgt.

Sept accessions représentant la diversité génétique du Sorgho (Sorghum bicolor) ont été cultivées dans une condition de carence (N⁻) et une condition non-limitante (N⁺) en nitrate. Les paramètres de croissance (taille de la plante et le nombre de feuilles), les paramètres physiologiques (teneur en nitrate, teneur en protéines, concentrations totales en carbone et azote) et l'activité de la glutamine synthétase (GS) ont été mesurés dans les feuilles et les racines des plantes de sorgho à trois stades précoces de développement végétatif (2, 4 et 6 semaines après germination). Les résultats montrent que : i) les valeurs obtenues pour l’ensemble des paramètres sont généralement plus faibles en situation de carence en nitrate ; ii) la taille des plantes et le nombre de feuilles sont plus grands sous un régime non-limitant ; iii) tous les paramètres étudiés sauf la teneur en Carbone, étaient sensibles à la disponibilité en azote. Cependant, les différents génotypes étudiés affichent de grandes variations dans leurs réponses aux deux conditions de cultures. On observe une variation de la taille des plantes entre les génotypes au cours du développement végétatif précoce, mais pas pour le nombre de feuilles. De même, on observe une grande variation dans les réponses physiologiques entre les différents génotypes. Des corrélations fortes et significatives ont été détectées entre la taille des plantes et la teneur en nitrate. Cependant ces corrélations varient selon les conditions de cultures et les génotypes étudiés. En outre, la teneur en nitrate et l'activité GS mesurés aux stades précoces de croissance, semblent être de bons marqueurs pour discriminer entre les différents génotypes pour leur aptitude à absorber ou assimiler les nitrates dans les deux conditions de cultures. L’expression dans les feuilles et racines de deux génotypes de sorgho, de deux copies d’un gène candidat pour l’efficacité d’utilisation d’azote, SbNRT1.1 codant pour un transporteur de nitrate, varie en fonction de la disponibilité en nitrate, de l’organe et de l’âge de la plante. Notre étude constitue une première contribution à l’analyse de l’efficacité d’utilisation d’azote chez le sorgho par une approche physiologique et une approche génétique. Les résultats obtenus ouvrent des perspectives pour de futures études fondamentales mais aussi des recherches finalisées qui conduiront à l’identification de génotypes valorisant mieux l’azote. / Seven accessions of Sorghum bicolor were grown with low (N⁻) and optimal (N⁺) nitrate supply. Growth parameters (plant height and leaf numbers), physiological parameters (nitrate, protein, total N and total C contents) and the activity of glutamine synthetase (GS) were studied in leaves and roots of sorghum plants at three time points of early vegetative growth (2, 4 and, 6 weeks post emergence). Plant height and leaf number were higher with nitrate supply. Except for carbon, all studied parameters were sensitive to N availability and values were typically lower when nitrate supply was low. However, different genotypes displayed considerable variation in their response to N regimes. Variation among genotypes during early vegetative development was observed for plant height, but not for leaf number. Likewise, physiological parameters varied among accessions. A significant and strong correlation, N- and accession-dependent, was detected between plant height and nitrate content. Moreover, nitrate content and GS activity at early growth stages appeared to be good markers to discriminate between nitrate uptake and assimilation capacities of different accessions under both N conditions. In some sorghum accessions, protein and total N content were indicative of high nitrate reduction and assimilation even under N limitation. Chlorophyll content was also sensitive to N availability. Furthermore, expression studies of SbNRT1.1gene copies in leaves and roots of two accessions reflected variability in expression dependent on nitrogen condition, plant organ, plant age, and gene of interest. This study is helpful to characterize different aspects of the N metabolism in sorghum and may aid in the identification of sorghum genotypes with enhanced nitrogen use efficiency, a trait that is of key interest in one of the most important crop plants in arid and semi-arid regions.

