Les cyanobactéries ont développé des mécanismes de photo-protection pour se prémunir des dommages causés par un excès de lumière. L’un d’eux repose sur l’activité de l’Orange Caroténoïde Protéine (OCP), protéine soluble qui attache un kéto-caroténoïde, l’hydroxy-echinenone. Sous illumination, l’OCP se photo-convertit en forme active et interagit avec les phycobilisomes pour dissiper l’énergie collectée sous forme de chaleur. En conséquence, l’énergie d’excitation reçue par les centres réactionnels et la fluorescence du complexe photosynthétique diminuent. L’OCP a aussi la faculté de neutraliser l’oxygène singulet pour lutter contre la photo-oxydation. J’ai développé un système d’expression hétérologue pour reconstituer la voie de biosynthèse de cette protéine dans E.coli. Ce système permet l’obtention d’une grande quantité d’OCP liant son caroténoïde in vivo. Grâce à ce système robuste et rapide, les OCPs de trois cyanobactéries : Synechocystis, Arthrospira et Anabaena ont été produites, liant différents caroténoïdes. Toutes les OCPs recombinantes sont photo-actives et capables de quencher la fluorescence des phycobilisomes in vitro. Elles possèdent toutes la faculté de neutraliser l'oxygène singulet quel que soit le caroténoïde lié. Ce système d'expression hétérologue nous a permis d’élucider les déterminants structurels impliqués dans la photo-activation et la structure de la forme active de l’OCP. Il constitue une avancée fondamentale dans l'étude des protéines à caroténoïde et dans la production d'antioxydants solubles qui présentent un grand intérêt pour l’industrie de la santé. / Cyanobacteria have developed some photo-protective mechanisms to protect themselves from stress caused by excess light. One of them relies on the activity of the soluble Orange Carotenoid Protein (OCP) that binds a keto-carotenoid, the hydroxyechinenone. Under illumination, the OCP gets photo-converted to an active form and can interact with phycobilisomes to dissipate the collected energy as heat. Consequently, the excitation energy arriving at the photosynthetic reaction centers and the phycobilisome fluorescence emission decrease. The OCP can also quench the singlet oxygen to fight against photo-oxidation. I developed a heterologous expression system in which the biosynthetic pathway of the OCP is built in E.coli. The expression system allows the production of a large amount of OCP binding its carotenoid in vivo. Thanks to this robust and fast expression system, OCPs from three different cyanobacteria: Synechocystis, Anabaena and Arthrospira were produced, binding different carotenoids. All recombinant OCPs are photoactive and able to induce a large phycobilisome fluorescence quenching. Moreover, they all have the ability to quench the singlet oxygen, whatever the bound carotenoid. This heterologous expression system allowed us to elucidate the structural determinants involved in the photo-activation and structure of the active form of the OCP. This work represents a fundamental advance in the study of caroteno-proteins and in the production of others soluble antioxidants that are of great interest to the health industry.

The world is currently faced with the enormous challenge of slowing down human triggered global warming. As the global energy demand increases, there is an urgent need for renewable and carbon-neutral fuel-sources. Isoprene and isobutene are crude-oil derived, short, volatile and reactive hydrocarbons that can be polymerised into longer chains to be used as jet fuel. Isoprene has previously been produced from the cyanobacterial strain Synechocystis sp. PCC 6803 but there has been no reported isobutene synthesis from any photosynthetic organism. This work aimed to synthesise isobutene in Synechocystis using a cytochrome P450 from Cystobasidium minutum with reported isobutene production capability. Substrate availability was to be provided through the insertion of two heterologous enzymes, IpdC from Salmonella typhimurium and PadA from Escherichia coli. Both IpdC and PadA were successfully expressed in Synechocystis but the functional activities of IpdC, PadA and the cytochrome P450 in Synechocystis remains undetermined. This project also had the aim to design and construct a photo-bioreactor and gas collection system capable of producing and harvesting isoprene directly from an engineered Synechocystis strain. Herein lies a description of a closed system photobioreactor connected to a cold-trap that was able to concentrate isoprene produced from Synechocystis to measurable amounts.

