Cette thèse aborde un point clé du couplage entre ces cycles: la dynamique des molécules azotées (AAs) des matières organiques du sol (MOS). Par des expériences d'incubation, nous avons estimé que les flux de biosynthèse des AAs par les micro-organismes du sol lors du processus de décomposition sont de l'ordre de 25% de la biomasse nouvellement formée. Le profil des AAs individuels biosynthétisés de novo est plus dépendant du type de sol que de la nature du substrat. Dans chaque sol, il est très similaire à celui des AAs des MOS. La biodégradation de matériaux végétaux marqués en 13C a révélé la transformation rapide des protéines végétales en matériaux microbiens. Ces résultats montrent que les AAs des MOS sont d'origine microbienne. Nous avons mesuré le renouvellement du C des AAs à long terme dans les horizons de surface de neuf sites présentant des végétations, climats et types de sol variés, en utilisant la technique de traçage par les abondances naturelles en 13C. L'âge moyen du carbone des AAs varie de 50 à 200 ans. Un modèle simple permet de discuter les hypothèses du recyclage des AAs des MOS par les micro-organismes. Les rapports isotopiques stables des AAs individuels ont été mesurés par chromatographie en phase gazeuse couplée à la spectrométrie de masse isotopique. À cette fin, nous avons développé une méthode d'étalonnage générique pour la détermination du rapport isotopique des composés spécifiques, par analyse de cultures microbiennes uniformément marquées. Au-delà des résultats présentés, l'étude apporte un large ensemble de données des AAs et examine les variations de l'abondance naturelle en 13C entre les AAs individuels. / We analyzed the coupled dynamics of C and N in Soil Organic Matter (SOM) through the dynamics of N-containing soil organic compounds (amino acids (AAs)) by tracing their carbon atoms. Stable isotope ratios of individual amino acids were measured by gas chromatography coupled with isotope ratio mass spectrometry. For this purpose, we developed a generic calibration method for compound-specific stable isotope ratio analysis, based on the analysis of uniformly labelled microbial cultures. We quantified the biosynthesis of AAs associated with the biodegradation process in four contrasted topsoils through short-term incubation experiments of 13C-labelled substrates. Amino acids-C accounts for ca. 25% of the newly-formed microbial biomass-C. The composition of the de novo biosynthesized individual amino acids was dependent on the soil type, and in each soil was similar to that of SOM amino acids. Biodegradation of 13C-labelled plant materials revealed the rapid conversion of plant proteins into microbial materials. These results together demonstrate that SOM amino acids are of microbial origin. We measured the dynamics of amino acids-C on the long term (decades to centuries) in nine sites using the natural 13C-labelling technique. On average, the age of AAs was equal or slightly inferior to that of bulk soil organic carbon, with mean ages ranging from 50 to 200 years. We built a conceptual model of AAs dynamics to discuss various hypotheses of AAs stabilization. Beyond these perspectives on C and N coupling in soil processes, the overall study brings a broad dataset of amino acids, as well as discuses variations of 13C natural abundance (δ13C) in-between individual amino acids.

Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.

Ischaemic stroke is a major cause of death and disability worldwide. Cerebral autoregulation, which can be impaired during acute stroke, and collateral flow to brain tissue through the circle of Willis, both play a role in preventing tissue infarction. The configuration of the arterial circle varies between individuals. Thus, personalised modelling of the cerebral arterial network, to determine the potential for collateral flow, can be of significant value in the clinical context of stroke. The interaction between autoregulation and collateral flow remains poorly understood. In this study, steady-state physiological models of the cerebral arterial network, including several common variants of the circle of Willis, were coupled to a spatially variable mathematical representation of cerebral autoregulation. The resulting model was used to simulate various arterial occlusions, as well as bilateral and unilateral impairment of autoregulation, in each structural variant. The work identified few circle of Willis variants that present either particularly high-risk or particularly low-risk of cerebral ischaemia. Instead it was found that most variants are dependent upon the bilateral function of autoregulation to facilitate collateral flow and preserve cerebral blood flows. When autoregulation was impaired unilaterally, downstream of an occlusion, blood flows in the contralateral hemisphere were preserved at the expense of the ipsilateral tissue at risk. Arterial network models have in the past been personalised using structural, rather than functional, angiography measurements. This thesis presents a novel model-based method for absolute blood volume flow rate quantification in short arterial segments using dynamic magnetic resonance angiography data. The work also investigated the additional information that can be obtained from such functional angiography. The flow quantification technique was found to accurately estimate flows in shorter arterial segments than an existing technique. However, improvements to noise performance, and strategies for rejection of contaminating signals from overlapping vessels within the imaging plane, are required before the technique can be applied to personalised cerebral arterial network modelling.