A síndrome de McCune Albrigth (SMA) é uma doença genética não hereditária, com incidência estimada entre 1/100.000 e 1/1.000.000 casos/ano. A SMA caracteriza-se clinicamente pela tríade: displasia óssea fibrosa (FD), manchas cutâneas café-com-leite e hiperfunção endócrina tais como: síndrome de Cushing, pseudo-puberdade precoce, hipertiroidismo, acromegalia. O diagnóstico da SMA clássica é usualmente baseado no quadro clínico associado a dosagens hormonais e exames de imagem, principalmente cintilografia do esqueleto. No entanto, quadros atípicos e formas parciais muitas vezes dificultam o diagnóstico preciso da síndrome. O objetivo deste estudo foi padronizar dentre as técnicas de PNA (peptide nucleic acid) e PCT em Tempo Real, para a detecção de polimorfismos de base única (SNPs), a técnica mais sensível para a discriminação das mutações ativadoras da subunidade da proteína G. Para este estudo foram selecionados 32 pacientes, 1 masculino e 31 femininos, com SMA, todos em seguimento no Hospital das Clínicas da Faculdade de Medicina da USP. Como resultado positivo, apresentamos nesse trabalho pela primeira vez o uso do RT-PCR genotipagem na detecção das mutações ativadoras da proteína G, em DNA extraído de tecidos afetados e em leucócitos de sangue periférico, sendo a técnica considerada sensível o suficiente para discriminar de forma simples e rápida as mutações ativadoras da PGs. Sugerimos nesse estudo o uso da técnica de discriminação alélica pelo sistema Taqman. Essa técnica possibilita a detecção destas mutações gsp no sangue periférico mesmo numa baixa porcentagem, uma vez que nem sempre o tecido afetado (gônada, osso, hipófise) é disponível. / The McCune-Albright Syndrome (MAS) is a genetic disease, with incidence estimated at 1/100.000 and 1/1000000 cases per year. MAS is clinically characterized by the triad: bone fibrous dysplasia (FD) café-au-lait skin spots and endocrine hyperfunction, such as: precocious puberty (PP), Cushing's syndrome, hyperthyroidism and acromegaly. The diagnosis of MAS is originally based on clinical characteristics associated with hormonal and imaging studies. However, atypical and partial forms often hamper the accurate diagnosis of the syndrome. For this study we selected 32 patients, 1male and 31 females, all being treated in Hospital das Clínicas, School of Medicine, University of São Paulo. As a positive result, we showed for the first time the use of Real Time PCR/genotyping for the detection of activating mutations of the stimulatory G protein, using blood leucocytes DNA. This technique was sensible and can bring fast results for the patient and the physician, making the diagnosis easier. Our study proposes the use of allelic discrimination by Taqman system, which can be used as a probe that allows the identification of specific genotypes. These techniques could help detect these mutations in peripheral blood when the affected tissue is not available.

A síndrome de McCune Albrigth (SMA) é uma doença genética não hereditária, com incidência estimada entre 1/100.000 e 1/1.000.000 casos/ano. A SMA caracteriza-se clinicamente pela tríade: displasia óssea fibrosa (FD), manchas cutâneas café-com-leite e hiperfunção endócrina tais como: síndrome de Cushing, pseudo-puberdade precoce, hipertiroidismo, acromegalia. O diagnóstico da SMA clássica é usualmente baseado no quadro clínico associado a dosagens hormonais e exames de imagem, principalmente cintilografia do esqueleto. No entanto, quadros atípicos e formas parciais muitas vezes dificultam o diagnóstico preciso da síndrome. O objetivo deste estudo foi padronizar dentre as técnicas de PNA (peptide nucleic acid) e PCT em Tempo Real, para a detecção de polimorfismos de base única (SNPs), a técnica mais sensível para a discriminação das mutações ativadoras da subunidade da proteína G. Para este estudo foram selecionados 32 pacientes, 1 masculino e 31 femininos, com SMA, todos em seguimento no Hospital das Clínicas da Faculdade de Medicina da USP. Como resultado positivo, apresentamos nesse trabalho pela primeira vez o uso do RT-PCR genotipagem na detecção das mutações ativadoras da proteína G, em DNA extraído de tecidos afetados e em leucócitos de sangue periférico, sendo a técnica considerada sensível o suficiente para discriminar de forma simples e rápida as mutações ativadoras da PGs. Sugerimos nesse estudo o uso da técnica de discriminação alélica pelo sistema Taqman. Essa técnica possibilita a detecção destas mutações gsp no sangue periférico mesmo numa baixa porcentagem, uma vez que nem sempre o tecido afetado (gônada, osso, hipófise) é disponível. / The McCune-Albright Syndrome (MAS) is a genetic disease, with incidence estimated at 1/100.000 and 1/1000000 cases per year. MAS is clinically characterized by the triad: bone fibrous dysplasia (FD) café-au-lait skin spots and endocrine hyperfunction, such as: precocious puberty (PP), Cushing's syndrome, hyperthyroidism and acromegaly. The diagnosis of MAS is originally based on clinical characteristics associated with hormonal and imaging studies. However, atypical and partial forms often hamper the accurate diagnosis of the syndrome. For this study we selected 32 patients, 1male and 31 females, all being treated in Hospital das Clínicas, School of Medicine, University of São Paulo. As a positive result, we showed for the first time the use of Real Time PCR/genotyping for the detection of activating mutations of the stimulatory G protein, using blood leucocytes DNA. This technique was sensible and can bring fast results for the patient and the physician, making the diagnosis easier. Our study proposes the use of allelic discrimination by Taqman system, which can be used as a probe that allows the identification of specific genotypes. These techniques could help detect these mutations in peripheral blood when the affected tissue is not available.

