Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-08-18T11:18:15Z No. of bitstreams: 1 texto completo.pdf: 483031 bytes, checksum: fdee08ea45b1d6006db79159f59bcb16 (MD5) / Made available in DSpace on 2016-08-18T11:18:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 483031 bytes, checksum: fdee08ea45b1d6006db79159f59bcb16 (MD5) Previous issue date: 2015-07-13 / Existe um crescente interesse na utilização de microalgas para a produção de biocombustíveis, alimentos e outros produtos de valor comercial. As microalgas possuem grande capacidade de fixar CO2 atmosférico e acumular carbono, sobretudo na forma de amido e lipídeos. Entretanto, ainda pouco é conhecida a regulação da biossíntese de amido e lipídeos ao longo do curso diário da fotossíntese e as relações das variações desses metabólitos com o crescimento e a produção final de biomassa. Assim, faz-se necessário uma maior compreensão das vias metabólicas e sua regulação para compreender a fisiologia e os mecanismos envolvidos na biossíntese de amido e lipídeos. Dessa forma o presente trabalho objetivou estudar a biossíntese e degradação do amido, de açúcares e lipídeos ao longo do dia. Foram selecionadas duas espécies de microalgas verdes com diferentes taxas de crescimento e contrastantes quanto a produção de amido e lipídeos totais. Com base nestes critérios foram utilizadas Chlamydomonas reinhardtii CC125, que possui um elevado acúmulo de amido e baixo teor lipídico, e Monoraphidium irregulare BR023, que possui menor conteúdo de amido e maior conteúdo de lipídeo. A cepa de M. irregulare apresentou ao final do cultivo 2,5x106 células/mL, sendo inferior ao verificado para o cultivo de C. reinhardtii (3,5x106 células/mL). Para proteínas foram encontrados valores médios inferiores em M. irregulare aos observadoss em C. reinhardtii. Comportamento similar entre as cepas foi observado para os teores de aminoácidos totais. Verificou-se menores taxas de síntese e degradação de amido para M. irregulare em ambas as fases quando comparado com as respectivas taxas para C. reinhardtii. Foram observadas maiores intensidades de fluorescência para lipídeos em M. irregulare, o que indica que esta espécie apresenta maior teor de lipídeo durante o cultivo em relação a C. reinhardtii. Conclui-se que: (i) os teores de proteínas e aminoácidos estão relacionados diretamente com a taxa de crescimento celular; (ii) lipídeo apresenta variação constante ao longo do dia; (iii) amido apresenta comportamento em ritmo circadiano; (iv) a taxa de degradação mais rápida do amido acompanha o aumento do crescimento, como em C. reinhardtii. / There is a growing interest in using microalgae as a resource for biofuel production, food and other value products. Microalgae have great ability to fix atmospheric CO2 and accumulate carbon, mostly in the form of starch and lipids. However, it is still little known regulation of starch biosynthesis and lipid along the daily course of photosynthesis and the relationship of variations of these metabolites to the growth and final biomass production. Thus, a greater understanding of metabolic pathways it is necessary and its regulation to understand the physiology and the mechanisms involved in the biosynthesis of starch and lipids. Thus the present study investigated the biosynthesis and degradation of starch, sugars and lipids throughout the day. Two species of green microalgae with different growth rates and contrasting as the production of starch and total lipids selected. Based on these criteria were used Chlamydomonas reinhardtii CC125, which has a high accumulation of starch and low- fat, and Monoraphidium irregulare BR023, which has a lower starch content and higher content of lipid. The strain of M. irregulare showed at the end of cultivation 2.5x106 cells/ml, being lower than that for the cultivation of C. reinhardtii (3,5x106 cells/ml). For proteins found average in M. irregulare average values lower to observed for the C. reinhardtii. Similar behavior was observed among strains for total amino acid content. It was found lower rates of starch synthesis and degradation M. irregulare in both phases compared to the respective rates for C. reinhardtii. There was higher fluorescence intensities for lipids in M. irregulare, indicating that this species has a higher lipid content during cultivation. It concludes that: (i) the levels of proteins and amino acids are directly related to the rate of cell growth; (ii) lipid presents constant change throughout the day; (iii) starch shows behavior circadian rhythm; (iv) the faster degradation rate of the starch accompanies increased growth, as in C. reinhardtii.

