In h?heren Pflanzen vermittelt der Blaulicht-sensitive Photorezeptor Phototropin verschiedene Reaktionen wie Phototropismus, Chloroplastendrehung und die ?ffnung von Schlie?zellen. Alle Reaktionen haben mit der Optimierung der pflanzlichen Lichtaufnahme und damit einer angepa?ten Photosynthese sowie der Vermeidung von Lichtsch?digung zu tun. In C.reinhardtii haben alle Phototropinreaktionen mit dem Sexualleben dieser Alge zu tun, d.h. Gametogenese, sexueller Kompetenz sowie der Keimung von Zygoten. Diese Doktorarbeit wurde Ende 2001 begonnen und verlief parallel zu den Arbeiten von Huang. Im Unterschied zu den Ergebnissen von Huang war das Chlamydomonas Phototropin in meinen H?nden unl?slich. Das Protein war nicht komplett membranassoziiert, sondern ein Teil des Proteins blieb immer l?slich. Die Phototropinmenge sowie die Verteilung waren in vegetativen Zellen, die unter Stark- oder Schwachlicht gewachsen waren, unterschiedlich. Deshalb wurde fš¹r diesen Unterschied Licht als wesentlicher Faktor verantwortlich gemacht. Jedoch zeigen verschiedene St?mme bei vegetativem Wachstum unter identischen Lichtbedingungen unterschiedliche Phototropinmengen. Das deutet auf weitere Faktoren hin, die Konzentration und Verteilung von Phototropin beeinflussen. In Chlamydomonas wurde neben dem Volll?ngenprodukt noch eine c-terminal verkš¹rzte Phototropinvariante gefunden. Licht wurde als Verursacher der Verkš¹rzung identifiziert. Aber nur lange Belichtungen von ca. 48 h fš¹hrten je nach Intensit?t zu klaren Abbaumustern. Fš¹r das Studium der Beteiligung des Phototropins am Sexualleben von Chlamydomonas, sollte ein Phot- Stamm generiert werden. Dazu wurde ein RNAi-Konstrukt hergestellt, mit dem es m?glich war, im Stamm cw15 arg- A das Phototropin bis auf 10% des Originalniveaus zu reduzieren. Leider funktionierte das Konstrukt in anderen St?mmen nicht zuverl?ssig. Im Stamm CC32pab1mt(+) konnte nur ein Klon mit einer Reduktion auf 15% des Originalniveaus erreicht werden. Au?erdem war die Silencing-Effizienz stark von den Wachstumsbedingungen abh?ngig. Die beste Reduktion wurde bei niedrigen Lichtintensit?ten gefunden. Es wurde ein weiterer Kreuzungstest etabliert, der fš¹r die Analyse der Zygotenkeimung verschiedene Vorteile gegenš¹ber bekannten Tests liefert. Aus den durchgefš¹hrten Tests konnte geschlossen werden, dass Licht auch ein wesentlicher Faktor fš¹r die Zygotenkeimung ist. Bei mittleren Lichtintensit?ten keimen Zygoten mit wenig Phototropin sp?ter. Starklicht kann diesen Mangel weitgehend kompensieren. Zur biochemischen Analyse des Phototropins in vitro war die Expression eines markierten Phototropins notwendig. Zur Analyse des Phototropinabbaus und fš¹r die sp?tere Reinigung wurde auch versucht, Phototropin in verschiedenen Gastorganismen zu exprimieren. In dieser Arbeit wurde Phototropin in Xenopus Oocyten und Diatom?en exprimiert. Diese Versuche haben best?tigt, dass das Phototropinabbauprodukt vom selben Gen wie das Volll?ngenprotein resultiert. Durch Expression der Phot-Mutante S57S/C250S konnte auch gezeigt werden, dass die Aktivierung des Phototropins keine Voraussetzung fš¹r den Abbau ist. Erstmalig konnte auch Phototropin als Fusionsprotein in Chlamydomonas exprimiert werden. Das Fusionsprotein wurde gereinigt und die Identit?t massenspektrometrisch verifiziert. Es wurde ein Stamm gefunden, der nur eine verkš¹rzte Variante des Phototropins exprimiert. Das Produkt war besser l?slich als die Volll?ngenversion. Eine Gro?produktion sollte fš¹r die Reinigung und nachfo! lgende Kristallisation angesetzt werden. Tandem Affinit?tsreinigungen sollten fš¹r die Identifizierung von Reaktionspartnern durchgefš¹hrt werden. / Abstract In higher plants, phototropin is in charge of phototropism (Liscum, 2002), chloroplast relocation (Wada et al., 2003) and stomatal opening (Schroeder et al., 2001). All its functions are connected with plants' modulation to the surrounding light conditions so that plants can make the best use of light for photosynthesis and dodge harmful strong light. In C.reinhardtii, all its reported functions are connected to the sexual life of this green alga, i.e. gametogenesis, maintenance of gamete competence and zygote germination. This PhD work started at the end of 2001 and went on in parallel with Huang's work. Different from Huang's observation that phototropin was insoluble (Huang et al., 2002), it was found that phototropin existed not only as a membrane associated protein, a portion of phototropin always remained soluble. Phototropin levels and distributions were different between vegetative cells grown in strong light or in darkness. Light was thought to be the essential factor that caused this difference. But, different strains grown vegetatively under same light conditions showed that the levels of phototropin and its distributions varied. This suggested the existence of other factors in the determination of its level and distribution. A C-terminus degradation product of phototropin was found as a stable component in C. reinhardtii. Light was found as a reason that caused the degradation. However, short time of illumination with strong light (up to 2 h) did not evoke the degradation machinery. In light gradient experiment, long time illumination (~48h) with different light intensity showed a clear degradation pattern. To study phototropin involvement in Chlamydomonas sexual life, a Phot1- strain was needed. An RNAi phototropin construct was made. It managed to reduce the level of phototropin in strain cw15 arg- A down to 10% of its original level. However, the construct did not work properly in other strains. Only one transformant of strain CC32pab1mt(+) with a reduction of the phototropin level to around 15% of the original level was found. Under different growth conditions, the silencing efficiency varied. The best silencing result appeared when cells were grown under low light conditions. A new mating assay was established in this work, which has many benefits over the traditional way of studying zygote germination. The conclusion was drawn from this assay that light was the primary