Chapter 2 The sea surface microlayer is a biogenic thin layer, comprising less than one millimeter of the ocean surface. This surface layer has gained much attention due to its dampening effect on ocean capillary ripples. The chemistry of the air-sea interface has been studied for decades; however, the structure and function of the marine bacterial community within the sea surface microlayer are still understudied. Although various sea surface microlayer sampling techniques were developed over the past decades, aseptic bacterial sampling in the open ocean is a rather challenging task. In this study, a new approach is presented. It is designed for bacterial sampling of the sea surface microlayer, which intends to reduce sampling contamination from the vessel, subsurface water and the investigators. A 47mm polycarbonate membrane was utilized at each sampling site. In addition, the metagenomic approach using the new generation 454 high-throughput DNA sequencing system was employed to compensate for the small sample size. Two sample sets were collected in summer 2010 and fall 2011 from the sea surface microlayer and underlying water (20 cm deep). A contamination assessment was carried out to determine that contamination might have been caused during the use of the sampling techniques. A total of 14,120 bacterial 16S rRNA gene sequences with an average length of 437.8 bp were obtained. A total of 1,254 Operational Taxonomic Units (OTUs) were constructed and 268 genera were identified. The results indicated that the bacterial compositions of the sea surface microlayer samples were distinct from those of the underlying water samples. This experiment demonstrated that the new generation sequencing platform and microbial metagenomics analysis software together served as powerful tools to gain a deeper understanding of microbial communities within the sea surface microlayer. Furthermore, it is suggested that the newly employed sampling methods could be used to obtain a snapshot of bacterial community structure as well as environmental conditions. Chapter 3 Synthetic aperture radar (SAR) remote sensing captures various fine-scale features on the ocean surface such as coastal discharge, oil pollution, vessel traffic, algal blooms and sea slicks. Although numerous factors potentially affect the SAR imaging process, the influence of biogenic and anthropogenic surfactants has been suggested as one of the primary parameters, especially under relatively low wind conditions. Surfactants have a tendency to dampen the short gravity-capillary ocean waves causing the sea surface to smoothen, thus allowing the radar to detect areas of surfactants. Surfactants are found in sea slicks, which are the accumulation of organic material shaped as elongated bands on the ocean’s surface. Sea slicks are often observable with the naked eye due to their glassy appearance and can also be seen on SAR images as dark scars. While the sources of surfactants can vary, some are known to be associated with marine bacteria. Countless numbers of marine bacteria are present in the oceanic environment, and their biogeochemical contributions cannot be overlooked. Not only do marine bacteria produce surfactants, but they also play an important role in the transformation of surfactants. In this study, we profiled the surfactant-associated bacteria composition within the biogenic thin layer of the ocean surface more commonly referred as the sea surface microlayer (SML). Bacterial samples were collected from the SML for comparative analysis from both within and outside of sea slick areas as well as the respective underlying subsurface water. The bacterial microlayer sampling coincided with SAR satellite, RADARSAT-2, overpasses to demonstrate the simultaneous in-situ measurements during a satellite image capture. The SML sampling method was designed to enable aseptic bacterial sampling. A 47 mm polycarbonate membrane was utilized at each sampling site to obtain a snapshot of the bacterial community structure at a specific space and time. Also, a new generation high-throughput sequencing method was employed to compensate for the small sample size acquired. A total of 27,006 nucleotide sequences (16S rRNA genes) with an average 437.8 bp in length were analyzed. The results revealed the presence of industrially important surfactant-producing marine bacteria, Acinetobacter, Bacillus, Corynebacterium and surfactant-degrading marine bacteria, Escherichia. In addition, Pseudomonas was detected which can be either a producer, decomposer or both. Recognizing that there is still a large number of marine bacterial species that have not been taxonomically classified nor recognized as surfactant-associated species, the effects on SAR imaging due to a high number of surfactant-associated marine bacteria is expected. This study has provided the basis for the biological importance for fine-scale synthetic aperture satellite imaging. Moreover, this new approach is expected to have applications in monitoring biological and chemical properties of the sea surface across the globe.

