Thesis advisor: Abhishek Chatterjee / Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2018-01-31T21:07:06Z No. of bitstreams: 1 Antonio Fabio Reis Figueiredo.pdf: 2917269 bytes, checksum: 41197e6d18b286701e9cf3cfa3160e77 (MD5) / Made available in DSpace on 2018-01-31T21:07:06Z (GMT). No. of bitstreams: 1 Antonio Fabio Reis Figueiredo.pdf: 2917269 bytes, checksum: 41197e6d18b286701e9cf3cfa3160e77 (MD5) Previous issue date: 2017-02-15 / The use of selected yeasts is an important aspect in the production of quality beers, since in addition to the production of ethanol and carbon dioxide they contribute significantly to the formation of secondary compounds, which confer the flavor of the beer. These yeasts also settle easily in the middle, avoiding undesired contaminations, in addition to generally presenting high rates of yield, productivity and fermentation efficiency. Selenium (Se) is a mineral that performs antioxidant functions, being essential as trace element to human health and its deficiency is correlated to the occurrence of several diseases. Due to these properties, the incorporation of Se into some food and / or food supplements associated with yeast is already a reality. Considering that beer is the most consumed alcoholic drink in the world, this research aimed to select yeasts to produce artisan beer supplemented with selenium. To do so, using the multivariate analysis technique, the selection of yeasts with potential for brewing was initially performed, and three strains (SC52, SC57 and SC82) were selected; Then applying the Doehlert factorial design and using the Response Surface Methodology technique, the optimization of the fermentation parameters (fermentation temperature and selenium concentration) for the production of beer supplemented with selenium was carried out. The yeasts SC52 and SC82 presented significant responses for the production of the supplemented beer, and the optimum values (Se concentration and fermentation temperature) were 11 mg L-1 and 25.0? C for yeast SC52 and 12 mg L-1 and 23.5? C for yeast SC82; (CSeSC52 and CSeSC82) were produced on a microbrewery scale, the fermentation parameters of the process were evaluated and total selenium and selenium species present in yeast and beers were quantified. The beers produced presented fermentative results within the commercial standards, being recommended by DDR (Recommended Daily Dose) of Se, a maximum consumption of approximately 600 mL of CSeSC52 beer and 740 mL of CSeSC82 beer. / O uso de leveduras selecionadas constitui aspecto relevante na produ??o de cervejas de qualidade, pois al?m da produ??o de etanol e g?s carb?nico contribuem significativamente para a forma??o de compostos secund?rios, os quais conferem o flavor da cerveja. Estas leveduras tamb?m se estabelecem facilmente no meio, evitando contamina??es indesejadas, al?m de apresentarem, geralmente, altas taxas de rendimento, produtividade e efici?ncia na fermenta??o. O sel?nio (Se) ? um mineral que desempenha fun??es antioxidantes, sendo essencial como elemento tra?o ? sa?de humana e a sua defici?ncia est? correlacionada ? ocorr?ncia de v?rias doen?as. Devido a estas propriedades, a incorpora??o do Se em alguns alimentos e/ou suplementos alimentares associados a leveduras j? ? uma realidade. Considerando que a cerveja constitui a bebida alco?lica mais consumida no mundo, esta pesquisa objetivou selecionar leveduras para produzir cerveja artesanal suplementada com sel?nio. Para tanto, a partir do uso da t?cnica de an?lise multivariada, inicialmente foi realizada a sele??o de leveduras com potencial para produ??o de cerveja, sendo selecionadas tr?s cepas (SC52, SC57 e SC82); em seguida, aplicando o planejamento fatorial Doehlert e utilizando a t?cnica de Metodologia de Superf?cie de Respostas, realizou-se a otimiza??o dos par?metros fermentativos (temperatura de fermenta??o e concentra??o de sel?nio) para a produ??o de cerveja suplementada com sel?nio. As leveduras SC52 e SC82 apresentaram respostas significativas para a produ??o da cerveja suplementada, sendo que os valores ?timos (concentra??o de Se e Temperatura de fermenta??o) obtidos foram de 11 mg L-1 e 25,0 ?C para a levedura SC52 e 12 mg L-1 e 23,5 ?C para a levedura SC82; por fim, foram produzidas duas cervejas artesanais suplementadas com sel?nio (CSeSC52 e CSeSC82) em escala de microcervejaria, sendo avaliados os par?metros fermentativos do processo e quantificados o sel?nio total e as esp?cies de sel?nio presentes nas leveduras e nas cervejas. As cervejas produzidas apresentaram resultados fermentativos dentro dos padr?es comerciais, sendo recomendado em virtude da DDR (Dose Di?ria Recomendada) de Se, um consumo m?ximo de aproximadamente 600 mL da cerveja CSeSC52 e 740 mL da cerveja CSeSC82.