factor that determines zygote germination; under moderate light conditions, zygotes with higher phototropin level would germinate earlier; strong illumination could compensate the difference caused by the low phototropin level in zygote germination. For phototropin function analysis in vitro, a Chlamydomonas strain that over-expressed phototropin was in need. To prove that the suspected degradation product originated from the phototropin gene and to purify phototropin for crystallization, trials to express C.r. phototropin cDNA in different organisms were also made. Phototropin was expressed in Xenopus oocytes and diatom in this work. The expression pattern confirmed that both the full-length and the suspected degradation product did originate from the same phototropin gene. It was also found that the degradation was independent on phototropin activation state by expressing C.r. Phot1 (C57S, C250S) in oocytes. For the first time, recombinant phototropin got expressed in Chlamydomonas by fusion expression strategy. The fusion product was purified and the identity was confirmed by Mass Spectrometry analysis. One strain which expressed only a C-terminus truncation version of the fusion product was found. Compared with the full length product, this mutant had better solubility and was easier to be purified. Large scale of purification should be performed to obtain enough material for crystallographic studies. Tandem affinity purification (TAP) tag should also be performed in Chlamydomonas proteomic studies about phototropin.

Os impactos ambientais causados pela queima dos combustíveis fósseis e pela sua manipulação, aliados ao crescente preço dos combustíveis, têm fomentado a procura de novos recursos renováveis e o desenvolvimento de novas tecnologias que suportem as necessidades desse mercado. Os biocombustíveis são recursos biodegradáveis e renováveis, que vêm se revelando uma alternativa economicamente viável. No entanto, a atual geração de biocombustíveis possui alguns pontos negativos, tais como: utilização de solos férteis e competição com a indústria de alimentos, uma vez que utiliza culturas como soja, milho e cana-de-açúcar, produtos de extrema importância econômica para seus países produtores. Por estes motivos, há um crescente interesse em explorar outras matérias-primas possíveis, em especial as voltadas exclusivamente para a geração de energia. Neste contexto, as microalgas vêm se mostrando uma opção bastante interessante. Estes organismos apresentam um alto potencial para tal, pois possuem alta taxa de crescimento e capacidade de produzir grande quantidade de óleo. Além disso, a produção do biocombustível por estes organismos pode ser otimizada tanto pela modificação das condições de cultivo (engenharia bioquímica), como através da manipulação genética das linhagens (engenharia genética). Neste trabalho, ambas as estratégias foram utilizadas com o intuito de se aumentar a quantidade de lipídeo produzido pela linhagem CC424 da microalga modelo Chlamydomonas reinhardtii. A via metabólica escolhida para a manipulação genética foi o ciclo do glioxilato, sendo as duas enzimas-chave desse ciclo, isocitrato liase (icl) e malato sintase (ms), os alvos. O plasmídeo pSL18 foi utilizado como vetor da transformação nas microalgas. Seis tipos de linhagens transformantes foram obtidas: duas delas subexpressando os genes icl e ms separadamente, duas subexpressando esses genes e duas contendo duplas transformações, ou seja, uma delas subexpressando ambos os genes ao mesmo tempo e a outra superexpressando os mesmos. Quando se subexpressou ambas as enzimas ao mesmo tempo, houve um aumento significativo na quantidade de lipídeos neutros da célula. Além disso, essa linhagem transgênica foi submetida à escassez de nitrogênio, o que acentuou ainda mais esse resultado. Enquanto em meio normal a diferença entre a quantidade de lipídeos foi de 1,5 vezes, em escassez de nitrogênio essa diferença foi de aproximadamente 3 vezes, corroborada pela diferença nos níveis de expressão gênica, que também foi em torno de 3 vezes. Além disso, a linhagem transgênica também mostrou um aumento em cada um dos ácidos graxos analisados individualmente, revelando uma grande quantidade de todos os tipos de C16 e C18, ácidos graxos importantes para que o biodiesel se adeque ao regulamento da Agência Nacional de Petróleo, Gás Natural e Biocombustíveis. Apesar de maior quantidade de lipídeos em relação à linhagem selvagem, a nova linhagem transgênica Dupla-ICL-MS-anti não mostrou nenhum efeito deletério crítico. Tanto a produção de biomassa, quanto a quantidade de clorofila a, proteínas totais e carboidratos totais se mantiveram estáveis após a introdução da mutação. Esses resultados sugerem que as enzimas do ciclo do glioxilato, sabidamente ligadas ao catabolismo de ácidos graxos, podem ser utilizadas como alvos promissores para a otimização de linhagens já utilizadas comercialmente na produção de biodiesel. / The environmental impacts caused by gases emitted from burning fossil fuels and their manipulation, combined with rising fuel prices, has stimulated demand for new renewable resources and developing new green technologies that support the industry and market needs. Biofuels are biodegradable and renewable resources, which come out to be an economically viable alternative. However, the current generation of biofuels has some disadvantages, such as: use of fertile soils and competition with the food industry, once it uses crops such as soybeans, corn and sugar cane, products of extreme economic importance to the producing countries. For these reasons, there is a growing interest in exploring other possible raw materials, especially those that are geared exclusively for power generation. In this context, microalgae have shown to be a very interesting option. These organisms have a high potential because they have fast growth rate and the ability to produce large amounts of oil. In addition, biofuel production by these organisms can be optimized for both the modification of culture conditions (biochemical engineering), and through the