Orientador: Luiz Fernando Rolim de Almeida / Resumo: O desenvolvimento das plantas é controlado por vários fatores ambientais dentre os quais a água, cujo papel vital determina o sucesso no estabelecimento vegetal. Embora as intervenções abióticas as quais estão sujeitas ao longo de seu desenvolvimento possam prejudicá-las, os eventos de estresse anteriores podem induzir seu metabolismo contra posteriores, influenciando respostas. Memória de estresse envolve múltiplas modificações em níveis fisiológicos, proteômicos, transcricionais e mecanismos epigenéticos em plantas em resposta às essas mudanças no ambiente. Neste estudo, sugeriu-se a hipótese de que as plantas de sorgo previamente expostas a ciclos de défice hídrico terão melhor desempenho fisiológico do que as que nunca enfrentaram esta condição, quando ambos grupos estão sujeitos à baixa disponibilidade de água. Este trabalho propôs identificar algumas das possíveis contribuições fisiológicas para com o desempenho fotoquímico de indivíduos da espécie Sorghum bicolor (L.) Moench submetidos a dois eventos de défice hídrico, em comparação com indivíduos expostos a apenas um evento, além do grupo controle mantido sob irrigação. As respostas fisiológicas observadas em plantas submetidas a défice hídrico recorrente indicam ajustes fisiológicos distintos das plantas do grupo submetido a esta condição apenas uma vez. Os resultados de relações hídricas, taxas fotossintéticas e conservação do aparato fotoquímico sugerem que experiência de estresse anterior pode desenvolver memória ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The development of plants is controlled by several environmental factors including water, whose vital role determines success in plant establishment. Notwithstanding the abiotic stresses that are subject throughout their development can harm them, stress events earlier tend to induce metabolism against further tensions in order to improve responses. Stress memory involves multiple changes in physiological, proteomic, transcriptional levels and epigenetic mechanisms in plants in response to these changes in the environment. In this study, the hypothesis was suggested that sorghum plants previously exposed to water deficit cycles will have a better physiological performance than those that never faced this condition, when both groups are subject to low water availability. This work proposed to identify some of the possible physiological contributions to the photochemical performance of individuals of the species Sorghum bicolor (L.) Moench submitted to two water deficit events, in comparison with individuals exposed to only one event, in addition to the control group kept under irrigation. The physiological responses observed in plants submitted to recurrent water deficit indicate physiological adjustments different from plants in the group submitted to this condition only once. The parameters of water relations, photosynthetic rates and conservation of the photochemical apparatus suggest that previous stress experience may develop physiological memory. The data suggest that th... (Complete abstract click electronic access below) / Mestre

Orientador: João Domingos Rodrigues / Resumo: Dois experimentos foram conduzidos em 2018 e 2019 para avaliar os efeitos de diferentes fungicidas de dois importantes grupos: estrobilurinas e carboxamidas, sobre a eficácia fotossintética e produtividade de plantas de tomate, inoculadas com Alternaria sp., em ambiente protegido na Fazenda Ituaú, Salto - SP, Brasil. O delineamento experimental aplicado em ambos os experimentos foi de blocos ao acaso, com oito tratamentos e cinco repetições: testemunha inoculada (água + inóculo), testemunha absoluta (água), azoxystrobina (50,0 g ha-1), boscalida (50,0 g ha-1), azoxystrobina (50,0 g ha-1) + boscalida (50,0 g ha-1), mandestrobina (194 g ha-1), impirfluxam (30 g ha-1), mandestrobina (194 g ha-1) + impirfluxam (30 g ha-1), aplicados em intervalos de 15 dias, totalizando 7 a 8 aplicações no ciclo da cultura. O efeito no desenvolvimento das plantas foi medido pela altura das plantas e produção dos cinco primeiros cachos. A eficiência fotossintética foi calculada pela medição das trocas gasosas, fluorescência da clorofila a, teores de pigmentos fotossintéticos, e produtividade. Os fungicidas apresentaram algumas melhorias nos parâmetros analisados, como melhorando os componentes da eficiência fotossintética e da produção de frutos comerciais, porém sem apresentar diferenças significativas em relação às testemunhas. Os resultados demonstram eficácia no controle de Alternaria sp. aplicando os fungicidas em tomateiro, e também apontam para efeitos adicionais em componentes da eficiênci... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Two experiments were carried out in 2018 and 2019 to evaluate the effects of the application of different fungicides from two important groups: strobilurins and carboxamides, on the photosynthetic efficiency and yield of tomato plants, inoculated with Alternaria sp., grown in greenhouse at Fazenda Ituaú, Salto - SP, Brazil. The experimental design applied in both trials was randomized blocks with eight treatments and five replicates: inoculated control (water + inoculum), absolute control (water), azoxystrobin (50,0 g ha-1), boscalid (50,0 g ha-1), azoxystrobin (50,0 g ha-1) + boscalid (50,0 g ha-1), mandestrobin (194 g ha-1), inpyrfluxam (30 g ha-1), mandestrobin (194 g ha-1) + inpyrfluxam (30 g ha-1), applied at intervals of 15 days, totalizing 7 or 8 applications in the crop cycle. The effect on plant development was measured by plant height and the production of the five initial bunches. The photosynthetic efficiency was calculated from measurements of gas exchange, chlorophyll a fluorescence, content of photosynthetic pigments, and tomato productivity were evaluated. All fungicides positively affected some evaluated parameters, improving photosynthetic efficiency components and commercial fruit production, however without significant difference to the controls. The results demonstrate the efficacy of the use of these fungicides in controlling Alternaria sp. in tomato and also appoint to some additional effects on photosynthesis efficiency components and antioxidant enzym... (Complete abstract click electronic access below) / Doutor

