O objetivo desta tese foi explorar o sistema de produção de proteínas heterólogas em microalga com ênfase em Chlamydomonas reinhardtii por meio de: (1) desenvolvimento de um fotobiorreator tubular fechado de escala laboratorial, utilizando técnicas de manufatura digital; (2) avaliação de 7 diferentes proteínas fluorescentes (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato e mCherry), como sistema reporter de secreção de proteínas em microalga; (3) avaliação do fotobiorreator desenvolvido utilizando cultivo de cepas recombinantes; (4) desenvolvimento de novos peptídeos sinais para secreção de proteínas em C. reinhardtii; (5) avaliação da produção de um biofármaco (hialuronidase) em microalgas, por meio da expressão de duas isoenzimas codificadas pelos genes HYA1 e SPAM1 em C. reinhardtii. O fotobiorreator tubular foi avaliado quanto a sua capacidade de resistir ao processo de esterilização por autoclavação e seu desempenho por meio do cultivo de cepa recombinante secretando mCherry. A fluorescência das proteínas fluorescentes foi medida por leitor de placas de fluorescência e visualizada intracelularmente por microscopia confocal de fluorescência. A atividade de hialuronidase foi determinada através de um ensaio enzimático turbidimétrico. O desenvolvimento do fotobiorreator resultou em um sistema fechado resistente a autoclavação, com capacidade de cultivo de cepas recombinantes de C. reinhardtii. Esse fotobiorreator proporcionou uma produtividade máxima de 10 mg/L.d de mCherry da cepa recombinante em sistema fechado, com velocidade específica de crescimento máxima de 1,27 d-1 para a cepa recombinante testada. Todas as proteínas fluorescentes avaliadas apresentaram capacidade de secreção por C. reinhardtii, com diferentes níveis de interferências em sua medição, permitindo a escolha da mCherry como proteína reporter. Entre os peptídeos sinais avaliados (quatro descritos na literatura - BiP, ARS1, CAH1 e IBP1 - e seis preditos), o peptídeo predito \"SP5\" foi o que apresentou maior capacidade de secreção, determinado por níveis de fluorescência no sobrenadante. A avaliação dos peptídeos sinais constatou a necessidade de explorar o desenvolvimento de sistemas de expressão (e.g. vetores de expressão) aliados a análises computacionais, como o SignalP 4.0. Por último, os dados desse estudo mostram que C. reinhardtii transformadas com o vetor de expressão foi capaz de produzir as duas isoformas de hialuronidase em sua forma ativa, evidenciando a capacidade desse sistema para a produção de biofármacos. Portanto, nesta tese o sistema de expressão de proteínas heterólogas baseado em microalgas foi explorado, atingindo os objetivos propostos. O fotobiorreator desenvolvido tem a capacidade de esterilização em escala laboratorial (1) e em cultivo com cepa recombinante propiciou elevada produtividade (3). As proteínas vermelhas fluorescentes apresentaram-se como as proteínas com menores interferências para estudos de secreção em C. reinhardtii (2). Além disso, o peptídeo predito SP5 apresentou o melhor desempenho na secreção de proteínas (4) e o vetor de expressão empregado permitiu a identificação de cepas produtoras de biofármaco hialuronidase (5). Portanto, o sistema de produção de proteínas heterólogas por microalgas é um sistema promissor e poderá permitir, utilizando sistemas de secreção, obter proteínas de alto valor comercial a baixos custos, empregando a secreção e técnicas de cultivo como a fermentação extrativa. / In this thesis, the heterologous protein production in microalgae with emphasis on Chlamydomonas reinhardtii was explored through: (1) development of a laboratory scale closed tubular photobioreactor using digital manufacturing techniques; (2) evaluation of different fluorescent proteins (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato and mCherry) as a reporter system for protein secretion in microalgae (3) evaluation of photobioreactor developed using recombinant