genetic manipulation of microalgae strains (genetic engineering). In this work, the two strategies have been used in order to increase the amount of lipid produced by the strain CC424 from the model organism Chlamydomonas reinhardtii. The metabolic route chosen for genetic manipulation is the glyoxylate cycle, and the two key enzymes of this cycle, isocitrate lyase (icl) and malate synthase (ms), the targets. The plasmid pSL18 was used as a vector of transformation in the microalgae. Six types of transformant strains were obtained, two of them overexpressing the ms and icl genes separately, two underexpressing these genes and two double transformations, one of them overexpressing both genes at the same time the other one underexpressing them. The strain underexpressing both enzymes at the same time, showed a significant increase in the amount of neutral lipids. In this mutant, the shortage of nitrogen led to an even greater increase in these lipids. While in normal media the difference between the amount of lipids was 1.5 times, under nitrogen starvation the difference was approximately 3 times, corroborated by the difference in gene expression levels, which was also about 3 times. Moreover, the mutant strain also showed an increase in each of the individual fatty acids analyzed, revealing a large amount in all kinds of C16 and C18 fatty acids, important for biodiesel that suits the regulation of Agência Nacional de Petróleo, Gás Natural e Biocombustíveis. Although the mutant Dupla-ICL-MS-anti produces higher amounts of lipids compared to the wild type, the strain showed no critical negative effects. Both the production of biomass and the amount of chlorophylla, total protein and total carbohydrates remained stable after the introduction of the mutation. These results suggest that the enzymes of the glyoxylate cycle, which are linked to the catabolism of fatty acids, can be used as promising targets for the optimization of strains already used commercially in the production of biodiesel.

Die vorliegende Dissertation beschäftigt sich mit den Faktoren, die das Wachstum und die Vertikalverteilung von Planktonalgen in extrem sauren Tagebaurestseen (TBS; pH 2-3) beeinflussen. Im exemplarisch untersuchten TBS 111 (pH 2.7; Lausitzer Revier) dominiert die Goldalge Ochromonas sp. in oberen und die Grünalge Chlamydomonas sp. in tieferen Wasserschichten, wobei letztere ein ausgeprägtes Tiefenchlorophyll-Maximum (DCM) ausbildet. Es wurde ein deutlicher Einfluss von Limitation durch anorganischen Kohlenstoff (IC) auf das phototrophe Wachstum von Chlamydomonas sp. in oberen Wasserschichten nachgewiesen, die mit zunehmender Tiefe von Lichtlimitation abgelöst wird. Im Vergleich mit Arbeiten aus neutralen Seen zeigte Chlamydomonas sp. erniedrigte maximale Wachstumsraten, einen gesteigerten Kompensationspunkt und erhöhte Dunkelrespirationsraten, was auf gesteigerte metabolische Kosten unter den extremen physikalisch-chemischen Bedingungen hinweist. Die Photosyntheseleistungen von Chlamydomonas sp. waren in Starklicht-adaptierten Zellen durch IC-Limitation deutlich verringert. Außerdem ergaben die ermittelten minimalen Zellquoten für Phosphor (P) einen erhöhten P-Bedarf unter IC-Limitation. Anschließend konnte gezeigt werden, dass Chlamydomonas sp. ein mixotropher Organismus ist, der seine Wachstumsraten über die osmotrophe Aufnahme gelösten organischen Kohlenstoffs (DOC) erhöhen kann. Dadurch ist dieser Organismus fähig, in tieferen, Licht-limitierten Wasserschichten zu überleben, die einen höheren DOC-Gehalt aufweisen. Da die Vertikalverteilung der Algen im TBS 111 jedoch weder durch IC-Limitation, P-Verfügbarkeit noch die in situ DOC-Konzentrationen abschließend erklärt werden konnte (bottom-up Kontrolle), wurde eine neue Theorie zur Entstehung der Vertikalverteilung geprüft. Grazing der phagotrophen und phototrophen Alge Ochromonas sp. auf der phototrophen Alge Chlamydomonas sp. erwies sich als herausragender Faktor, der über top-down Kontrolle die Abundanz der Beute in höheren Wasserschichten beeinflussen kann. Gemeinsam mit der Tatsache, dass Chlamydomonas sp. DOC zur Wachstumssteigerung verwendet, führt dies zu einer Akkumulation von Chlamydomonas sp. in der Tiefe, ausgeprägt als DCM. Daher erscheint grazing als der Hauptfaktor, der die beobachtete Vertikalschichtung der Algen im TBS 111 hervorruft. Die erzielten Ergebnisse liefern grundlegende Informationen, um die Auswirkungen von Strategien zur Neutralisierung der TBS auf das Nahrungsnetz abschätzen zu können. / In this thesis, I investigated the factors influencing the growth and vertical distribution of planktonic algae in extremely acidic mining lakes (pH 2-3). In the focal study site, Lake 111 (pH 2.7; Lusatia, Germany), the chrysophyte, Ochromonas sp., dominates in the upper water strata and the chlorophyte, Chlamydomonas sp., in the deeper strata, forming a pronounced deep chlorophyll maximum (DCM). Inorganic carbon (IC) limitation influenced the phototrophic growth of Chlamydomonas sp. in the upper water strata. Conversely, in deeper strata, light limited its phototrophic growth. When compared with published data for algae from neutral lakes, Chlamydomonas sp. from Lake 111 exhibited a lower maximum growth rate, an enhanced compensation point and higher dark respiration rates, suggesting higher metabolic costs due to the extreme physico-chemical conditions. The photosynthetic performance of Chlamydomonas sp. decreased in high-light-adapted cells when IC limited. In addition, the minimal phosphorus (P) cell quota was suggestive of a higher P requirement under IC limitation. Subsequently, it was shown that Chlamydomonas sp. was a mixotroph, able to enhance its growth rate by taking up dissolved organic carbon (DOC) via osmotrophy. Therefore, it could survive in deeper water strata where DOC concentrations were higher and light limited. However, neither IC limitation, P availability nor in situ DOC concentrations (bottom-up control) could fully explain the vertical distribution of Chlamydomonas sp. in Lake 111. Conversely, when a novel approach was adopted, the grazing influence of the phagotrophic phototroph, Ochromonas sp., was found to exert top-down control on its prey (Chlamydomonas sp.) reducing prey abundance in the upper water strata. This, coupled with the fact that Chlamydomonas sp. uses DOC for growth, leads to a pronounced accumulation of Chlamydomonas sp. cells at depth; an apparent DCM. Therefore, grazing appears to be the main factor influencing the vertical distribution of algae observed in Lake 111. The knowledge gained from this thesis provides information essential for predicting the effect of strategies to neutralize the acidic mining lakes on the food-web.

