Plant secondary products such as flavonoids have a variety of roles in plants including UV protection, antifeedant activity, pollinator attraction, stress response, flavor, and many more. These compounds also have effects on human physiology. Glucosylation is an important modification of many flavonoids and other plant secondary products. In grapefruit, glucosylation is important in the synthesis of the bitter compound naringin and several flavonoid glucosyltransferase (GT) enzymes have been characterized from young grapefruit leaf tissue. To study structure and function of flavonoid GTs, it is necessary to isolate cDNA’s that can be cloned and manipulated. In prior work, the plant secondary product glucosyltransferase (PSPG) box was used to identify putative GT clones. We report on results from experiments to test the hypothesis that PGT clones 4 and 11 are plant secondary product GTs, specifically flavonoid GTs. Previously, PGT 4 was cloned into a bacterial expression system, however all protein was localized into inclusion bodies and GT activity could not be tested. For this work, recombinant PGT 4 and PGT 11 were transformed into yeast and the proteins expressed and screened for glucosyltransferase activity with a variety of flavonoid substrates including flavanones, flavones, and flavonols.

Glucosyltransferases (GTs) are enzymes which perform glucosylation reactions, which involve attaching a UDP-activated glucose molecule to acceptor molecules specifi c to the enzyme. The enzyme which our lab focuses its research on is a fl avonol-specifi c 3-OGT found in Citrus paradisi, or grapefruit (Cp3GT). This enzyme is part of the class of enzymes known as fl avonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Cp3GT in an attempt to discover the residues important for substrate and regiospecifi city. In this study, we are testing the basis of substrate septicity of Cp3GT. We hypothesize that incorporation of fi ve amino acids specifi c to Citrus sinensis GT (CsGT) into Cp3GT at 308th position may facilitate mCp3GT to use anthocyanidins as one of the substrates. We report our fi ndings thus far concerning the addition of specifi c residues to the Cp3GT’s amino acid sequence based on an alignment with the sequence of a putative fl avonoid GT found in Citrus sinensis.

Flavonoids are a class of plant metabolites with a C6-C3-C6 structure. They are responsible for a large range of biological functions including UV protection, pigmentation, and anti-microbial properties. Citrus paradisi, the grapefruit, contains a wide variety of flavonoids, including the target flavonols which are characterized by a hydroxyl group at the C3 position. A glucose molecule is added to flavonols by 3-Oglucosyltransferases (3-O-GTs). C. paradisi F3-O-GT only glucosylates flavonols; however, Vitis vinifera (grape) 3-O-GT can accept both flavonols and anthocyanidins. The two enzymes have some identity with one another but sequence alignment pinpointed several areas of non-homology. Homology modeling using the crystallized structure of the V. vinifera 3-GT revealed sites within the non-homologous areas that could influence the binding site most directly. The 382 site was of particular interest with arginine in C. paradisi changed to tryptophan in V. vinifera, a much bulkier and non-charged amino acid. Site-directed mutagenis was performed to form the R382W mutant line and transformed into yeast for expression after induction with methanol. Western blot was used to determine the optimal protein induction time, after which the cells were harvested and broken to extract the proteins. Isolation and purification of the protein in question allows for enzyme analysis. This is performed by measuring incorporation of radioactive glucose onto various substrates from each flavonoid class. High counts indicate that the enzyme is active upon the substrate while low counts indicate little to no activity. Characterization will also be performed by varying reaction conditions. Thus, the optimal pH, temperature, substrate quantity, enzyme quantity, and reaction duration can be determined for this specific mutant. These experiments will determine if the R382W mutation has a significant impact on the substrate specificity or reaction conditions for the enzyme. A change in activity to include other classes of flavonoids besides flavonols indicates that the mutation site has a direct impact on the conformation of the binding site. Failure of the mutation to change substrate specificity still provides valuable information for the structure and function of the enzyme. This has implications for engineering enzymes to perform specific functions.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-08-09T15:54:02Z No. of bitstreams: 1 DissDMC.pdf: 1717798 bytes, checksum: fff511d67fecb091c6e4ccf4fa3cd068 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-09T17:30:02Z (GMT) No. of bitstreams: 1 DissDMC.pdf: 1717798 bytes, checksum: fff511d67fecb091c6e4ccf4fa3cd068 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-09T17:30:08Z (GMT) No. of bitstreams: 1 DissDMC.pdf: 1717798 bytes, checksum: fff511d67fecb091c6e4ccf4fa3cd068 (MD5) / Made available in DSpace on 2017-08-09T17:33:58Z (GMT). No. of bitstreams: 1 DissDMC.pdf: 1717798 bytes, checksum: fff511d67fecb091c6e4ccf4fa3cd068 (MD5) Previous issue date: 2016-12-19 / Não recebi financiamento / This study presents a rescue of the studies graduate institutionalization in Brazil, since its emergence, discussing its trajectory, as well the expansion of the Studies Graduate National System. As a special focus, it was adopted a clipping about its evolution in the Federal University of São Carlos (UFSCar), which is a strategic part of Ministry of Education expansion policy context. The present research was developed through a dense bibliographical revision and an empirical investigation, that permitted to present the history of its implementation since 1930s, considering the appearance of the first universities in the Brazil. In front of this expansion, it was given a focus in the evolution in numbers of UFSCar's studies graduate programs, showing the growing and the challenges faced in the last decades. Being the Studies Graduate be a strategic factor for the development of a country, it is demonstrated through results of this study the necessity of propositions and adoptions of future actions in order that allow a quantitative and qualitative growth of the graduate studies in the UFSCar and at Brazil level. / O estudo apresenta um resgate da institucionalização da pós-graduação no Brasil, desde seu surgimento, discorrendo sobre sua trajetória, bem como a expansão do Sistema Nacional de Pós-Graduação. Como foco especial, foi adotado um recorte sobre sua evolução na Universidade Federal de São Carlos (UFSCar), a qual faz parte estratégica do contexto da política do Ministério da Educação. A presente pesquisa se desenvolve por meio de uma densa revisão bibliográfica e também uma investigação empírica, que permite apresentar um histórico da implementação da pós-graduação brasileira desde a década de 1930, considerando o surgimento das primeiras universidades no País. Frente a esta expansão, enfatizou-se a evolução dos programas de pós-graduação na UFSCar, mostrando seu crescimento e os desafios enfrentados nas últimas décadas. Sendo a pós graduação um fator estratégico para o país, demonstrou-se através dos resultados deste estudo a necessidade da proposição e adoção de ações futuras, de forma que possibilitem um crescimento quantitativo e qualitativo da pós-graduação na UFSCar e a nível de Brasil.