strains culture; (4) development of new signals peptides for protein secretion in C. reinhardtii (5) expression evaluation of a biopharmaceutical (Hyaluronidase) in microalgae, through the expression of two isoenzymes encoded by the HYA1 and SPAM1 genes in C. reinhardtii. The tubular photobioreactor was evaluated for its ability to resist sterilization process by autoclaving and its performance by culturing recombinant strain secreting mCherry. Fluorescence of fluorescent proteins was measured by fluorescence plate reader and observed intracellularly by confocal fluorescence microscopy. The hyaluronidase activity was determined by a turbidimetric enzymatic assay. The development of the photobioreactor resulted in a closed system resistant to autoclaving, capable of culturing recombinant strains of C. reinhardtii. This recombinant strain achieved a maximum productivity of 10 mg/L.day of mcherry in the closed system, with a maximum growth rate of 1.27 d-1 for the recombinant strain tested. All the fluorescent proteins evaluated had C. reinhardtii secretion capacity, with different interference levels in their measurement, allowing the selection of mCherry as a reporter protein. Among the evaluated peptides (four described in the literature - BiP, ARS1, CAH1 and IBP1 - and six predicted), the predicted peptide \"SP5\" was the one that presented greater capacity of secretion, determined by levels of fluorescence in the supernatant. The results of this study point out the need to explore the development of biological systems (i.e., expression vectors) allied to computational analysis. Finally, the data from this study showed that C. reinhardtii could produce the two isoforms of hyaluronidase in its active form, evidencing the capacity of this system to produce biopharmaceuticals. Therefore, in this thesis the heterologous protein expression system based on microalgae was explored, reaching the proposed objectives. The developed photobioreator has sterilization capabilityin laboratorial scale (1) and in culture with recombinant strain had high productivity (3). The red fluorescent proteins was found as the most suitable proteins for studies of secretion in C. reinhardtii with lower interference levels(2). In addition, the predicted SP5 peptide showed the best performance in protein secretion (4) and the expression vector employed allowed the identification of strains producing biopharmaceutical hyaluronidase (5). Therefore, the system of heterologous proteins production by microalgae is promising and will allow, using secretion systems, to obtain proteins of high commercial value at low costs, using secretion and cultivation techniques such as extractive fermentation.

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2017-08-18T21:04:45Z No. of bitstreams: 1 Jonas F Recalcatti 2016.pdf: 1372488 bytes, checksum: dc974cf30808e2ac26e1121f83b9cf4a (MD5) / Made available in DSpace on 2017-08-18T21:04:45Z (GMT). No. of bitstreams: 1 Jonas F Recalcatti 2016.pdf: 1372488 bytes, checksum: dc974cf30808e2ac26e1121f83b9cf4a (MD5) Previous issue date: 2016-09-08 / The necessity of alternative energy sources is an example of action that may contribute to diminish environment related issues. Thus, biofuels rise as an option that fit to sustainable development, because they are produced from renewable sources of energy in order to reduce harmful gas emissions to the environment. Studies approach the potential of micro-algae to produce biodiesel, a biofuel obtained from the oil of oilseeds and micro-algae. The micro-algae consist of a variety of autotrophic, prokaryotic or eukaryotic organisms. Their simple structure allows them to easily convert solar energy into chemical energy. The products of this conversion are been used to obtain biomass from micro-algae and, consequently, products of commercial applications. Some micro-algae contain high levels of lipid, showing the potential of these microorganisms