Os impactos ambientais causados pela queima dos combustíveis fósseis e pela sua manipulação, aliados ao crescente preço dos combustíveis, têm fomentado a procura de novos recursos renováveis e o desenvolvimento de novas tecnologias que suportem as necessidades desse mercado. Os biocombustíveis são recursos biodegradáveis e renováveis, que vêm se revelando uma alternativa economicamente viável. No entanto, a atual geração de biocombustíveis possui alguns pontos negativos, tais como: utilização de solos férteis e competição com a indústria de alimentos, uma vez que utiliza culturas como soja, milho e cana-de-açúcar, produtos de extrema importância econômica para seus países produtores. Por estes motivos, há um crescente interesse em explorar outras matérias-primas possíveis, em especial as voltadas exclusivamente para a geração de energia. Neste contexto, as microalgas vêm se mostrando uma opção bastante interessante. Estes organismos apresentam um alto potencial para tal, pois possuem alta taxa de crescimento e capacidade de produzir grande quantidade de óleo. Além disso, a produção do biocombustível por estes organismos pode ser otimizada tanto pela modificação das condições de cultivo (engenharia bioquímica), como através da manipulação genética das linhagens (engenharia genética). Neste trabalho, ambas as estratégias foram utilizadas com o intuito de se aumentar a quantidade de lipídeo produzido pela linhagem CC424 da microalga modelo Chlamydomonas reinhardtii. A via metabólica escolhida para a manipulação genética foi o ciclo do glioxilato, sendo as duas enzimas-chave desse ciclo, isocitrato liase (icl) e malato sintase (ms), os alvos. O plasmídeo pSL18 foi utilizado como vetor da transformação nas microalgas. Seis tipos de linhagens transformantes foram obtidas: duas delas subexpressando os genes icl e ms separadamente, duas subexpressando esses genes e duas contendo duplas transformações, ou seja, uma delas subexpressando ambos os genes ao mesmo tempo e a outra superexpressando os mesmos. Quando se subexpressou ambas as enzimas ao mesmo tempo, houve um aumento significativo na quantidade de lipídeos neutros da célula. Além disso, essa linhagem transgênica foi submetida à escassez de nitrogênio, o que acentuou ainda mais esse resultado. Enquanto em meio normal a diferença entre a quantidade de lipídeos foi de 1,5 vezes, em escassez de nitrogênio essa diferença foi de aproximadamente 3 vezes, corroborada pela diferença nos níveis de expressão gênica, que também foi em torno de 3 vezes. Além disso, a linhagem transgênica também mostrou um aumento em cada um dos ácidos graxos analisados individualmente, revelando uma grande quantidade de todos os tipos de C16 e C18, ácidos graxos importantes para que o biodiesel se adeque ao regulamento da Agência Nacional de Petróleo, Gás Natural e Biocombustíveis. Apesar de maior quantidade de lipídeos em relação à linhagem selvagem, a nova linhagem transgênica Dupla-ICL-MS-anti não mostrou nenhum efeito deletério crítico. Tanto a produção de biomassa, quanto a quantidade de clorofila a, proteínas totais e carboidratos totais se mantiveram estáveis após a introdução da mutação. Esses resultados sugerem que as enzimas do ciclo do glioxilato, sabidamente ligadas ao catabolismo de ácidos graxos, podem ser utilizadas como alvos promissores para a otimização de linhagens já utilizadas comercialmente na produção de biodiesel. / The environmental impacts caused by gases emitted from burning fossil fuels and their manipulation, combined with rising fuel prices, has stimulated demand for new renewable resources and developing new green technologies that support the industry and market needs. Biofuels are biodegradable and renewable resources, which come out to be an economically viable alternative. However, the current generation of biofuels has some disadvantages, such as: use of fertile soils and competition with the food industry, once it uses crops such as soybeans, corn and sugar cane, products of extreme economic importance to the producing countries. For these reasons, there is a growing interest in exploring other possible raw materials, especially those that are geared exclusively for power generation. In this context, microalgae have shown to be a very interesting option. These organisms have a high potential because they have fast growth rate and the ability to produce large amounts of oil. In addition, biofuel production by these organisms can be optimized for both the modification of culture conditions (biochemical engineering), and through the genetic manipulation of microalgae strains (genetic engineering). In this work, the two strategies have been used in order to increase the amount of lipid produced by the strain CC424 from the model organism Chlamydomonas reinhardtii. The metabolic route chosen for genetic manipulation is the glyoxylate cycle, and the two key enzymes of this cycle, isocitrate lyase (icl) and malate synthase (ms), the targets. The plasmid pSL18 was used as a vector of transformation in the microalgae. Six types of transformant strains were obtained, two of them overexpressing the ms and icl genes separately, two underexpressing these genes and two double transformations, one of them overexpressing both genes at the same time the other one underexpressing them. The strain underexpressing both enzymes at the same time, showed a significant increase in the amount of neutral lipids. In this mutant, the shortage of nitrogen led to