Univ., Diss.--Regensburg, 2000.

abstract: In oxygenic photosynthesis, Photosystem I (PSI) and Photosystem II (PSII) are two transmembrane protein complexes that catalyze the main step of energy conversion; the light induced charge separation that drives an electron transfer reaction across the thylakoid membrane. Current knowledge of the structure of PSI and PSII is based on three structures: PSI and PSII from the thermophilic cyanobacterium Thermosynechococcus elonagatus and the PSI/light harvesting complex I (PSI-LHCI) of the plant, Pisum sativum. To improve the knowledge of these important membrane protein complexes from a wider spectrum of photosynthetic organisms, photosynthetic apparatus of the thermo-acidophilic red alga, Galdieria sulphuraria and the green alga, Chlamydomonas reinhardtii were studied. Galdieria sulphuraria grows in extreme habitats such as hot sulfur springs with pH values from 0 to 4 and temperatures up to 56°C. In this study, both membrane protein complexes, PSI and PSII were isolated from this organism and characterized. Ultra-fast fluorescence spectroscopy and electron microscopy studies of PSI-LHCI supercomplexes illustrate how this organism has adapted to low light environmental conditions by tightly coupling PSI and LHC, which have not been observed in any organism so far. This result highlights the importance of structure-function relationships in different ecosystems. Galdieria sulphuraria PSII was used as a model protein to show the amenability of integral membrane proteins to top-down mass spectrometry. G.sulphuraria PSII has been characterized with unprecedented detail with identification of post translational modification of all the PSII subunits. This study is a technology advancement paving the way for the usage of top-down mass spectrometry for characterization of other large integral membrane proteins. The green alga, Chlamydomonas reinhardtii is widely used as a model for eukaryotic photosynthesis and results from this organism can be extrapolated to other eukaryotes, especially agricultural crops. Structural and functional studies on the PSI-LHCI complex of C.reinhardtii grown under high salt conditions were studied using ultra-fast fluorescence spectroscopy, circular dichroism and MALDI-TOF. Results revealed that pigment-pigment interactions in light harvesting complexes are disrupted and the acceptor side (ferredoxin docking side) is damaged under high salt conditions. / Dissertation/Thesis / Ph.D. Biochemistry 2010

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-04-26T09:56:41Z No. of bitstreams: 1 texto completo.pdf: 848309 bytes, checksum: f73dc789b087c3df42f96258e81b0812 (MD5) / Made available in DSpace on 2016-04-26T09:56:41Z (GMT). No. of bitstreams: 1 texto completo.pdf: 848309 bytes, checksum: f73dc789b087c3df42f96258e81b0812 (MD5) Previous issue date: 2015-02-19 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Microalgas têm atraído interesse como fonte potencial de matérias-primas para produção de biocombustíveis e de subprodutos de interesse (e.g., bioetanol e proteínas). Entretanto, a produção em escala comercial ainda é economicamente inviável, principalmente, pelo pouco conhecimento sobre a regulação do crescimento, a biossíntese e armazenamento de lipídios em microalgas.Neste estudo, avaliou-se o potencial de 10 cepas de algas coletadas na região de Viçosa – MG, com o objetivo de avaliar o crescimento e caracterizar o metabolismo em autotróficas de crescimento. Entre as cepas estudadas, duas cepas de Scenedesmus(BR003 e BR024) apresentaram os maiores valores para produtividade de biomassa, que associado a quantidades intermediárias de lipídios garantiram uma alta produtividade de lipídios em relação às outras cepas. Essas duas cepas também se destacaram entre as demais em relação ao perfil de ácido graxo, apresentando altos teores dos ácidos graxos C16:0 e C18:1 e baixos teores dos poliinsaturados. No entanto, o perfil graxo das cepas Chlamydomonas sp. BR020 e Chlorella vulgaris BR017 apresentou altos teores de ácido α-linolénico (C18:3) e esta cepa também apresentou maiores teores de proteínas, constituindo- se em importantes cepas para o conceito de biorefinária. Verificou-se também que a maioria dos metabólitos (aminoácidos e ácidos orgânicos) avaliados apresentaram correlação negativa com a biomassa produzida e positiva com o conteúdo de lipídios. Tomados em conjunto, esses resultados indicam que a baixa produção de biomassa esta relacionada com acúmulo de aminoácidos (isoleucina, leucina e valina), intermediários do ciclo dos ácidos tricarboxílicos (citrato, succinato e fumarato), que frequentemente estão associados com acúmulo de óleo na célula. Entretanto, apesar da proximidade filogenética de algumas cepas em termos de gênero ou espécie, algumas cepas apresentaram comportamentos distintos o que não permitiu o agrupamento das mesmas, demonstrando, portanto, a necessidade de uma caracterização mais detalhada desses organismos. / Microalgae have attracted interest as a potential source of raw materials for biofuels and by-products of interest such as bioethanol and protein. However, the production on a commercial scale is still costly mainly by little knowledge about the regulation of growth, biosynthesis and lipid storage in microalgae. In this study, we evaluated the potential of 10 strains of algae collected in Viçosa region - MG, with the aim of evaluating the growth and characterize the metabolism in autotrophic conditions. Among the studied strains, two strains of Scenedesmus (BR003 and BR024) had the highest values for biomass productivity, which associated with intermediate amounts of lipids guaranteed high productivity of lipids compared to other strains. In addition these two strains differed among the others in relation to the fatty acid profile, with high levels of C16 and C18: 1 fatty acidsand low levels of the polyunsaturated fatty acids. However, the fatty acid profile of Chlamydomonas sp., BR020, and Chlorella vulgaris,BR017, showed high levels of α-linolenic acid (C18: 3) and this strain also exhibited higher protein levels, suggesting that these are important strains for the biorefinery concept. It was also observed that most of the evaluated metabolites (amino acids and organic acids) were negatively correlated with biomass and positively with lipid contents. Taken together, these results indicate that low biomass production is related to the levels of amino acids (isoleucine, leucine and valine),intermediates of the tricarboxylic acid cycle (citrate, succinate and fumarate), which are often associated with oil accumulation in the cell. However, despite the phylogenetic proximity in terms of genus or species showed, some of the strains displayed differential behavior which did not allow grouping of the strain in the same cluster, thus indicating the need for a deepercharacterizationof these organisms.

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.

No description available.

Eukaryotic flagella and cilia are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella and cilia contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins, located all along the length of the axonemal structure. Fluorescent ATP analogs have been a useful tool to study ATPase activity of various motor proteins. \acrfull{mant} has been previously used to probe the activity of various ATPases, including dynein. It has been shown by various authors, that MANT-ATP supports dynein activity as well as the axonemal beat. However, direct observations of binding to the axonemal structure were not previously reported. Using highly sensitive fluorescent microscopy to monitor the binding of the fluorescent ATP analog, I probed dynein activity directly in the immobilized intact axoneme for the first time. To understand these kinetics a kinetic model was developed. By fitting this model to experimental data I was able to identify ATP-binding sites with distinct kinetic properties in the axoneme. I report a turnover rate of k = 0.02 s−1 at 1μM mant-ATP for dynein. Moreover, I discovered that there is binding of the ATP analog to the axoneme with a much higher rate of k = 11 s−1 at 1μM mant-ATP. By the application of this method to axonemes with reduced dyneins, it has been identified that the slow rate belongs to dynein.

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