to produce biofuels. In this way, the objective of this paper was to study the micro-algae Chlamydomonas reinhardtii cultivation, growing with and without nitrogen, for the extraction and quantification of oil from this biomass in different temperatures and the analysis of the composition of the fatty acids produced. From the results obtained with the extraction, it has been seen that the deletion of nitrogen influence lipids productivity, rising the levels of the lipids on the micro-algae biomass when compared to lipids levels of the biomass cultivated with nitrogen, showing better outcomes when extracted at the temperature of 45º C (7% with nitrogen and 11% without nitrogen), independently of cultivation conditions. The deletion of nitrogen on the cultivation does not interfere significantly with the composition of the fatty acids in the oil of the biomass from the micro-algae. What predominates in its composition are saturated fatty acids (C 16:0) and mono-unsaturated (C 14:1, C 16:1 and C 20:1), propitious to biodiesel production. The results obtained show that the biomass Chlamydomonas reinhardtii micro-algae has the potential to produce biodiesel, when cultivated on the conditions tested on this paper. / A necessidade de fontes alternativas de energia são exemplos de ações que podem contribuir para amenizar os problemas relacionados ao meio ambiente. Assim, os biocombustíveis surgem como uma opção que se ajusta ao desenvolvimento sustentável, pois estes são produzidos a partir de fontes de energia renováveis de forma a diminuir a emissão de gases nocivos ao ambiente. Estudos abordam as potencialidades de microalgas para a produção de biodiesel, um biocombustível obtido a partir do óleo de diversas oleaginosas e microalgas. As microalgas consistem em uma variedade de organismos autotróficos, procarióticos ou eucarióticos, a estrutura simples das microalgas permite que elas convertam facilmente a energia solar em energia química. Desta forma, o trabalho teve como objetivo estudar o cultivo da microalga Chlamydomonas reinhardtii, em meios de cultivo com e sem nitrogênio, para extração e quantificação do óleo desta biomassa em diferentes temperaturas e análise da composição dos ácidos graxos produzidos. A microalga Chlamydomonas reinhardtii foi cultivada em meio com e sem nitrogênio. Após a recuperação da biomassa, esta foi analisada quanto à matéria orgânica e inorgânica, teor de clorofila a e b e por fim, foi obtido e quantificado o teor lipídico presente na biomassa, analisando-se o perfil de ácidos graxos presentes por cromatografia gasosa. A partir dos resultados obtidos com a extração, observou-se que, a supressão de nitrogênio influencia na produtividade de lipídeos, aumentando os teores destes na biomassa da microalga, quando comparado ao teor lipídico da biomassa cultivada com nitrogênio, apresentando maiores rendimentos quando extraído na temperatura de 45º C (7% CN e 11% SN), independente da condição de cultivo. A supressão de nitrogênio no meio de cultivo não interfere significativamente na composição dos ácidos graxos no óleo da biomassa algácea, predominando em sua composição ácidos graxos saturados (C 16:0) e mono-insaturados (C 14:1, C 16:1 e C 20:1), propícios para a produção de biodiesel. Os resultados obtidos demonstram que a biomassa microalga Chlamydomonas reinhardtii apresenta potencial para produção de biodiesel, quando cultivada nas condições testadas neste estudo.