an even greater increase in these lipids. While in normal media the difference between the amount of lipids was 1.5 times, under nitrogen starvation the difference was approximately 3 times, corroborated by the difference in gene expression levels, which was also about 3 times. Moreover, the mutant strain also showed an increase in each of the individual fatty acids analyzed, revealing a large amount in all kinds of C16 and C18 fatty acids, important for biodiesel that suits the regulation of Agência Nacional de Petróleo, Gás Natural e Biocombustíveis. Although the mutant Dupla-ICL-MS-anti produces higher amounts of lipids compared to the wild type, the strain showed no critical negative effects. Both the production of biomass and the amount of chlorophylla, total protein and total carbohydrates remained stable after the introduction of the mutation. These results suggest that the enzymes of the glyoxylate cycle, which are linked to the catabolism of fatty acids, can be used as promising targets for the optimization of strains already used commercially in the production of biodiesel.

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T15:05:51Z (GMT). No. of bitstreams: 1 Almeida_GustavoPereirade_M.pdf: 7665145 bytes, checksum: 3fef8dc5d333834f8117015fea3b10ef (MD5) Previous issue date: 2012 / Resumo: A produção de energia por meio de fontes renováveis é uma exigência atual para se atingir uma economia sustentável. Os organismos fotossintetizantes surgem nesse contexto como ferramentas importantes na produção de compostos carbônicos ricos em energia, com destaque para microalgas em que tais compostos podem atingir até 80% do peso seco. Entretanto, um fator ainda desfavorável para sua utilização é o seu baixo rendimento na produção de biomassa. A espécie Chlamydomonas reinhardtii, por exemplo, é capaz de duplicar apenas algumas vezes durante 24 horas. As vias que controlam o crescimento celular, portanto, são alvos promissores para modificação genética. Dentre essas vias, à via de sinalização sensível à rapamicina aparece como um controlador central. Com o intuito de entender melhor como esse controle é exercido ao nível da expressão gênica global, foi utilizado a ferramenta de sequenciamento de RNA em larga escala para obtenção dos transcriptomas de culturas (sincronizadas) sob inibição dessa via e na condição controle, em oito momentos ao longo de um ciclo celular de 24h. O controle exercido por essa via sobre o metabolismo e sobre o ciclo celular foi o foco das análises. Foi encontrado que a inibição da via da TOR é capaz de gerar uma resposta de direcionamento parcial do metabolismo para a produção de TAG em detrimento de moléculas complexas como proteínas. Esse direcionamento foi considerado parcial devido à ocorrência concomitante de reações catabólicas. Outros dados obtidos sugerem que a via da TOR, além de regular o metabolismo de uma maneira geral e diversas funções celulares, também exerce influência sobre o progresso do ciclo celular e sua inibição resulta no atraso do desenvolvimento das fases do ciclo. Diversos fatores reguladores da transcrição envolvidos no desenvolvimento, no crescimento e na regulação do ciclo celular, foram encontrados diferencialmente expressos e constituem possíveis genes chave no controle do crescimento. Eles representam alvos em potencial para modificação genética com intuito de otimizar as taxas de crescimento na primeira etapa do sistema de produção. Na busca de alternativas aos processos atuais de indução do acúmulo de cadeias carbônicas, os efeitos da combinação rapamicina e via da TOR representam uma abordagem interessante para pesquisas futuras para viabilização da utilização de microalgas como fonte de energia. Este estudo possibilitou um melhor entendimento da atuação da via da TOR no crescimento e progresso do ciclo celular em C. reinhardtii ao nível de expressão gênica / Abstract: The energy production through renewable sources is an actual demand for achieving a sustainable economy. In this context, photosynthesizing organisms come to light as important tools for the production of energy-rich carbonic compounds, especially the microalgae, in which these compounds can reach up to 80% of the dry weight. However, an unfavorable factor for its utilization is the low yield of biomass production. The species Chlamydomonas reinhardtii, for instance, is capable of achieving only some duplication after 24 hours. The pathways that control cell growth are therefore promising targets for genetic modification. Among them, the rapamycin-sensitive signaling pathway emerges as a central controller. With the aim of better understanding how this control is fulfilled by the means of global gene expression, the high throughput RNA sequencing technology was used. With it, the synchronized cultures transcriptome under the inhibition of this pathway and in the control condition, of eight points during a cellular cycle of 24 hours, were obtained. The metabolism and the cell cycle control by the TOR pathway was the main focus of the analysis. It was found that the inhibition of this pathway is capable to partially draw the metabolism towards TAG production to the detriment of producing more complex chains as proteins. This directing was considered partial due to the concomitant occurrence of catabolic reactions. Other data suggested that the TOR pathway, apart from the metabolism regulation in a general way and regulation of many other cellular functions, also influence the cell cycle progression and its inhibition retards the development of cell phases. Several transcription regulators involved in development, growth and cell cycle regulation were found out to be differentially expressed and are likely to constitute key genes in growth control. They represent potential targets for genetic modification aiming the optimization of growth rate in the first step of the production system. In the search for alternatives to the current process of inducing carbon chain accumulation, the effects of the combination between rapamycin and TOR pathway represent an interesting approach for