Processos fermentativos que visam um salto tecnológico na produção de etanol, que potencialize o processo fermentativo, destaca-se a fermentação extrativa a vácuo. Assim buscou-se selecionar linhagens tolerantes à alta pressão osmótica para serem empregadas em processo de fermentação extrativa. Foram realizadas seleções a partir de 444 cepas de leveduras oriundas da biodiversidade das Usinas Brasileiras, que fazem parte do banco de leveduras do Centro de Tecnologia Canavieira (CTC), tendo como base o meio formulado com melaço e vinhaça para simular a fermentação extrativa. A seleção se baseou em submeter as linhagens às fermentações em condições crescentes de estresse osmótico, buscando destacar linhagens com a tolerância desejada. Para tal, as linhagens em mistura foram submetidas à propagação durante 93 gerações em meios com crescentes quantidades de melaço e vinhaça. Em etapa seguinte, 90 cepas foram isoladas e avaliadas mediante o crescimento (μmax e biomassa) em condição de estresse osmótico e genotipadas. O método usado na genotipagem foi o PCR - microssatélite, o qual permitiu verificar se as cepas resultantes da seleção eram cepas industriais (BG-1, CAT-1, PE-2 e SA-1) ou cepas selvagens e/ou predominantes. Foram utilizados 5 pares de primers representando 5 diferentes loci. A genotipagem e a avaliação do crescimento em condições de estresse osmótico, baseado em características genéticas e fisiológicas, permitiu identificar 24 linhagens com potencial de tolerância a pressão osmótica. As cepas com desempenho superior foram submetidas à avaliação de crescimento em placa e as sete mais tolerantes à pressão osmótica foram selecionadas e utilizadas em reciclos fermentativos empregando-se mosto constituído de melaço e vinhaça. A linhagem com as melhores características para a fermentação com alta pressão osmótica foi avaliada em condições de fermentação extrativa à vácuo. A linhagem selecionada, denominada de F1-5, mostrou-se com grande potencial para a fermentação extrativa quando comparada com a referência CAT-1. / Fermentative processes that aim for technology innovations in the ethanol production, which improve the fermentative process, emphasizing the in vacuum extractive fermentation. So we have selected strains tolerant for high osmotic pressure to be in extractive fermentation process for ethanol production. We 444 yeast strains from Brazilian Mills biodiversity, which Sugarcane Technology Center\'s (CTC) yeast bank, based on a medium formulated with molasses and vinasse, to simulate the extractive fermentation. The selection was based on submiting strains to the fermentation in in high osmotic stress condition, trying to feature strains with desired tolerance, so, the mixed strains were submitted to propagation for 93 generations in medium with increasing amounts of molasses and vinasse. In the following step, 90 strains were isolated and evaluated by growth (μmax and biomass) in osmotic stress condition and genotyped. The method used in genotyping was the PCR - microsatellite, which enable to estimate if resulting selection strains were from industrial strains (BG-1, CAT-1, PE-2 and SA-1) or wild strains and / or prevalent. 5 pairs of primers were used representing 5 different loci. The genotyping and the growth evaluation in osmotic stress conditions allowed identify strains based on genetic and physiological characteristics, making possible to identify 24 strains with a potential tolerance to osmotic pressure. The strains with better performance were submitted to evaluation growth on boards and the 7 most tolerant to osmotic pressure were selected and used in fermentative recycles using medium containing molasses and vinasse. The strain with the best features in the fermentation in high osmotic pressure was evaluated in vacuum extractive fermentation conditions. The selected strain, named F1-5, proved to have a great potential for extractive fermentation when compared to the reference CAT-1.