In my thesis I present data collected from a long-term selection experiment using the freshwater model organism Chlamydomonas reinhardtii. The selection experiment was designed to disentangle the effects of the number of multiple environmental drivers (MEDs) and the identity of those environmental drivers including high CO2, high temperature, general nutrient depletion, reduced light intensity, reduced phosphate availability, the addition of a herbicide, UV radiation and reduced pH. Using up to eight environmental drivers, I show how simple organisms such as C. reinhardtii evolve in response to MEDs. The first step in this investigation is to examine the short-term response of MEDs. Data collected at the beginning of the selection experiment will provide insight into the early stages of microevolution by investigating key differences in the short-term (plastic) responses to few vs. many MEDs. Here, I focus on how the data collected from the responses to single environmental drivers can help us predict the responses to MEDs by using ecological models (additive, comparative, multiplicative). I show that the short-term plastic responses to single environmental drivers can predict the effect of MEDs using the comparative model because the response is largely driven by the single dominant driver present. I also demonstrate the importance of the number of environmental drivers (NED) for making predictions from the single environmental drivers and show that predictions become more reliable as the NED increases. The results gathered from short-term responses provide evidence that single environmental driver studies are useful for predicting the effect of MEDs. After evolution, I found that the strength of selection varies with NED in a predictable way, which connects the NED to the evolutionary response (size of the direct response) through the strength of selection. Here, I used statistical models to quantify the effect of NED on the evolutionary response to MEDs and then interpreted this by considering the possible genetic constraints on adaptation to MEDs. A subset of populations evolved in environments with five environmental drivers and all populations evolved in the single environmental driver environments are used to examine how adapting to single vs. many environmental drivers affect local adaptation. I examine how populations selected in environments with one environmental driver, five environmental drivers and the evolved control, differ in their response to new environments with the same NED, environments with different NED, and a novel environment. I found that there is a relationship between local adaptation and the strength of selection in the local environment and patterns of local adaptation are affected by the NED of new environments. Lastly, I present the phenotypic consequences of evolution under MEDs. I found that before evolution, measures of chlorophyll content and cell size decline with increasing NED. However, after evolution the relationship between chlorophyll content and cell size with NED is weaker because populations converge on the same phenotypes as they evolve. I also present a case-study of how mass spectrometry methods can be used to better understand underlying molecular mechanisms of two phenotypes (chlorophyll positive and chlorophyll negative cells). This selection experiment is a good example of how laboratory investigations and model organisms can be used to design experiments with enough replication to have high statistical power in order to make more accurate predictions on the short- long-term effects of MEDs. Whilst there have been some studies on the effects of MEDs, these studies rarely have more than three environmental drivers (sometimes 5 environmental drivers) and there are only a handful of long-term MED studies. This study can be used to develop a priori hypotheses for investigating how environmental change will shape natural microbial communities, and is especially useful for organisms where long-term studies with multiple environmental drivers are unfeasible.

Abstract Collagen prolyl 4-hydroxylases (C-P4Hs) and related enzymes catalyze the hydroxylation of certain proline residues in animal collagens and plant hydroxyproline-rich proteins, respectively. Animal C-P4Hs and their isoenzymes have been characterized to date from humans, rodents, insects and nematodes. Most of the animal C-P4Hs are α2β2 tetramers in which protein disulphide isomerase (PDI) serves as the β subunit, but the nematode C-P4Hs characterized so far have unique molecular compositions. Two P4Hs have been cloned from the plant Arabidopsis thaliana and one from the Paramecium bursaria Chlorella virus-1, these being monomeric enzymes. This thesis reports on the identification of a large P4H family in the green alga Chlamydomonas reinhardtii and the cloning and characterization of one member, Cr-P4H-1. This is a soluble monomer that hydroxylates in vitro several peptides representing sequences found in C. reinhardtii cell wall proteins. Lack of its activity led to a defective cell wall structure, indicating that Cr-P4H-1 is essential for proper cell wall assembly and that the other P4Hs cannot compensate for the lack of its activity. Two C. elegans genes, Y43F8B.4 and C14E2.4, predicted to code for C-P4H α subunit-like polypeptides were analyzed. Three transcripts were generated from Y43F8B.4, one of them coding for a functional C-P4H α subunit named PHY-4.1. C14E2.4 turned out not to be a C-P4H α subunit gene, as a frame-shift led to the omission of codons for two catalytically critical residues. PHY-4.1 formed active tetramers and dimers with PDI-2 and had unique substrate requirements in that it hydroxylated certain other proline-rich sequences besides collagen-like peptides. Inactivation of the Y43F8B.4 gene led to no obvious morphological abnormalities. Spatial expression of the phy-4.1 transcript and PHY-4.1 polypeptide was localized to the pharynx and the excretory duct. Taken together, these data indicate that PHY-4.1 is not involved in the hydroxylation of cuticular collagens but is likely to have other substrates in vivo. Cloning and characterization of the PHY-1 and PHY-2 subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in assembly properties between the C. elegans and C. briggsae PHY-2 subunits in spite of their high amino acid sequence identity. Genetic disruption of C. briggsae phy-1 resulted in a less severe phenotype than that observed in C. elegans, evidently on account of its more efficient assembly of the C. briggsae PHY-2 to an active C-P4H explaining the milder phenotype. Rescue of C. elegans and C. briggsae phy-1 mutants was achieved by injection of a wild-type phy-1 gene from either species.