Dans le contexte éco-environnemental actuel, l’Humanité est à la recherche d’alternatives pour remplacer les carburants fossiles responsables du changement climatique affectant le quotidien un peu plus chaque année. Inspiré par la Nature et le processus de la photosynthèse, un nouveau domaine à l’interface entre chimie, physique et ingénierie émergea dans les années 70 : la photosynthèse artificielle. Depuis, de nombreux systèmes catalytiques ont été rapportés mais leur intégration à l’échelle commerciale n’est pas encore d’actualité. De nombreux défis demeurent afin d’améliorer les performances, coupler les différentes réactions et s’assurer de la stabilité à long terme des dispositifs. Dans le cadre de cette thèse de doctorat, nous nous sommes attachés à explorer la chimie de coordination de complexes de ruthénium et de rhénium, omniprésents dans la littérature mais dont les propriétés peuvent être encore largement modulées par le design de nouveaux ligands. Dans un deuxième temps, les polyoxométallates, composés inorganiques à mi-chemin entre oxydes et molécules aux propriétés fascinantes, ont été recrutés pour développer des dyades covalentes, stratégie permettant de stabiliser un chromophore dans les conditions photocatalytiques. / In the current ecological and environmental context, Mankind needs to find alternative energy sources to replace fossil fuels, as their combustion is a major cause of the current global climate change affecting our daily life a bit more each year. Inspired by Nature and the photosynthetic process, a new area at the interface between chemistry, physics and engineering has emerged in the 70s: artificial photosynthesis. Since then, many catalytic systems have been reported but their integration at commercial scale has not yet been achieved. Several challenges remain to improve their performances, ensure efficient coupling between the different reactions and enhance the long-term stability of these devices. Within this doctoral thesis, we have focused on exploring the coordination chemistry of ruthenium and rhenium complexes, ubiquitous in the literature though their properties can still be vastly tuned by designing new ligands. In a different approach, polyoxometalates, which are inorganic compounds half-way between oxides and molecules with fascinating properties, were recruited to develop covalent dyads, a strategy enabling stabilization of the dye under photocatalytic conditions.

Brassinosteroids are plant hormones which are also known for their pleiotropic effects on plants exposed to various biotic and abiotic stress factors. The aim of this study was to evaluate the role of 10-8 M 24-epibrassinolide in Zea mays L. and Vicia faba L., grown under conditions of drought stress (6, 10, 14, 18 days) with an emphasis on different answer to their drought sensitive and resistant genotypes. Net photosynthetic rate (PN) and transpiration rate (E), stomatal conductance (gs), osmotic potential (ψs), proline (Pro) and malondialdehyde (MDA) content and membrane injury index (MI) were measured in stressed plants and plants grown under controlled conditions. The most important factor influencing these parameters was drought, which led to a gradual decrease of PN, E, gs, ψs and increase of Pro, MDA a MI. The effect of 24-epibrassinolid was significant rather exceptionally and in these cases, more pronounced response was observed in drought stressed plants compared with plants grown under controlled conditions. Plants showed intraspecific variability in their reactions, which in sensitive and tolerant genotypes were not clear and differed depending on the studied parameters and conditions of plant cultivation.