future research intending to turn the utilization of microalgae as an energy source into a feasible option. This study enabled a better understanding of the role of the TOR pathway in growth and cell cycle progression of C. reinhardtii at the level of gene expression / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Acteurs des mécanismes moléculaires de signalisation cellulaire, les espèces réactives de l'oxygène (ROS) et les espèces réactives de l'azote (RNS) agissent comme des molécules signal transférant des informations extracellulaires ou intracellulaires et induisant des réponses spécifiques. Les ROS/RNS agissent principalement via un ensemble de modifications post-traductionnelles réversibles des résidus thiols sur les protéines parmi lesquelles la nitrosylation et la glutathionylation apparaissent comme des éléments jouant un rôle important dans de nombreux processus cellulaires fondamentaux et impliqués dans nombre de maladies humaines. Bien que présents chez les organismes photosynthétiques, ces modifications ont été moins étudiées. Mon projet était d'étudier, in vivo, chez l'algue Chlamydomonas reinhardtii, la dynamique de la nitrosylation et de la glutathionylation, en utilisant une combinaison d'approches multidisciplinaires incluant protéomique, biochimie et biologie moléculaire. En réponse au stress nitrosatif, 492 protéines S-nitrosylées in vivo et 392 sites de nitrosylation ont été identifiés par spectrométrie de masse. Ces protéines participent à un large éventail de processus biologiques tels que la photosynthèse et la réponse au stress. Avec une stratégie similaire, l’analyse de la glutathionylation en réponse à des stresses physiologiques de forte lumière et de choc thermique, a révélé des voies spécifiques de réponse au stress. En parallèle, la dépendance redox des mécanismes moléculaires sous-jacents a été examinée pour la GAPDH cytoplasmique et l’isocitrate lyase, mais aussi la triosephosphate isomérase et la phosphoglycérate kinase chloroplastiques. / Actors of the molecular mechanism of cell signaling, reactive oxygen species (ROS) and reactive nitrogen species (RNS) act as signaling molecules to transfer extracellular or intracellular information and elicit specific responses. ROS/RNS mainly act through a set of reversible post-translational modifications of thiol residues on proteins among which nitrosylation and glutathionylation have emerged as key elements playing a major role in numerous fundamental cell processes and implicated in a broad spectrum of human diseases. Despite ROS and RNS are present in photosynthetic organisms, such modifications have been less studied. My project was to investigate in the green algae Chlamydomonas reinhardtii, the in vivo dynamics of nitrosylation and glutathionylation, using a combination of multidisciplinary approaches including proteomic, biochemistry and molecular biology. In response to nitrosative stress, 492 in vivo s-nitrosylated proteins and 392 sites of nitrosylation were identified by mass spectrometry. These proteins were found to participate in a wide range of biological processes and pathway such as photosynthesis, stress response and carbohydrate metabolism. Employing a similar strategy, analysis of glutathionylation in response to physiological stresses, specifically high light and heat stress revealed specific stress dependent targeted pathways. In a second part, the redox dependence of the underlying molecular mechanisms was examined for the cytoplasmic GAPDH and ICL, but also the chloroplastic TPI and PGK. This work has highlighted the existence of a strong interplay between these redox modifications. a complex redox network

Dans notre contexte moderne, les besoins en carburant demandent une innovation constante en terme de sources d'énergie. La dominance actuelle des carburants d'origine fossile dans les modes de transport personnels tels que l'aviation peut être renversée à condition de disposer de carburants efficaces riches en molécules énergétiques comme les hydrocarbures long. Dans le cas où les marchés économiques augmentent le prix des énergies fossiles, une source d'énergie renouvellable seront les plantes. Les plantes qui semblent les plus prometteuses sont les algues. En effet, ces cellules vertes produisent des lipides et ne nécessitent pas l'utilisation de terres arables si fortement prisées dans d'autres secteurs de l'agriculture. Cette thèse examine donc les conditions requises pour la productionde lipides neutres par Chlamydomonas reinhardtii. Pour commencer, un mutant est caractérisé en comparaison avec le type sauvage: sa concentration en amidon et lipides neutres est plus elevée sous illumination forte et normale. La caractérisation genomique a mis en lumière 41 gènes manquants. A la place, cette étude présente les résultats liés à l'effet de l'exposition de la souche sauvage à une forte illumination d'une part, et à une carence en azote d'autre part. Pendant les périodes de luminosité intense, les niveaux de TAG sont d'abord élevés. Dans le cas de carence en azote, l'augmentation du niveau de TAG commence seulement après l'épuisement total d'azote. L'expression du MLDP est plus notable dans le cas d'une carence en azote que d'une forte luminosité. Afin de révéler l’effet de la forte lumière seule, les corps lipidiques ont été isolés et comparés. / Neutral lipid accumulation by microalgae has recently regained considerable interest as these organisms are considered as a promising feedstock for the production of renewable biodiesel. Nitrogen deprivation is well described as a trigger for neutral lipid accumulation in various species of microalgae including Chlamydomonas. However nitrogen deprivation provokes a stop in protein synthesis and cell division, therefore limiting microalgal biomass productivity. In order to elucidate mechanisms of lipid accumulation in Chlamydomonas reinhardtii, a mutant exhibiting elevated TAG levels is characterized. The mutant exhibits reduced chlorophyll butelevated starch and neutral lipids under strong illumination in replete medium. Genomic characterization has revealed 41 missing genes. This mutant has highlighted the link between luminosity and TAG biosynthesis. To gain insights into the differences in molecular mechanisms behind oil accumulation processes under nitrogen starvation to that of high light, lipidomic changes in separate wildtype cultures were observed. Results showed that despite intracellular TAGs were found to accumulate to lower levels in response to high light in comparison to nitrogen deprivation; the TAGs productivity was higher due to a persistent biomass production. Furthermore differences in both the lipid and protein composition were observed in lipidomes and proteomes determined by pure extracts of lipid bodies isolated from both conditions revealing differences in lipid bodies isolated from different conditions.