This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled. / Entwicklung und Anwendung von Methoden zur multiparallelen Analyse von Metaboliten in der einzelligen Grünalge Chlamydomonas reinhardtii, einem der wichtigsten Modellorganismen der Zellbiologie, sind Gegenstand dieser Arbeit. Metabolomanalyse, die umfassende Analyse von Veränderungen der Konzentrationen von Stoffwechselprodukten durch Umweltreize oder genetische und entwicklungsbedingte Signale, ist ein wichtiges Komplement anderer Genomanalysemethoden, um die Integration von Genen, Proteinen und Metaboliten in ein nahtloses und dynamisches Netzwerk zur Aufrechterhaltung der Lebensfunktionen eines Organismus zu verstehen. Die Methode wurde im Hinblick auf schnelle Inaktivierung enzymatischer Aktivität, Maximierung der Extraktionskapazität und Behandlung großer Probenmengen optimiert. Im Ergebnis der Probenaufarbeitung, Extraktion und Analyse mittels Gaschromatographie und Time-Of-Flight-Massenspektrometrie konnten mehr als 800 analytische Signale in Einzelproben dargestellt werden, von denen über 100 identifiziert werden konnten. Die Arbeit stellt methodische Innovationen zur systematischen Erkennung von Artefakten in GC-MS Chromatogrammen und Werkzeuge zur Anwendung der Hauptkomponentenanalyse auf Metabolom-Daten vor. Zellen unter Stickstoff- (N), Phosphor- (P), Schwefel- (S), oder Eisen- (Fe) Mangel zeigen deutliche Unterschiede in ihrer Metabolitenausstattung. Die Anpassung an die einzelnen Nährstoffmangelsituationen ist durch spezifische Änderungen einer Reihe von Metaboliten zentraler Prozesse des Primärstoffwechsels gekennzeichnet. Die Konzentrationsänderungen von Substraten für die Stickstoffassimilation und den oxidativen Pentosephosphatweg deuten darauf hin, dass die Fähigkeit zur schnellen Aktivierung der N-Assimilation auch unter Bedingungen herabgesetzter Stoffwechsel- und Wachstumsaktivität aufrechterhalten wird. Die Akkumulation von 4-Hydroxyprolin unter Schwefelmangel könnte im Zusammenhang stehen mit der Degradation von Proteinen der Chlamydomonas-Zellwand, deren wesentlicher Bestandteil hydroxyprolinreiche Glykoproteine sind und die unter Schwefelmangel aktiv umgebaut wird. Die Anpassung an Schwefelmangel wurde mit größerer zeitlicher Auflösung in Wildtyp-Zellen und Zellen des sac1-Stammes untersucht. SAC1 ist ein zentraler Regulator der Schwefelmangelantwort in Chlamydomonas. Zeitgleiche Ab- und Zunahme von Metaboliten ist ein charakteristisches Element der Anpassung an Schwefelmangel in Wildtypzellen. Die Reaktion von SAC1-Mutanten auf Schwefelmangel ist durch weit reichenden Verlust zur Steuerung von Prozessen gekennzeichnet, die normalerweise zur vorübergehenden oder dauerhaften Anreicherung bestimmter Metabolite führen. Die Verfügbarkeit von Chlamydomonas-Stämmen mit fehlender Enzymaktivität für fast jeden der Schritte der Argininbiosynthese eröffnet die Möglichkeit, das Potential der Metabolitenanalyse zur Untersuchung der Regulation der Aminosäurebiosynthese in photosynthetischen Eukaryoten zur Anwendung zu bringen. Drei Stämme, mit fehlender Aktivität für N-Acetylglutamat-5-phosphat Reduktase (arg1), N2 Acetylornithin-Aminotransferase (arg9) beziehungsweise Argininosuccinat Lyase (arg2) wurden in Bezug auf die Aktivierung ihrer endogenen Argininbiosynthese nach Entzug externer Argininquellen analysiert. Die einzelnen enzymatischen Blocks konnten durch Precursor-Anreicherung, wie die Anhäufung von Argininosuccinat in arg2-Zellen, und Erschöpfung von Intermediaten nachgelagerter Reaktionen, beispielsweise die deutliche Abnahme von N2-Acetylornithin, Ornithin und Argininosuccinat in arg9-Zellen charakterisiert werden. Das unerwartete Vorhandensein von zum Teil das Wildtyp-Niveau überschreitender Mengen von N2-Acetylornithin, Citrullin und Argininosuccinat, die Produkte bzw. Substrate dem enzymatischen Block nachgelagerter Reaktionen in arg1-Zellen sind, bot eine Erklärung für eine noch vorhandene Restkapazität zum Wachstum des arg1-Stamms auch ohne äußere Arginingabe. Der Nachweis dieser Verbindungen sowie die ungewöhnliche Anreicherung von N-Acetylglutamat, der ersten Verbindung, die das Glutamat-Gerüst für die Ornithin- und Argininsynthese bindet, in arg1-Zellen könnte auf alternative Reaktionen, möglicherweise unter Beteiligung von Ornithin-Aminotransferase, zur Synthese von Ornithin hindeuten, die in Erscheinung treten, wenn die Synthesekette nach Acetylierung von Glutamat blockiert ist.