Das Enzym Glutaminsynthetase (GS) wird in Organen mit niedriger enzymatischer Aktivität in zumeist allen Zellen exprimiert. Auf der anderen Seite ist die Expression in Geweben mit hoher Aktivität auf spezialisierte Zellen beschränkt. So findet man in der Säugerleber Expression der GS nur in Hepatozyten, die in ein bis drei Zellreihen um die Zentralvenen lokalisiert sind. In der vorliegenden Arbeit wurde die Frage gestellt, ob der zwischen verschiedenen Spezies hoch konservierte 3’-Bereich der nicht-translatierten Region des GS-Gens an der Regulation der Expression und der Zonierung beteiligt ist. Hierzu wurden Reportergenstudien, transiente Transfektionen sowie Northern-Blot-Experimente unter Verwendung von primären Hepatozyten aus dem periportalen und perizentralen Bereich der Rattenleber durchgeführt. Die Ergebnisse der Arbeit lassen eine über das 3’-Ende vermittelte selektive Destabilisierung der GS-mRNA in periportalen (GS-negativen) Hepatozyten vermuten. Zudem zeigte sich, dass die Wechselwirkung des 3’-UTRs mit Bereichen des 5’-UTRs, bzw. dem GS-Promotor für die eigentliche Regulation verantwortlich ist. Es lässt sich vermuten, dass eine posttranskriptionale Regulation neben den in den letzten Jahren aufgeklärten Mechanismen der Regulation der Transkription mit zur Feinsteuerung der Expression der GS beiträgt.

L’épuisement professionnel est un problème de santé psychologique qui constitue un phénomène grandissant dans la population générale. La prévention de ce trouble devrait constituer une priorité dans les organisations et pourrait être améliorée par une meilleure identification des facteurs de risque en jeu. Bien qu’il existe des facteurs de risque organisationnels et individuels de l’épuisement professionnel, seuls les premiers faisaient jusqu’ici l’objet d’un questionnaire validé servant à les mesurer. La présente étude a effectué la validation d’un questionnaire mesurant les facteurs de risque individuels de l’épuisement professionnel : le Style personnel au travail (SPT). La cohérence interne, la validité factorielle ainsi que la validité convergente ont été vérifiées. Ce dernier type de validité a été examiné en comparant le questionnaire SPT à une mesure de l’épuisement professionnel, le Maslach Burnout Inventory - General Survey (MBI-GS) ainsi qu’à une mesure des facteurs reliés à la personnalité normale, le Sixteen Personality Factor Questionnaire (16PF). L’étude visait également à déterminer si le questionnaire SPT était complet, c’est-à-dire s’il semblait comprendre l’ensemble des principaux facteurs de risque individuels de l’épuisement professionnel. Un échantillon constitué de 230 travailleurs à temps complet issus de différentes organisations a répondu à trois questionnaires en format électronique : le SPT, le MBI-GS et le 16PF. Une analyse de fidélité par bissection, une analyse en composantes principales, des corrélations ainsi que des régressions multiples hiérarchiques ont été conduites afin d'atteindre les objectifs formulés. Les résultats appuient d’abord la bonne cohérence interne du questionnaire SPT et soutiennent la structure factorielle en six facteurs. Ils fournissent également plusieurs indices quant à la validité convergente du questionnaire en mettant en évidence plusieurs liens corrélationnels entre les facteurs du SPT et ceux du MBI-GS, d’une part, et du 16PF, d’autre part. Les résultats permettent finalement de considérer l’ajout potentiel d’un facteur de risque, issu du 16PF, au questionnaire SPT : la Stabilité émotionnelle. Les avantages et inconvénients d’un tel ajout sont discutés. La présente étude comporte certaines limites, concernant entre autres la taille et la composition de l’échantillon ainsi que la méthode utilisée. Néanmoins, les retombées de cette recherche sont importantes. En effet, l’étude fournit plusieurs indices quant à la validité du questionnaire SPT, qui permet désormais de mesurer les facteurs de risque individuels de l’épuisement professionnel conjointement avec l’AMT, un questionnaire validé mesurant les facteurs de risque organisationnels du trouble. Il est ainsi possible d’obtenir, en une seule passation, un portrait plus complet des principaux facteurs de risque reliés à l’épuisement professionnel. Ce portrait permettra également le développement d’outils de prévention efficaces et ciblés, dans le but de réduire l’incidence de ce trouble chez les travailleurs.

