The extent to which evolution is repeatable has important implications. If evolution is highly repeatable, the trajectories and outcomes of evolution in different lineages will always be the same. On the other hand, if evolution is not repeatable, then trajectories and outcomes will be diverse. Thus, the repeatability of evolution affects our understanding of the nature of biodiversity and can inform the extent to which evolutionary theory can be used to make predictions. The repeatability of evolution depends on the relative contribution of selection, chance, and history. To determine what factors affect the importance of chance and history during evolution, I propagated replicated populations of the unicellular green alga Chlamydomonas reinhardtii in controlled environments. I measured the change in fitness after a few hundred generations and determined how much variation had arisen among replicate populations and among populations with different histories. I applied a similar approach to study the importance of history in extinctions, and measured rates of extinction in populations with different histories. I found that evolution is much less repeatable in small than in large populations because history is more constraining and selection less efficient in small than in large populations. There is also a significant effect of sex and recombination on the repeatability of evolution at the fitness level, but this effect is highly dependent on the environment of selection. Sex can increase the importance of chance or history in some environments, but lower their importance in others, thereby leading to convergence or divergence depending on the environment. Thirdly, I found that the importance of history during evolution does not appear to come from the accumulation of past evolutionary selection pressures, but rather comes from only the most recent selection pressure as it determines genetic correlations for growth between different environments and the amount of genetic variance. Finally, I found that extinction risks are extremely high during continuous environmental deterioration, although a history of sexual reproduction and phenotypic plasticity play an important role in adaptation. By focusing not solely on the effect of treatments on mean trait values, but also on the variance that arises in our evolution experiments, we can gain a better understanding of the contribution that chance and history make to evolution. The repeatability of evolution can therefore inform us about the adaptive vs. stochastic nature of the diversity we see today, and about the specificity or generality of evolutionary outcomes.

As the worldwide demand for recombinant proteins and valuable metabolites continues to grow, and as the biological toolset at our disposal continues to expand, the development of novel, robust, and effective platforms for the production of these bioproducts represents an area of ever-increasing interest. Although many such bioprocesses are currently economically viable, many more, though holding considerable promise, remain uncompetitive. The development of novel, more productive systems increases the versatility and industrial applications of bioprocesses. The work described in this thesis explores several aspects of bioprocessing, both on the upstream side, concerned with the development of novel recombinant protein expression platforms or the isolation of novel genes with products possessing characteristics of interest, and on the downstream side, through the improvement of fermentation-based bioprocesses. Thirty-six homoplasmic recombinant strains of the microalga Chlamydomonas reinhardtii were developed having integrated genes for phytase or xylanase under the control of psbA and psbD promoters, codon optimized using novel algorithms, at two different genetic loci, in chloroplasts, to be used as novel animal feed additives. Enzyme production was characterized, and results, when compared to other published work in this field, may provide insight into the factors impacting recombinant protein production in microalgae. Using a “bio-prospecting approach”, the microflora of the digestive tract of a Canadian beaver was screened for cellulase-producing microorganisms. Although the screening approach did successfully identify a novel β-glucosidase gene from an isolated strain of Bacillus thuringiensis, the sequence was not significantly different from those already characterized. Two bioprocessing studies were performed to improve recombinant protein production in Pichia pastoris. In the first, the composition of standard Basal Salt Medium (BSM) was systematically optimized for the production of recombinant phytase, and the optimized media produced significantly more enzyme than the standard one, while also containing significantly reduced concentrations of KH2PO4 and MgSO4·7H2O (27.9 g/l and 4.8 g/l respectively), lowering the price of process inputs. The second was based on the screening of unconventional carbon sources for candidates that could sustain the growth and enzyme production using the same P. pastoris strain. Fructose and ethanol have shown to be viable alternatives to glucose or glycerol as sole carbon sources, and provide flexibility in terms of process design.

This thesis studies the effects of various concentrations of sodium chloride on growth and the content of secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii. The total content of phenolic compounds, flavonoids and total antioxidant capacity was analyzed by spectrophotometry. In the context of growth, also the content of photosynthetic pigments was determined. Further, the amount of selected metabolites was determined by HPLC/MS-MS. Cultivation of microalgae with increased NaCl inhibited cell growth and production of photosynthetic pigments. Conversely, higher levels of NaCl have proven to stimulate the synthesis of complete phenolic compounds and flavonoids. Similarly, the amount of phenolic acids was significantly influenced by the effect of increasing NaCl concentration, while the total antioxidant capacity of the microalgae also increased. These results confirm the involvement of phenolic compounds in the defense mechanism of unicellular algae Ch. reinhardtii against the observed stress factor.

Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa. / This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass.

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.

Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa. / This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass.

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.

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.

The aim of this research was to increase the rate of cell division in algae by exploring the effect of combinations of lasers of various wavelengths. Literature search has identified a gap in knowledge of the potential for increase in efficiency of the electron transition between photosystem II and photosystem I. This through the use of several wavelengths of blue and or red lasers, including 405 nm, 450, and 473 nm, 635 nm, 650 nm, 680 nm, 685 nm and 700 nm to generate photons with energies more closely matching the absorption spectra of algae receptors known as pigments. This investigation underpins the realisation that photons emanating from a specific laser are absorbed by algae pigments because there is a much closer match between the emission spectrum of the laser and the absorption spectrum of the pigments within the photosystems of algae. This research examined all of the available laser wavelengths in particular combinations; the resultant data contributed to the assembly of a matrix that illustrates the most appropriate laser combinations that promote cell division within algae. Chlamydomonas reinhardtii algae cells successfully grew and divided under exposure to both the blue laser, red laser and that of white light LED when each was applied individually or combined in a sequence. The order of the sequence of using the red and blue lasers in specific cases was important. The pH was maintained between 6.9 and 7.7, with temperatures maintained between 19.00 and 25.00 ºC. For the blue lasers, the laboratory results were as follows, (irradiation time was 12 hours every time): • 405 nm blue laser produced 1.8 x cell division of the white light LED. • For 450 nm blue laser: the white light LED produced 1.5 x cell division of the blue laser 450 nm. • 473 nm blue laser produced 2 x cell division of the white light LED. • 405 nm blue laser produced 3.6 x cell division of natural day light. • 450 nm blue laser produced 1.4 x cell division of natural day light. • 473 nm blue laser produced 4 x cell division of natural day light. For the red lasers, the laboratory results were as follows, (irradiation time was 12 hours every time): • For 635 nm red laser: the white light LED produced 4 x cell division of the red laser 635 nm. • 650 nm red laser produced 1.96 x cell division of the white light LED. • 680 nm red laser produced 2.3 x cell division of the white light LED. • For 685 nm red laser: white light LED produced 1.22 x cell division of the red laser 685 nm. • 700 nm red laser produced 1.35 x cell division of the white light LED. • For 635 nm red laser: the natural day light produced 2 x cell division of the red laser 635 nm. • 650 nm red laser produced 3.9 x cell division of natural day light. • 680 nm red laser produced 4.6 x cell division of natural day light. • 685 nm red laser produced 1.6 x cell division of natural day light. • 700 nm red laser produced 2.7 x cell division of natural day light. For the combination of blue and red lasers, the laboratory results were as follows, (irradiation time was 12 hours every time): • First combination: 405 nm blue laser followed by a combination of 680 nm and 700 nm red lasers produced 4.86 x cell division of the white light LED. • Second combination: 473 nm blue laser followed by a combination of 680 nm and 700 nm red lasers produced 4.66 x cell division of the white light LED. • Third combination: a combination of 680 nm and 700 nm red lasers produced 4.43 x cell division of the white light LED.