The transcriptional regulation of the cellular mechanisms involves many different components and different levels of control which together contribute to fine tune the response of cells to different environmental stimuli. In some responses, diverse signaling pathways can be controlled simultaneously. One of the most important cellular processes that seem to possess multiple levels of regulation is photosynthesis. A model organism for studying photosynthesis-related processes is the unicellular green algae Chlamydomonas reinhardtii, due to advantages related to culturing, genetic manipulation and availability of genome sequence. In the present study, we were interested in understanding the regulatory mechanisms underlying photosynthesis-related processes. To achieve this goal different molecular approaches were followed. In order to indentify protein transcriptional regulators we optimized a method for isolation of nuclei and performed nuclear proteome analysis using shotgun proteomics. This analysis permitted us to improve the genome annotation previously published and to discover conserved and enriched protein motifs among the nuclear proteins. In another approach, a quantitative RT-PCR platform was established for the analysis of gene expression of predicted transcription factor (TF) and other transcriptional regulator (TR) coding genes by transcript profiling. The gene expression profiles for more than one hundred genes were monitored in time series experiments under conditions of changes in light intensity (200 µE m-2 s-1 to 700 µE m-2 s-1), and changes in concentration of carbon dioxide (5% CO2 to 0.04% CO2). The results indicate that many TF and TR genes are regulated in both environmental conditions and groups of co-regulated genes were found. Our findings also suggest that some genes can be common intermediates of light and carbon responsive regulatory pathways. These approaches together gave us new insights about the regulation of photosynthesis and revealed new candidate regulatory genes, helping to decipher the gene regulatory networks in Chlamydomonas. Further experimental studies are necessary to clarify the function of the candidate regulatory genes and to elucidate how cells coordinately regulate the assimilation of carbon and light responses. / Pflanzen nutzen das Sonnenlicht um Substanzen, sogenannte Kohlenhydrate, zu synthetisieren. Diese können anschließend als Energielieferant für das eigene Wachstum genutzt werden. Der aufbauende Prozess wird als Photosynthese bezeichnet. Ein wichtiges Anliegen ist deshalb zu verstehen, wie Pflanzen äußere Einflüsse wahrnehmen und die Photosynthese dementsprechend regulieren. Ihre Zellen tragen diese Informationen in den Genen. Die Pflanzen nutzen aber in der Regel nicht alle ihre Gene gleichzeitig, die sie zur Anpassung an Umwelteinflüsse besitzen. Zu meist wird nur eine Teilfraktion der gesamten Information benötigt. Wir wollten der Frage nachgehen, welche Gene die Zellen für welche Situation regulieren. Im Zellkern gibt es Proteine, sogenannte Transkriptionsfaktoren, die spezifische Gene finden können und deren Transkription modulieren. Wenn ein Gen gebraucht wird, wird seine Information in andere Moleküle übersetzt (transkribiert), sogenannte Transkripte. Die Information dieser Transkripte wird benutzt um Proteine, Makromoleküle aus Aminsäuren, zu synthetisieren. Aus der Transkription eines Gens kann eine große Zahl des Transkripts entstehen. Es ist wahrscheinlich, dass ein Gen, dass gerade gebraucht wird, mehr Transkriptmoleküle hat als andere Gene. Da die Transkriptionsfaktoren mit der Transkription der Gene interferieren können, entwickelten wir in der vorliegenden Arbeit Strategien zur Identifikation dieser im Zellkern zu findenden Proteine mittels eines „Proteomics“-Ansatzes. Wir entwickelten weiterhin eine Strategie zur Identififikation von Transkripten Transkriptionsfaktor-codierender Gene in der Zelle und in welche Menge sie vorkommen. Dieser Ansatz wird als „Transcript-Profiling“ bezeichnet. Wir fanden Zellkern-lokalisierte Proteine, die als Signalmoleküle funktionieren könnten und Transkripte, die bei unterschiedlichen Umweltbedingungen in der Zelle vorhanden waren. Wir benutzten, die oben genannten Ansätze um die einzellige Grünalge Chlamydomonas zu untersuchen. Die Informationen, die wir erhielten, halfen zu verstehen welche Transkriptionsfaktoren notwendig sind, damit Chlamydomonas bei unterschiedlichen Umweltbedingungen, wie z.B. unterschiedliche Lichtintensitäten und unterschiedlicher Konzentration von Kohlenstoffdioxid, überlebt.