The metabolism of plants grown in the cultivation medium is influenced by its composition and availability of CO2. In this project the effect of cultivation medium on the activity phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-malic enzyme (NADP-ME, EC 1.1.1.40), pyruvate, phosphate dikinase (PPDK, EC 2.7.9.1) and enzymes of nitrogen metabolism: nitrate reductase (NR, EC 1.7.1.1), glutamine synthetase (GS, EC 6.3.1.2), glutamate synthase (GOGAT, EC 1.4.1.13) and glutamate dehydrogenase (GDH, EC 1.4.1.2) was studied. The tobacco plant Nicotiana tabacum L., cv. Petit Havana SR1 were grown in vitro in containers fitted with a filter, which caused limited access of CO2. The cultivation medium was modified Murashige-Skoog agar with decreased amounts of phosphate, nitrate, ammonium, or with casein as source of nitrogen and with or without of 1.5% sucrose as additional carbon source Activity of PEPC was higher in plants grown in medium in the presence of sucrose. Reduced concentrations of phosphates, nitrates or NH4 + ions or when casein was the only source of N in medium caused decreased activity of PEPC. Under these conditions, also activity of NADP-ME and of enzymes of nitrogen metabolism: NR, GS decreased. Activity of all enzymes was also negatively affected by limited CO2. On the other...

El medio interestelar (MIE) no sólo no es homogéneo sino que además posee una compleja topología que se manifiesta por la presencia de una variedad de estructuras tales como arcos, gusanos, cáscaras y supercáscaras. En particular, las supercáscaras se encuentran entre los objetos más enigmáticos e interesantes del MIE de una galaxia. Las mismas se detectan mayoritariamente en la emisión de la distribución de hidrógeno neutro (HI) como mínimos en la emisión de HI rodeados, total o parcialmente, por ”paredes” de mayor emisión. Las supercáscaras pueden expandirse a velocidades de varias decenas de kilómetros por segundo. Con tiempos dinámicos de vida del orden de decenas de millones de años, las supercáscaras sobrevivirían a las estrellas de gran masa que pudieran haberle dado origen (si ese hubiese sido el mecanismo que las originó), por lo que las mismas podrían ser usadas como registros fósiles para estudiar los efectos de formación estelar en la Vía Láctea. En esta Tesis se ha realizado un nuevo catálogo de candidatas a supercáscaras utilizando una combinación de un método automático de detección más uno visual. Una particularidad que tiene nuestro algoritmo de búsqueda automática es que es capaz de detectar estructuras que no están completamente cerradas, o sea, que no están completamente rodeadas por paredes de emisión de HI. Este hecho permitió estudiar el porcentaje de estructuras que tienen su lado ”abierto” hacia al halo de la Galaxia, lo cual convertiría a estas estructuras en candidatas a objetos identificados como ”chimeneas” galácticas. Se han detectado un total de 575 estructuras en la parte externa de la Galaxia a las cuales se les han determinado algunos parámetros físicos como por ejemplo: las distancias, dimensiones, edades dinámicas, velocidades radiales de los centroides, rangos de velocidades donde se detectan. A cada estructura se le ajustó una elipse la cual tiene como parámetros los semiejes mayor y menor y el ángulo de inclinación del semieje mayor con respecto al plano de la Galaxia. Se ha determinado la distribución de las supercáscaras en la Vía Láctea, así como también las principales propiedades estadísticas de los parámetros (tamaños lineales, velocidades de expansión, distancias galactocéntricas, dimensiones, edades dinámicas) encontrados para las mismas. Se ha comparado el catálogo obtenido con catálogos similares realizados por otros autores. También se ha analizado la posible presencia, en el interior de las estructuras, de objetos estelares que pudieron haberle dado origen. Por último se estudió en detalle una de las estructuras catalogadas, GS 100-02-41, la cual presenta evidencia de formación estelar inducida.