Processos fermentativos que visam um salto tecnológico na produção de etanol, que potencialize o processo fermentativo, destaca-se a fermentação extrativa a vácuo. Assim buscou-se selecionar linhagens tolerantes à alta pressão osmótica para serem empregadas em processo de fermentação extrativa. Foram realizadas seleções a partir de 444 cepas de leveduras oriundas da biodiversidade das Usinas Brasileiras, que fazem parte do banco de leveduras do Centro de Tecnologia Canavieira (CTC), tendo como base o meio formulado com melaço e vinhaça para simular a fermentação extrativa. A seleção se baseou em submeter as linhagens às fermentações em condições crescentes de estresse osmótico, buscando destacar linhagens com a tolerância desejada. Para tal, as linhagens em mistura foram submetidas à propagação durante 93 gerações em meios com crescentes quantidades de melaço e vinhaça. Em etapa seguinte, 90 cepas foram isoladas e avaliadas mediante o crescimento (μmax e biomassa) em condição de estresse osmótico e genotipadas. O método usado na genotipagem foi o PCR - microssatélite, o qual permitiu verificar se as cepas resultantes da seleção eram cepas industriais (BG-1, CAT-1, PE-2 e SA-1) ou cepas selvagens e/ou predominantes. Foram utilizados 5 pares de primers representando 5 diferentes loci. A genotipagem e a avaliação do crescimento em condições de estresse osmótico, baseado em características genéticas e fisiológicas, permitiu identificar 24 linhagens com potencial de tolerância a pressão osmótica. As cepas com desempenho superior foram submetidas à avaliação de crescimento em placa e as sete mais tolerantes à pressão osmótica foram selecionadas e utilizadas em reciclos fermentativos empregando-se mosto constituído de melaço e vinhaça. A linhagem com as melhores características para a fermentação com alta pressão osmótica foi avaliada em condições de fermentação extrativa à vácuo. A linhagem selecionada, denominada de F1-5, mostrou-se com grande potencial para a fermentação extrativa quando comparada com a referência CAT-1. / Fermentative processes that aim for technology innovations in the ethanol production, which improve the fermentative process, emphasizing the in vacuum extractive fermentation. So we have selected strains tolerant for high osmotic pressure to be in extractive fermentation process for ethanol production. We 444 yeast strains from Brazilian Mills biodiversity, which Sugarcane Technology Center\'s (CTC) yeast bank, based on a medium formulated with molasses and vinasse, to simulate the extractive fermentation. The selection was based on submiting strains to the fermentation in in high osmotic stress condition, trying to feature strains with desired tolerance, so, the mixed strains were submitted to propagation for 93 generations in medium with increasing amounts of molasses and vinasse. In the following step, 90 strains were isolated and evaluated by growth (μmax and biomass) in osmotic stress condition and genotyped. The method used in genotyping was the PCR - microsatellite, which enable to estimate if resulting selection strains were from industrial strains (BG-1, CAT-1, PE-2 and SA-1) or wild strains and / or prevalent. 5 pairs of primers were used representing 5 different loci. The genotyping and the growth evaluation in osmotic stress conditions allowed identify strains based on genetic and physiological characteristics, making possible to identify 24 strains with a potential tolerance to osmotic pressure. The strains with better performance were submitted to evaluation growth on boards and the 7 most tolerant to osmotic pressure were selected and used in fermentative recycles using medium containing molasses and vinasse. The strain with the best features in the fermentation in high osmotic pressure was evaluated in vacuum extractive fermentation conditions. The selected strain, named F1-5, proved to have a great potential for extractive fermentation when compared to the reference CAT-1.

Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...