Im Rahmen des ersten Teils der vorliegenden Doktorarbeit konnten zwei nicht-essentielle (rps15, rpl36) und fünf essentielle (rps3, rps16, rpl22, rpl23, rpl32) im Plastom von Nicotiana tabacum kodierte Proteine des plastidären Ribosoms bezüglich ihrer Essentialität charakterisiert werden. Diese Gene wurden durch gezielte Knockout-Experimente inaktiviert und die resultierenden Effekte untersucht. Die Ergebnisse lassen einen Rückschluss auf die Lokalisation der Gene der insgesamt sieben untersuchten ribosomalen Proteine zu, die im Plastom mehrerer parasitischer, Plastiden-besitzender Spezies nicht mehr nachweisbar sind. Im Fall von rps15 könnte tatsächlich ein Verlust des Genes stattgefunden haben, im Fall der restlichen Gene ist eher mit einem Transfer in den Nukleus zu rechnen (rpl36 ausgenommen). Dies würde bedeuten, dass die Geschwindigkeit der erfolgreichen Etablierung eines Gentransfers in vielen parasitischen Spezies gegenüber grünen Pflanzen stark erhöht ist. Alle in E. coli nicht-essentiellen Proteine mit Homologen in Plastiden (rps15, rpl33, rpl36) sind auch dort, trotz ~1,5 Milliarden Jahren getrennter Evolution, nicht essentiell. Dieses Ergebnis bestätigt den schon früher festgestellten hohen Konservierungsgrad der bakteriellen und plastidären Translationsmaschinerien. Die Phänotypen der KO-Pflanzen der nicht-essentiellen Gene (rps15, rpl36) weisen auf eine interessante Rolle von S15 während der Ribosomenassemblierung hin und im Fall von L36 auf eine wichtige funktionelle Rolle im Plastiden-Ribosomen sowie auf eine Involvierung der Plastidentranslation in der Generierung eines retrograden Signals, welches die Blattform zu beeinflussen im Stande ist. Des Weiteren konnte eine Verbindung der Translationsaktivität mit der Ausbildung von Seitentrieben hergestellt werden, die vermutlich auf veränderte Auxinsynthese im Chloroplast zurückzuführen ist. Aus dem Folgeprojekt, bei dem Doppel-KO-Pflanzen nicht-essentieller ribosomaler Proteine erzeugt wurden, lässt sich auf eine relativ große Plastizität der Architektur von Plastidenribosomen schließen. Im zweiten Teil der Arbeit konnte erfolgreich ein Hochdurchsatz-Screeningsystem zur semiquantitativen Analyse von 192 verschiedenen miRNAs aus Chlamydomonas reinhardtii etabliert werden. Es gelang durch die Untersuchung von 23 verschiedenen Wachstums- und Stressbedingungen sowie Entwicklungsstadien mehrere miRNAs zu identifizieren, die eine differenzielle Expression zeigen sowie unter allen untersuchten Bedingungen konstant bleibende miRNAs nachzuweisen. Dadurch konnten mehrere vielversprechende Kandidaten-miRNAs ausgemacht werden, die nun eingehender untersucht werden können. / Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway. In the second project a qRT-PCR based plattform for the analysis of miRNAs in Chlamydomonas reinhardtii has been developed. 20 different growth conditions have been scanned.

When microalgae are exposed to changing environmental conditions, e.g., light-dark cycles or oscillations in nutrient availability (CO2, nitrogen, phosphate or silicate) they respond with metabolic changes in the carbon allocation pattern. Short time regulations in the time range of few seconds to minutes can be mirrored best by mass spectroscopy based metabolomics. However, these snap shots do not reflect the alterations in the carbon flow to the cellular macromolecules like protein, carbohydrate or lipid. In this review it is shown how the combination of FTIR spectroscopy and Chla-in-vivo-fluorescence based electron transport rates can reveal changes in the metabolic flux rates of carbon during a shift of the environmental conditions. The review will demonstrate in which time range FTIR spectroscopy can deliver significant information and how FTIR spectroscopy data can synergistically support metabolome analysis by mass-spectroscopy.