Durant longtemps la spectroscopie RMN en solution a été limitée à de petits objets biologiques. Aujourd'hui, il est clairement reconnu que la stratégie du marquage isotopique spécifique de groupements méthyle dans une protéine perdeutérée a considérablement repoussé la frontière de cette technique. En effet, des protéines aussi grande que 1 MDa ont pu être récemment étudiées par RMN. Cependant, cette stratégie présente un inconvénient important lié au nombre réduit de sondes protonées. Dans ce contexte, le projet de thèse vise a développer de nouvelles méthodes pour faire face à cette rareté d'information structurale, en s'appuyant sur le marquage simultané (combinatoire) de plusieurs groupements méthyle afin d'augmenter le nombre de sondes. Pour un marquage combinatoire optimal, le choix des acides aminés à marquer et les précurseurs à utiliser ainsi que le protocole de leur incorporation doit être judicieusement étudié. Dans le présent travail, un nouveau protocole a été mis en place pour un marquage AbId1(LV)proS optimisé, exempt de toutes fuites isotopiques. En comparaison avec le marquage “AbId1LV standard", le modèle proposé permet la diminution d'un facteur de 2 le nombre de signaux RMN des Leu et Val et améliore par un facteur de 4 l'intensité des nOes à long portée qui sont expérimentalement détectable. Par ailleurs, ce protocole permet également la suppression des corrélations parasites, particulièrement nocives pour les études structurales basées sur la détection / analyse de nOes. Afin d'exploiter les spectres RMN obtenus en utilisant le protocole ci-dessus mentionné, l'attribution des signaux des méthyles est obligatoire. Deux stratégies ont été donc proposées. La première s'applique aux systèmes dont le poids moléculaire ne dépasse pas les 100 kDa (e.g. MSG). Elle se repose sur la linéarisation du marquage isotopique des acides aminés permettant ainsi l'utilisation de l'expérience 13C-TOCSY pour attribuer de manière régio et stéréo-spécifique les méthyles de l'isoleucine, leucine et valine en une seule étape. En ce qui concerne la seconde, adaptée aux protéines supra-moléculaire (> 100 kDa), c'est une optimisation de l'approche SeSAM (Sequence-Specific Assignment of Methyl groups by Mutagenesis) précédemment décrite dans la littérature. En effet, grâce au milieu de culture enrichi, mit au point pour le marquage spécifique de l'Ala, le volume minimal de culture requis a été considérablement diminué. Ceci a permis par conséquence de produire les protéines dans des plaques de 24 puits et de les purifier dans des plaques de 96 puits, raccourcissant ainsi le temps global de préparation des échantillons. Il a été estimé que l'utilisation de cette version améliorée de SeSAM offre la possibilité d'attribuer environ 100 méthyles en 2 semaines, dont 4 jours de temps de RMN, en consommant moins de 2 k € de matériaux isotopiques. Pour illustrer la pertinence du marquage isotopique et la protonation sélectifs des méthyles, de façon combinée ou pas, de nombreuses applications ont été présentés, à savoir l'étude en temps réel des processus d'auto-assemblage d'une protéine supramoléculaire (PhTET-2, ~ 0,5 MDa) par RMN. Le marquage combinatoire des protéines (82 kDa et 0,5 MDa) pour la détection de nOes longue portée (jusqu'à 10 Å et 8 Å respectivement) a également été étudié. Cette même approche a également été utilisée pour le filtrage de nOes inter-monomères à long portée, qui sont particulièrement importants pour le calcul de structure, dans des systèmes symétrique et homo-oligomèriques (PhTET-2). / Solution NMR spectroscopy has been limited to small biological objects for a long time. Nowadays, it is unequivocally recognized that the strategy of specific isotope labeling of methyl groups in a perdeuterated protein has significantly extended the frontier of this technique. Indeed, proteins as large as 1 MDa could be investigated by NMR. Conversely, this strategy presents an important drawback consisting of the drastically reduced number of protonated probes. The project of this thesis falls within the framework of developing new methodologies to cope with this scarce structural information, relying on the simultaneous labeling of several methyl groups to increase the number of probes. For optimized combinatorial labeling, the choice of the ensemble of amino acids to label simultaneously and the precursors as well as the protocol for their incorporation have to be carefully studied. In this work, a new protocol was introduced for the scrambling-free and optimized isotopic labeling of AbId1(LV)proS methyl groups. In comparison to the “standard AbId1LV” labeling scheme, the proposed pattern induces a 2-fold decrease of number of Leu and Val NMR signals and enhances the intensity of detectable long-range nOes by a factor 4. The described protocol also permits the suppression of spurious correlations, especially harmful for structural studies based on detection/analysis of nOes. To make an efficient use of the obtained high quality NMR spectra using this protocol, assignment of the methyl groups signals is mandatory. Two strategies were then proposed. The first is suitable for systems whose molecular weight does not exceed 100 kDa. It relies on the use of isotopically linearized precursors (with different isotope topologies to discriminate each methyl group) to assign in a regio- and stereo-specific manner the isoleucine, leucine and valine methyl groups in a single step, employing an optimized “out and back” 13C-TOCSY pulse sequence. While the second, adapted to supra-molecular proteins (> 100 kDa), consists of optimizing the previously reported SeSAM approach (Sequence-Specific Assignment of Methyl groups by Mutagenesis). Indeed, thanks to the developed enriched culture medium for the specific labeling of Ala, the minimal required culture volume was significantly decreased, enabling the proteins expression in 24 well plates and their parallel purification in 96 well plates. This improved SeSAM version was estimated to allow the assignment of ca. 100 methyl cross-peaks in 2 weeks, including 4 days of NMR time and less than 2 k€ of isotopic materials. To illustrate the pertinence of using selectively protonated methyl groups, either in a single or combined fashion, several applications were presented, namely the real-time NMR study of self-assembly process of a ~0.5 MDa supra-molecular protein (PhTET-2). The use of combinatorial labeling for the detection of long-range nOes to up to 10 Å (8 Å) in proteins of 82 kDa (respectively 0.5 MDa) was also investigated. This same approach was exploited for the filtering of inter-monomeric long-range nOes in the same symmetrical and homo-oligomeric PhTET-2 protein.