Tubulin post-translational modifications (tPTMs) are currently studied as vital, yet obscure, cytoskeletal regulators. Their regulatory function relies on the spatiotemporal control over the activity of multiple tubulin modifying enzymes that functionalize microtubules, enabling their differentiation. The cilium, one of the organelles with the richest tPTMs diversity, has been studied with determination for the last decades, allowing the interrogation of the molecular processes that give rise to its function. The inner structure of this thin organelle, the axoneme, comprises a microtubule scaffold periodically decorated with macromolecular complexes whose characterization has been achieved with pseudoatomic detail. The molecular distribution and mechanism of action of tPTMs in cilia remain elusive. Using a combination of immunolabelling and cryoelectron tomography we interrogated the molecular function two tPTMs in the axonemal context. We showed that tubulin polyglycylation spanned most of the microtubular surface and was required for axonemal dynein activity regulation and male fertility. Additionally, there was an enrichment of polyglutamylation on a single microtubule protofilament, forming a pattern complementary to that of polyglycylation, that was required for proper coupling of microtubule sliding and bending forces during ciliary beating.

Die Grünalge Chlamydomonas reinhardtii ist ein Modellorganismus, der nach der Entdeckung von zwei Photorezeptoren, den Kanalrhodopsinen, eine Schlüsselrolle bei der Entwicklung der Optogenetik spielte. Kanalrhodopsine sind lichtinduzierte Ionenkanäle, die für das photoinduzierte Verhalten von Chlamydomonas verantwortlich sind. Aufgrund der Herausforderungen bei der gentechnischen Modifizierung in diesem Organismus konnte ihre Funktion in Phototaxis und photophober Reaktion bisher nicht umfassend untersucht werden. In dieser Arbeit präsentiere ich eine CRISPR-Cas9-basierte Technik, die einen zielgerichteten Austausch einzelner Nukleotide in einem ausgewählten Gen in vivo ermöglicht. Hierfür wird gezielt ein DNA-Doppelstrangbruch innerhalb des Gens induziert und anschließend wieder durch Aktivierung des Homologie-gesteuerten Reparaturmechanismus der Alge behoben, bei dem eine ausgewählte Punktmutation integriert wird. Diese Technik habe ich anschließend verwendet, um die Funktion der Kanalrhodopsine ChR1 und ChR2 in vivo aufzuklären. Dazu habe ich die codierenden Gene jeweils im Chlamydomonas Wildtyp-Stamm ausgeschaltet und Punktmutationen im verbleibenden Kanalrhodopsin eingeführt. Die so erzeugten Kanalmutanten weisen Veränderungen in ihrer Photozykluskinetik und Ionenselektivität auf, die das lichtabhängige Verhalten der Alge beeinflussen sollten. Die Ergebnisse zeigten zum einen, dass sowohl ChR1 als auch ChR2 Photorezeptoren sind, die die Phototaxis steuern, und zum anderen, dass eine Erhöhung der ChR2-Expression nach Deletion von ChR1 die phototaktische Aktivität der Algen wiederherstellt. Darüber hinaus wiesen die mutierten Chlamydomonas-Stämme mit veränderter Photozykluskinetik und reduzierter Kalzium-Permeabilität eine fast 100-fache Verringerung der Photosensitivität, eine verminderte photophobische Reaktion und schnellere Lichtadaptation auf. Zudem führte die Umkehr der Selektivität vom Channelrhodopsin von Kationen zu Anionen zum kompletten Verlust der Photoreaktion der Algen. Nicht zuletzt konnte diese Studie die Bedeutung der Proton-Leitfähigkeit der Kanalrhodopsine für das photoinduzierte Verhalten von Chlamydomonas aufzeigen. / The green alga Chlamydomonas reinhardtii is a model organism that played a key role in the development of optogenetics, after discovery of two photoreceptors called channelrhodopsins. Channelrhodopsins are light-gated ion channels that are responsible for photo-induced behavior of Chlamydomonas. Untill now, their functionality in algal photoresponses such as phototaxis and photophobic reaction has not been extensively studied, primarily due to the challenges connected to genetic editing in the organism. In this work, I presented a CRISPR-Cas9-based technique allowing a targeted exchange of single nucleotides in a gene of interest in vivo. It is based on targeted induction of DNA double-stranded breaks in the gene and on subsequent engagement of homologous recombination to repair the damage and integrate a selected point mutation. To elucidate the function of channelrhodopsins ChR1 and ChR2 in vivo, I created channelrhodopsin single knockouts in the wild-type Chlamydomonas strain and integrated point mutations in the remaining channelrhodopsin gene. The selected mutations affected photocycle kinetics and ion selectivity. It was shown that, first, both ChR1 and ChR2 are photoreceptors that mediate phototaxis and second, the upregulation of ChR2 upon the deletion of ChR1 rescues phototactic activity of the algae. Further, the mutant Chlamydomonas strains with altered photocycle kinetics and lower calcium permeability exhibited nearly 100-fold reduction of photosensitivity, a diminished photophobic reaction and faster light adaptation rates. Moreover, the conversion of channelrhodopsin selectivity to anions aborted algal photoresponse. In addition, the study highlighted the importance of proton conductance in the photo-induced behavior of Chlamydomonas.