The aim of this master thesis was to test and compare two available methods of genetic transformation (biolistics, electroporation) of the plastid genome of green algae Chlamydomonas reinhardtii. For biolistic transformation a wide range of experimental parameters which generally influence ptDNA transformation efficiency was optimized: physiological condition of acceptor cells, type and size of microparticles, pressure of propulsion gas (helium), length of projectile trajectory, transformation of cells directly on selective medium (containing spectinomycin 150 ?g/ml) or on nonselective medium (without spectinomycin) and form of transforming DNA. In contrast to nuclear transformation ptDNA transformation of the experimental object C. reinhardtii by means of electroporation hadn´t been described. Hence a wide range of values of different physical parameters which can significantly influence the transformation efficiency was tested.

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.

L’agent pathogène Phytophthora parasitica est un oomycète qui a des effets dévastateurs sur l’agriculture et les écosystèmes naturels. En tant qu'organisme hémi-biotrophe, il infecte les racines des plantes en établissant d'abord un contact intime avec les cellules hôtes (biotrophie) avant de les tuer (nécrotrophie) et de terminer son cycle d'infection. Pour contrôler ces processus, les oomycètes sécrètent des protéines effectrices, qui sont internalisées dans les cellules végétales par un motif de translocation (appelé RxLR-EER) pour manipuler la physiologie et les réponses immunitaires de l'hôte. Les études des échanges moléculaires entre Phytophthora parasitica et la plante qui ont été menées par le laboratoire d'accueil ont permis d'identifier un effecteur RxLR, dénommé Avh195. La séquence en acides aminés de l'effecteur est caractérisée par la présence de cinq motifs AIM (« ATG8 Interacting Motive ») qui indiquent une interaction potentielle avec la protéine centrale de l’autophagie, ATG8. Avh195 co-localise avec la fraction membranaire de l'ATG8, et un système double-hybride en levure permettant la détermination d’interactions entre protéines membranaires, a confirmé une interaction non sélective entre Avh195 et plusieurs isoformes d'ATG8. La caractérisation de la perturbation de l'autophagie dépendante de Avh195 a été réalisée dans l'algue unicellulaire Chlamydomonas reinhardtii après génération de lignées transgéniques surexprimant l'effecteur. Les analyses par cytométrie de flux ont révélé que Avh195 ne modifie pas la physiologie et la « fitness » de l'algue dans des conditions de croissance normales et pendant l'autophagie induite par la rapamycine. La microscopie électronique à transmission a révélé que l'effecteur provoque dans les cellules de l’algue un retard dans le flux autophagique, se traduisant par une réduction de la coalescence et de la clairance des vacuoles et une forte accumulation d'amidon dans les chloroplastes. Cependant, ce phénotype est transitoire et seulement légèrement lié aux modifications de la régulation transcriptionnelle de la machinerie autophagique. L'analyse de la fonction effectrice chez les plantes a montré que Avh195 retarde le développement de la mort cellulaire hypersensible, déclenchée par un éliciteur d’oomycète. Cette activité dépend de trois AIM sur cinq, ce qui renforce encore l’importance de l’interaction Avh195-ATG8 pour la fonction de l’effecteur. La surexpression stable d'Avh195 chez A. thaliana a permis de déterminer que l'effecteur n'altère pas les réponses immunitaires des plantes, mais favorise globalement le développement de l'agent pathogène, accélérant le passage de la biotrophie à la nécrotrophie au cours de l'infection. À notre connaissance, le travail présenté dans cette thèse représente la première preuve qu'un effecteur d’oomycète possède une activité transitoire, ciblant de manière non sélective la protéine ATG8 dans différents organismes photosynthétiques pour ralentir le flux autophagique, favorisant ainsi le mode de vie hémi-biotrophe d'un agent pathogène. / The plant pathogen Phytophthora parasitica is an oomycete with devastating impact on both agriculture and natural ecosystems. As a hemi-biotrophic organism it infects the roots of plants first establishing an intimate contact with host cells (biotrophy) before killing them (necrotrophy) and completing its infection cycle. To control these processes, oomycetes secrete effector proteins, which are internalized in plant cells by a translocation motif (called RxLR-EER) to manipulate the physiology and the immune responses of the host. Studies of the molecular