Au cours de ces travaux de thèse, la synthèse de BODIPYs émettant dans le rouge et le proche infrarouge a été réalisée. Un premier travail d’extension de la conjugaison électronique (soit par introduction de groupements aromatiques sur le corps du BODIPY, soit par dimérisation) a permis d’atteindre les basses énergies du spectre électromagnétique. La validation d’une méthode de synthèse de BODIPYs à l’aide d’orthoesters a permis une mise en oeuvre aisée et une nette amélioration de leurs rendements. Parmi les fluorophores obtenus, les dithiényl-BODIPYs émettant dans la fenêtre thérapeutique (650-900 nm) présentent des propriétés optiques et électrochimiques intéressantes. Ils ont été utilisés pour des applications dans le domaine biologique. Après leur solubilisation dans les milieux aqueux par introduction de groupements ammoniums, la détection d’un acide aminé, la cystéine, a pu être réalisée par spectroscopies d’absorption et de fluorescence mettant en évidence un système ratiométrique. De plus, le marquage d’une protéine modèle, le sérum d’albumine bovine, permet d’envisager l’utilisation de ce type de BODIPYs pour l’imagerie biologique grâce à un rendement quantique de fluorescence élevé en milieu physiologique. / During this thesis the synthesis of red and near-infrared emitting BODIPYs was realized. First, the electronic conjugaison was extended by introducing aromatic groups on the BODIPY core or by dimerization in order to archieve these low energies of the electromagnetic spectrum. The validation of a synthetic method of BODIPYs with orthoester allowed to have an efficient synthesis with a clear enhancement of the reaction yield. Among the obtained dyes dithienyl-BODIPYs which display an emission in the therapeutic window (650-900 nm) present interesting optical and electrochemical properties. They have been used for applications in the biological field. After their water-solubilization with ammonium groups the detection of an amino-acid (the cysteine) was archieved with a ratiometric system observed in absorption and fluorescence spectrocopies.Moreover the labelling of a model protein, the bovin serum albumine, allows to consider the use of this type of BODIPYs in biological imaging because of a red emission and a high quantum yied in physiologic medium.

Our common future involves many important challenges. People and nature need to improve the relationship in order to reach an ecologically sustainable development. In a society where consumption of products steadily increases, the consumer awareness about social and environmental issues connected to the products becomes an importent factor. More and more companies choose to work more actively with these issues and more and more products get labelled by some of the eco labelling organisations. From the cradle to the gate means that a study has been done on a part of a products life cycle. In this master thesis a pair of cotton trousers has been followed from the cotton field and through the manufacturing chain in order to sees how much resource that are connected to the cultivation and to the production. Methological approach has been Life Cycle Inventory (LCI) according to ISO 14040. The empirical material is collected in South India, in an area known for its intense cotton manufacturing. Studies like this can be a good way of showing the environmental impacts of a certain product. LCI can for example work as a criterion for eco labelling, but the methodology could also support the overall environmental work in companies.