Le processus photosynthétique se traduit par un flux d’électron impliquant différents complexes de la membrane thylacoïdale. Ce flux peut adopter deux chemins différents : le transfert d’électron linéaire (Merchant, Prochnik et al. 2007) à travers lequel les électrons sont transférés de l’eau oxydée au niveau du Photosystème II (PSII), au NADPH réduit par le PSI ; et le transfert d’électron cyclique autour du Photosystème I (PSI) et du complexe cytochrome b6f. Ces flux d’électrons sont couplés à un pompage de proton du stroma vers le lumen générant une différence de potentiel transmembranaire, permettant la synthèse d’ATP (Allen 2002). La coexistence de ces deux flux est considéré comme nécessaire à la fixation et la métabolisation des molécules de dioxyde de carbone (Seelert, Poetsch et al. 2000 ; Munekage, Hashimoto et al. 2004) dans un rapport stricte ATP / NADPH. Cette coexistence qui semble essentiel soulève la question des mécanismes qui prévalent à l’implication des mêmes acteurs photosynthétiques, dans une même membrane, dans l’un ou l’autre mode de transfert d’électron. Chez l’algue verte Chlamydomonas reinhardtii, nous avons démontré que la commutation entre les deux transferts était dépendante de l’état redox des cellules, mais contrairement à ce qui avait été suggéré dans les études précédentes (Bulté, Rebeillé et al. 1990 ; Finazzi, Rappaport et al. 2002) indépendante du phénomène de transition d’état (Takahashi, Clowez et al. 2013), qui implique la migration latérale des complexes antennaires au sein de la membrane. L’association de ces antennes au Photosystème I conduirait à la séquestration, dans une même entité biochimique, des différents acteurs du mode cyclique. Cette formation de supercomplexe dans les conditions anoxiques, à fait l’objet d’une étude fonctionnelle in vitro, laissant quelques questions ouvertes sur leurs capacités fonctionnelles. Ce travail de thèse présente aussi la caractérisation d’une limitation transitoire des accepteurs du Photosystème I, en début d’anoxie pendant laquelle il n’est pas possible d’observer d’oxydation de P700, à 705 nm. Ce phénomène dû à la recombinaison de charge est créé par un engorgement du pool de NADPH. L’oxydation spontanée du PSI au bout d’un certain temps d’anoxie implique l’induction de l’hydrogénase, acceptant les électrons du PSI. Il reste possible d’induire cette évolution de l’oxydation de P700 lorsque les cellules sont constamment sous illumination dans les conditions anoxiques, impliquant cette fois ci, la voie de l’ATP chloroplastique. L’ATP synthétisé à la lumière permettrait la consommation de NADPH via le cycle de Benson Calvin. / The photosynthetic process relies on an electron flow involving several complexes in the thylakoid membranes of photosynthetic organisms. This flux can follow two possibly competing pathways: the linear electron transfer through which electrons are transferred from water (which is oxidized) to NADP+ (which is reduced), which is coupled to the generation of a transmembrane potential difference allowing the synthesis of ATP (Allen 2002); the cyclic pathway (around PSI and Cytochrome b6f complex) which only allows the production of ATP. These two pathways are thought to be essential for the reduction of CO2 and must likely coexist to allow the photosynthetic ATP/NADPH ratio to meet the requirement of the reduction of CO2 into carbohydrates (Seelert, Poetsch et al. 2000 ; Munekage, Hashimoto et al. 2004). This mere statement raises the question of the mechanisms that prevail in the implication of the same actors, within the same membrane, in either one of the two functional modes. In the green algae Chlamydomonas reinhardtii, our results show that the regulation of cyclic electron transfer is controlled by the redox poise and not by the lateral migration of antennae (Takahashi, Clowez et al. 2013), and disprove with the conclusion drawn from previous studies (Bulté, Rebeillé et al. 1990 ; Finazzi, Rappaport et al. 2002) according to which state transition would determine this switch. The association of these antennae to Photosystem I would promote the sequestration, within a single unit, of all the actors of the cyclic mode. Functional studies, in vitro, of supercomplex formation under anoxic conditions, questions on their functional capacities. This PhD work presents also the characterization of transient ‘’acceptor side limitation’’ of PSI, upon the onset of anoxia where it is not possible to observe an oxidation of P700 in 705 nm. This phenomenon due to the charge recombination is created by an accumulation of NADPH. The spontaneous oxidation of the PSI acceptor pool, after some time under anoxia, involves the hydrogenase induction, accepting the electrons from NADPH. It’s also possible to induce this PSI oxidation as soon as cells are constantly under illumination, involving chloroplast ATP pathway. ATP synthesised in the light, allow the consumption of NADPH through Benson-Calvin cycle.

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京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20099号 / 生博第359号 / 新制||生||47(附属図書館) / 33215 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 福澤 秀哉, 教授 佐藤 文彦, 教授 河内 孝之 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20212号 / 理博第4297号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 准教授 小山 時隆, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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