Much success was already achieved for the development of efficient oxyfunctionalization bioprocesses by the application of oxygenases in heterotrophic whole-cell host systems. However, several restrictions such as the technically limited O2 supply and carbohydrate-based electron supply still limit their implementation on an industrial scale concerning production rates and costs. The use of phototrophic organisms as whole-cell biocatalysts for oxygenase-based biotransformations provides an alternative and promising technology for the eco-efficient production of oxyfunctionalized value-added chemicals. While numerous cyanobacterial or microalgal bioprocesses were already developed for CO2-derived fermentations, biotransformation processes relying on the generation of activated reduction equivalents as well as O2-derived from photosynthetic water oxidation are rare. In this context, research mainly focuses on the demonstration of engineered catalysts with emphasis on the production of hydrogen. Yet, an integrated bioprocess design for the application of phototrophic organisms in redox biotransformations beyond the proof-of-concept catalyst development is lacking. This thesis aims at the integrated application of biotechnological methods and strategies for the development of eco-efficient photosynthesis-driven oxyfunctionalization processes. The main research question combines the conceptual evaluation of photosynthetic electron and O2 supply with the technical applicability of cyanobacteria as phototrophic host organisms in a hydrocarbon oxyfunctionalization bioprocess. Using a guide of integrated bioprocess design, biocatalyst, reaction, and process engineering tools are applied for the establishment of new, photosynthesis-driven bioprocesses.

Die Dissertation beschäftigt sich mit der Untersuchung und Entwicklung definiert beleuchteter Photobioreaktorsysteme (klassische Systeme wie Schüttelkolben und Blasensäule als auch neuartige Konzepte) für reaktionskinetische Untersuchungen. Diese dienen zur Untersuchung des Einflusses des Lichts auf das Wachstum phototropher Organismen, als Modellorganismus wurde das Cyanobakterium Arthrospira platensis ausgewählt.

La photosynthèse oxygénique fournit l’énergie pour presque toutes les formes de vie sur Terre. Les premières étapes sont réalisées par quatre complexes membranaires. Deux sont des photosystèmes (PS) divisés en un core et une antenne qui, chez les plantes, est composée des protéines Lhca et Lhcb respectivement pour le PSI et le PSII. Les Lhcb les plus abondantes forment les hétérotrimères LHCII. Pour optimiser leur croissance, les organismes photosynthétiques doivent réguler précisément l’absorption de la lumière. Dans cette thèse, nous nous sommes focalisés sur un de ces mécanismes chez Arabidopsis thaliana : les transitions d’état. Cette régulation intervient en lumière faible ou fluctuante pour répondre à un déséquilibre d’excitation des photosystèmes. Elle implique la phosphorylation de Lhcb1 et Lhcb2, deux des isoformes composant le LHCII, et leur déplacement du PSII au core du PSI où elles agissent comme antenne. Récemment, l’attachement d’un LHCII additionnel à l’antenne du PSI a été rapporté, sans indice sur le rôle de la phosphorylation dans la liaison. Nous nous sommes attachés à quantifier la phosphorylation du LHCII dans les supercomplexes PSI et PSII et à déterminer les rôles respectifs de Lhcb1 et Lhcb2 dans les transitions d’état. Nous avons établi que la phosphorylation d’un seul Lhcb2 est suffisante pour l’attachement d’un LHCII au core du PSI. Nous avons ensuite isolé un complexe PSI-LHCII2 et déterminé que la liaison du second LHCII implique aussi la phosphorylation d’un seul Lhcb2. Ce travail apporte de nouvelles preuves des rôles divergents des isoformes Lhcb dans la régulation de la photosynthèse, qui sont discutées ici à la lumière de leur évolution. / Oxygenic photosynthesis directly or indirectly provides energy for almost all forms of life on earth. The first steps are performed by four membrane complexes. Two of them are photosystems (PS) organized in a core complex and an antenna system, which is composed in green organisms by Lhca and Lhcb proteins for PSI and PSII, respectively. The most abundant Lhcb proteins form the LHCII heterotrimers. To optimize growth, photosynthetic organisms have to precisely regulate their light harvesting. In this thesis, we focused on one of these mechanisms in Arabidopsis thaliana: state transitions. This regulation occurs in low or fluctuating light to answer for the imbalance of excitation between photosystems. It involves the phosphorylation of Lhcb1 and Lhcb2, two of the isoforms composing LHCII. The phosphorylated LHCII trimers detach from PSII and bind to PSI core and act as an antenna for it. Recently the attachment of additional LHCII trimers to PSI on its antenna side has been reported, with no clue of the role of phosphorylation in this binding. We set on quantifying the LHCII phosphorylation of purified PSI and PSII supercomplexes to determine the respective roles of the Lhcb1 and Lhcb2 isoforms in state transitions. We established that the phosphorylation of a single Lhcb2 protein is sufficient for the binding of PSI core. We then isolated a PSI-LHCII2 supercomplex, and determined that the binding of the additional trimer also involves the phosphorylation of a single Lhcb2 isoform per trimer. This work brings new evidences for the divergent roles of the Lhcb isoforms in light harvesting and its regulation, which are discussed here in the light of their evolution.