Die einzellige Grünalge C. reinhardtii dient als Modellorganismus und ist aufgrund des GRAS-Status, des schnellen, kostengünstigen Wachstums und der Möglichkeit posttranslationaler Modifikationen im Kerngenom, als Expressionssystem für beispielsweise orale Impfstoffe sehr interessant. Herausforderungen sind die im Vergleich zu konventionell verwendeten Expressionssystemen sehr geringen Expressionsraten im Kerngenom. Daher sollten in dieser Arbeit neuartige, teils induzierbare, Promotorkonstrukte verwendet und mittels Luciferase-Reporter auf ihre Expressionssteigerung hin getestet werden. Zunächst wurden ausgewählte Promotoren des Chlorella-Virus-1 (PBCV-1) gewählt, diese führten allerdings zu keiner Expression. Außerdem wurden synthetische, aneinandergereihte Hitzeschockelemente mit dem endogenen RBCS2-Promotor fusioniert und die Expressionsraten analysiert. Dabei ergab sich bei der Kombination aus dem synthetischen Hitzeschockelement in achtfacher Wiederholung (HSE8x) mit RBCS2 nach der Hitzeinduktion eine Steigerung der Expressionsrate um das bis zu dreifache. Die Basalexpression war hierbei bei HSE1x-RBCS2 am höchsten und erreichte Expressionslevels, welche um das fünffache höher lagen als die Positivkontrolle HSP70A-RBCS2. Mittels Chromatinimmunopräzipitation mit dem Antikörper gegen HSF1 konnte gezeigt werden, dass eine Bindung an das synthetische Hitzeschockelement vorliegt und deshalb die Expression über die konventionelle Hitzeschockantwort in Chlamydomonas reinhardtii funktioniert. Im Gegensatz zur Luciferase benötigen Fluoreszenzproteine als Reporter kein Substrat. Infolge dieses Vorteils und der Möglichkeiten der FACS-Analyse und Fluoreszenzmikroskopie wurde tagRFP als neuer Reporter in C. reinhardtii etabliert. Die Erhöhung der Expressionsrate durch neue Promotorkombinationen und die Anwendung von tagRFP als neuen Fluoreszenzreporter bedeuten wichtige Schritte in der Etablierung von C.reinhardtii als Expressionssystem für Produkte in der Biotechnologie. / The unicellular green alga C.reinhardtii which is used as a model organism could be an interesting expression system for oral vaccines. This is because the alga is generally regarded as safe, it shows fast growth rates and culturing is cheap. Furthermore it offers the possibility of posttranslational modifications. Challenges lie in the low expression rates in the nuclear genome when compared to other expression systems. Therefore the first step in this work was to test whether selected Chlorella virus PBCV-1 promoters, do lead to enhanced expression rates, but there was no detectable expression. Furthermore synthetic repeats of heat shock elements were used in combination with the endogenous RBCS2-promoter and analysed for expression rates via reporter measurements. The combination of synthetic heat shock elements in eightfold repeats in combination with RBCS2 enhanced expression rates of luciferase after heat shock up to threefold in comparison with the up to now strongest known promoter combination HSP70A-RBCS2. Basalexpression turned out to be best for the HSE1x-RBCS2 promoter and reached expression levels fivefold higher compared to HSP70A-RBCS2. To examine if the artificial heat shock elements (HSEs) are bound by the heat shock factor 1 in C. reinhardtii a ChIP assay with HSE8x-RBCS2 and the antibody of HSF1 of C. reinhardtii was done. It could be shown that HSF1 binds HSEs and therefore one can explain the heat shock inducibility of HSEs is regulated via the conserved heat shock response. In contrast to luciferase fluorescence reporters do not need substrate. Because of this advantage and the possibility of FACS analyses and fluorescence microscopy tagRFP was established as new reporter in C.reinhardtii. Enhancement of expression rates through new constitutive and inducible promoter combinations and the possible use of tagRFP as new fluorescence reporter are significant steps in establishing C.reinhardtii as expression system for products in biotechnology.