Em sistemas de plantio direto, o material vegetal incorporado libera fósforo solúvel no solo, porém, sua absorção por plantas subseqüentes ainda é pouco conhecida. Parte do fósforo nos tecidos vegetais está na forma de compostos insolúveis, que precisam ser mineralizados para serem absorvidos pelas plantas. Os objetivos deste trabalho foram estabelecer a melhor época de aplicação de 32P para marcação de plantas de adubo verde; determinar as formas de P nas plantas; determinar as principais formas de fósforo no solo após a incorporação de diferentes espécies de adubo verde; estimar a eficiência dos adubos verdes no fornecimento de P para plantas de arroz, e determinar a relação entre as formas de P nas plantas de adubo verde e sua mineralização no solo. Os experimentos foram conduzidos em duas etapas. Na Etapa 1 foram marcadas com 32P plantas de aveia-preta, milheto e nabo forrageiro na semeadura e aos 14, 28 e 42 dias. Estas plantas foram incubadas e utilizadas como adubo verde no cultivo de arroz. As plantas de arroz apresentaram eficiência de utilização de P semelhantes para adubos verdes marcados até 28 dias, podendo ser utilizada uma atividade 75% menor para marcação dessas espécies. Na etapa 2 foram cultivadas plantas de aveia-preta, crotalária e nabo forrageiro marcadas com 32P aos 14 dias. Foi verificado que a maioria do P nas plantas está na forma solúvel, correspondendo em média 67% do 32Pt. Essas plantas foram incubadas por 20, 40 e 60 dias e cultivadas plantas de arroz. A eficiência de utilização do P proveniente de adubo verde foi maior para incubação por 20 e 60 dias, diminuindo consideravelmente para incubação por 40 dias. Foram determinadas as frações de P no solo incubado com adubo verde nos diferentes períodos de incubação, utilizando o método de Hedley. A fração Pi-resina e P-NaHCO3, que correspondem ao P-lábil, apresentaram as maiores atividades específicas, demonstrando que a maior parte do P solúvel do adubo verde são encontrados nessas frações. A fração Po-NaOH apresentou as maiores concentrações de P, enquanto as menores foram encontradas na fração P-HCl / In no-tillage systems, plant materials incorporated into soil release soluble phosphorus, but its absorption by subsequent plants is not completely clarified. A fraction of phosphorus in vegetal tissues is in the form of insoluble species, which require mineralization to be absorbed by plants. The aims of this work were to establish the best period for 32P application for green manure labeling; to determine the forms of P in plants and in soil after incorporation of different green manures; to estimate the efficiency of green manures for supplying phosphorus to rice plants and to determine the relationship between the P species in green manure and their mineralization in soil. The experiments were carried out in two steps. In the first, plants of black oat, millet and turnip were labeled with 32P at sowing and after 14, 28 and 42 days. These plants were incubated and used as green manure for rice plants. The rice plants showed similar utilization efficiencies of P form green manure plants labeled up to 28 days, making possible the use of a 75% lower activity to label these plants. In step two, black oat, sunnyhemp and turnip plants labeled with 32P after 14 days from sowing were grown. It was verified that the more significant fraction of P in plants is in the soluble form, corresponding in average 67% of 32Pt. These plants were incubated for 20, 40 and 60 days before culturing rice plants. The utilization efficiency of P from green manure sources was higher when the material was incubated for 20 and 60 days, being considerably reduced for 40 days incubation. The P-fractions in the soil incubated with green manure in different periods were determined by the Hedley method. The Pi-resin and P-NaHCO3 fractions, which correspond to labile P, showed the highest specific activities, because the most significant part of the soluble P was found in these fractions. The Po-NaOH fraction showed the highest concentrations of P, while the lowest amounts were in the P-HCl fraction