exchanges between Phytophthora parasitica and the plant that were conducted by the hosting laboratory led to the identification of an RxLR effector, designed to as Avh195. The amino acid sequence of the effector is characterized by the presence of five AIMs (ATG8 interacting motifs), that indicate a potential interaction with the autophagic core protein, ATG8. Avh195 colocalizes with the membrane-bound fraction of ATG8, and a yeast two-hybrid system, which allows to determine interactions between membrane proteins, confirmed a non-selective interaction between Avh195 and several ATG8 isoforms. The characterization of Avh195-dependent autophagy perturbation was carried out in the unicellular alga Chlamydomonas reinhardtii after generation of transgenic lines overexpressing the effector. Analyses by flow cytometry revealed that Avh195 does not modify the physiology and fitness of the alga, both under normal growth conditions and during rapamycin-induced autophagy. Transmission electron microscopy of cells revealed that the effector provokes a delay in the autophagic flux, manifested as a reduced coalescence and clearance of autophagic vacuoles and a strong accumulation of starch in chloroplasts. However, this phenotype was transient and only slightly related to modifications in the transcriptional regulation of the autophagic machinery. The analysis of effector function in planta showed that Avh195 delays the development of hypersensitive cell death, which is triggered by an oomycete elicitor. This cell death-delaying activity is dependent on three out of five AIMs, further consolidating the importance of the Avh195-ATG8 interaction for the function of the effector. The stable overexpression of Avh195 in A. thaliana allowed to determine that the effector does not impair plant defense responses, but overall promotes the development of the pathogen, accelerating the switch from biotrophy to necrotrophy during infection. To our knowledge, the work presented in this thesis represents the first evidence for an oomycete effector to possess a transitory activity, which targets in a non-selective manner the protein ATG8 in different organisms from the green lineage to slow down autophagic flux, thus promoting the hemibiotrophic life style of a pathogen.

With a surge in future demand for hydrogen as a renewable fuel, the specific aim of this study was to develop a novel strategy in photosynthetic hydrogen production from green algae, which is one of the cleanest processes among existing hydrogen-production methodologies currently being explored. The novel strategy designed was a spectral-selective PSI-activation/PSII-deactivation protocol that would work to maintain a steady flow of electrons in the electron transport system in the light-dependent part of photosynthesis for delivery of electrons to hydrogenase for photo-hydrogen production. The strategy would work to activate PSI to assist in driving the electron flow, while partially deactivating PSII to a degree that it would still supply electrons, but would limit its photosynthetic oxygen production below the respiratory oxygen consumption so that an anoxic condition would be maintained as required by hydrogenase. This study successfully showed that the implementation of the spectral-selective PSIactivation/ PSII-deactivation strategy resulted in actual and relatively sustained photohydrogen production in Chlamydomonas reinhardtii cells, which had been dark-adapted for three hours immediately prior to exposure to a PSI-spectral selective radiation, which had a spectral peak at 692 nm, covering a narrow waveband of 681-701 nm, and was applied at 15 W m⁻². The optimal condition for the PSI-spectral-selective radiation (692 nm) corresponded with low cell density of 20 mg chlorophyll L⁻¹ ("chl" henceforth) with cells grown at 25⁰C. At this condition, the PSI-spectral-selective radiation induced the maximal initial hydrogen production rate of 0.055 mL H² mg⁻¹ chl h⁻¹ which statistically the same as that achieved under white light of 0.044 mL H² mg⁻¹ chl h⁻¹, a maximal total hydrogen production of 0.108 mL H² mg⁻¹ chl which significantly exceeded that under white light of 0.066 mL H² mg⁻¹ chl, and a maximal gross radiant energy conversion efficiency for hydrogen production of 0.515 μL H² mg⁻¹ chl L⁻¹ that statistically matched that under white light of 0.395 μL H² mg⁻¹ chl L⁻¹. The study also successfully demonstrated the reversibility feature of the novel strategy, allowing for the cells to alternately engage in photo-hydrogen production and to recover by simply switching on or off the PSI-spectral-selective radiation.