Orientador: Paulo Alexandre Monteiro de Figueiredo / Resumo: Diante da iminente escassez de fósforo (P) para utilização agrícola, surge a necessidade de estudos voltados para o melhor entendimento de estratégias de suprimento deste elemento. Estudos recentes demonstram que a atividade hormonal potencializa processos em que o P é vital para ocorrência no metabolismo, destacando os análogos de Brassinoesteróides (BR) pela influência sobre o desenvolvimento vegetal. São escassos os estudos sobre a interação P e BR e sua aplicação em grandes culturas consumidoras de fósforo, como é o caso da cana-de-açúcar. Assim, o objetivo do presente estudo foi avaliar o efeito da aplicação de doses do análogo de BR 24-epibrassinolide (0; 0,5; 1,0; 2,0 mg. L-1), associado à presença ou ausência de fertilização fosfatada sobre os parâmetros de crescimento, fosfatase, micorrização e fotossíntese de plantas jovens de cana-de-açúcar. O experimento foi conduzido em casa de vegetação, em DBC com fatorial 4x2 com seis repetições. A aplicação dos BR proporcionou um incremento nos valores de massa seca radicular até o ponto máximo da curva de 1,27 mg.L-1. A interação P com BR incrementou respectivamente em 30% e 48% os valores de massa seca aérea nas doses 0,5 e 1,0 mg.L-1 do hormônio; e apresentou uma correlação de Pearson positiva das doses de BR com adição de P sobre essa variável. A presença de P estimulou todas as variáveis de crescimento da cana-de-açúcar. O BR desencadeou um pico significativo na atividade da enzima fosfatase ácida na dose de 1,0 mg. L-1 ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In view of the imminent shortage of phosphorus (P) for agricultural use, increase the need for studies aimed at better understanding the supply strategies of this element. Recent studies show that hormonal activity potentiates processes in which it is vital for the occurrence of metabolism, highlighting the analogues of Brassinoesterteroids (BR) for their influence on plant development. There are few studies on P and BR interaction and their application in large phosphorus-consuming crops, such as sugar cane. Thus, the objective of the present study was to evaluate the effect of applying doses of the 24-epibrassinolide dialogue (0; 0.5; 1.0; 2.0 mg. L-1), associated with the presence or absence of phosphate fertilization on the growth, phosphatase, mycorrhization and photosynthesis parameters of young sugarcane plants. The experiment was conducted in the greenhouse, in the DBC with a 4x2 factorial with six replications. The application of BR provides an increase in the values of root dry mass up to the maximum point of the curve of 1.27 mg. L-1. A P interaction with BR increases, respectively, by 30% and 48% the values of air dry mass at doses 0.5 and 1.0 mg.L-1 of the hormone; and showed a positive Pearson correlation of BR doses with the addition of P on this variable. The presence of P stimulates all sugar cane growth variables. BR triggered a significant peak of acid phosphatase enzyme activity at a dose of 1.0 mg. L-1 and did not affect root mycorrhizal colonization. The... (Complete abstract click electronic access below) / Mestre