Evidências apontam para o envolvimento da peroxidação lipídica em diversas patologias. Os hidroperóxidos de lipídios (LOOH) são os produtos primários da peroxidação lipídica e sua decomposição resulta em produtos de maior reatividade e toxicidade, como os radicais peroxila. Esses radicais desempenham papel importante na propagação da peroxidação lipídica e também podem gerar oxigênio molecular singlete (1O2) por meio da combinação de dois radicais peroxila. Neste trabalho investigamos a possibilidade dos LOOH, em particular dos hidroperóxidos de ácido linoléico (LAOOH), de servirem como fonte 1O2 na presença de oxidantes de relevância biológica como metais, peroxinitrito ou ácido hipocloroso. A formação de 1O2 foi claramente demonstrada na reação de LAOOH com esses oxidantes pelas detecções (i) da emissão bimolecular na região espectral do vermelho (λ>570 nm), (ii) da emissão monomolecular no infravermelho-próximo (λ=1270 nm), (iii) do espectro de emissão no infravermelho, e (iv) da intensificação e supressão da luminescência na presença de D2O e azida, respectivamente. Além disso, os mecanismos de reação foram estudados utilizando LAOOH marcados com oxigênio-18 (LA18O18OH) e captadores químicos específicos para 1O2 aliada à tecnica de detecção por HPLC acoplada à espectrometria de massa. Os resultados mostraram a formação de 1O2 marcado [18(1O2) ] na reação de LA18O18OH com os três oxidantes, revelando que os átomos de oxigênio do 1O2 são derivados do hidroperóxido. Em conjunto, as evidências obtidas levam à conclusão de que os LOOH podem servir como fontes potenciais de 1O2 em sistemas biológicos em situações onde haja a coexistência de LOOH e metais, peroxinitrito ou ácido hipocloroso. / Evidences point to the involvement of lipid peroxidation in several diseases. Lipid hydroperoxides (LOOH) are the primary products of lipid peroxidation and their decomposition generates more reactive and toxic compounds, such as peroxyl radicals. These radicals play an important role in the propagation of lipid peroxidation and may also generate singlet molecular oxygen (1O2) by the combination of two peroxyl radicals. In this study we have investigated the possibility of LOOH, in particular linoleic acid hydroperoxide (LAOOH), to be a source of 1O2 in the presence of biologically relevant oxidants such as, metal ions, peroxynitrite or hypochlorous acid. The formation of 1O2 was clearly demonstrated in the reaction of LAOOH with all the three tested oxidants by detecting: (i) the dimol light emission in the red spectral region (λ>570 nm), (ii) the monomol light emission in the near-infrared region (λ=1270 nm), (iii) the infrared light emission spectrum, and (iv) the enhancing effect of deuterium oxide and the quenching effect of azide on light emission. Furthermore, the mechanism was studied using LAOOH labeled with 18-oxygen isotope (LA18O18OH) and specific 1O2 chemical traps in combination with HPLC coupled to mass spectrometry detection. The results have showed the formation of 18-oxygen labeled 1O2 [18(1O2) ] in the reaction of LA18O18OH with the three oxidants, indicating that oxygen atoms in 1O2 are derived from the hydroperoxide. Altogether, the obtained evidences lead to the conclusion that LOOH may serve as a potential source of 1O2 in biological systems, in situations where LOOH can interact with metals, peroxynitrite or hypochlorous acid.

Made available in DSpace on 2014-10-09T12:23:11Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:29Z (GMT). No. of bitstreams: 1 00636.pdf: 4746331 bytes, checksum: 49aa6e0eb3bdd9da261862a04bcb4b58 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Quimica, Universidade de Sao Paulo - IQ/USP

Efforts to reduce negative impacts from consumption and production include voluntary market-based initiatives. Examples are the concept of eco-labelling and the concept of green procurement. These have emerged as policy instruments with great potentials to steer product innovation and purchasing decisions in a sustainable direction. This potential has been recognized by the United Nations, the European Union, the Organization for Economic Cooperation and Development and national governments through, e.g., various programmes and schemes. The aim of this thesis is to assess current criteria development processes within eco-labelling and green procurement from a strategic sustainability perspective and to describe possible improvement potentials from such a perspective to make these instruments more supportive of sustainable product and service innovation. A previously published framework for strategic sustainable development, including a definition of sustainability and generic guidelines to inform strategies towards sustainability, is adapted and used for this purpose. Criteria development processes in two Swedish eco-labelling programmes and at a governmental expert body for green procurement are studied. This includes interviews with criteria developers, studies of process documents and a case study at the governmental expert body for green procurement in which two criteria development processes were shadowed. The result reveals several strengths but also gaps and thus potentials for improvement. The criteria development processes and the resulting criteria mostly concern the current market supply and a selection of current environmental impacts outside the context of long-term objectives. Neither sustainability nor any other clearly defined long-term objective is agreed upon, and the criteria are not structured to support procurers, suppliers and product developers in a systematic and strategic stepwise approach towards sustainability. Recommended improvements include a more thorough sustainability assessment, communication of clearer objectives, broader competence in the criteria development groups and more emphasis on the dialogue and interaction between key actors. This includes an extended view on both the product concept and actors involved. Based on this, a new criteria development prototype is suggested, which aims at widening the scope from some currently known product impacts to the remaining gap to sustainability. During its further development and implementation, the criteria development prototype will be tested in successive iterations of action research together with experienced practitioners within eco-labelling and green procurement.