Ce travail prend pour thème général les relations entre, d’un côté, les segments actifs et liés à la science et à l’université au Brésil et, de l’autre, le champ de la « politique » ainsi que les politiques publiques qui en proviennent. Pour comprendre ces rapports, il faut mettre en relation des processus sociaux plus amples, comme la constitution du travail scientifique en tant que pratique sociale et professionnelle légitime, dotée d’un règlement minimum, de la reconnaissance mutuelle et d’un certain degré de consensus entre ses participants pour ce qui est des valeurs et intérêts qui confèrent à la fois une singularité et une différenciation sociale à ce groupe et servent de référence aux disputes internes. La création et la consolidation d’institutions gouvernementales ayant joué un rôle actif dans la structuration des masters/doctorats ont également été mises en relief du fait qu’elles expriment, dans des logiques bureaucratiques et administratives, les arrangements et les rapports de force mis en oeuvre par les secteurs gouvernementaux et l’université. En ce sens, nous avons pris le 4e Plano Nacional de Pós-graduação (Plan national de master/doctorat) comme une expression et une référence fondamentale des valeurs hégémoniques orientant les politiques et, en même temps, comme plan d’action formalisé, délimitant le cours de l’action gouvernementale. Sachant que l’un des principes de formation et de gestion des politiques de financement de la science est la « participation » des agents liés à l’université (soit, le « public cible » des politiques), l’analyse s’est concentrée sur les conceptions et représentations sociales du rôle des scientifiques et chercheurs dans les processus de décision, sur leurs critères et conditions d’accès dans les arènes d’influence et les espaces de pouvoir et de contrôle bureaucratique de l / O presente trabalho tem como foco geral as relações entre os segmentos atuantes e ligados à ciência e à universidade no Brasil e o campo da “política”, bem como as políticas públicas que daí advêm. Na compreensão destas relações estão interligados processos sociais mais amplos, como a constituição do trabalho científico como prática social e profissional legítima, dotada de um regramento mínimo, do reconhecimento mútuo e de algum grau de consenso entre os seus praticantes acerca dos valores e interesses que, ao mesmo tempo, conferem singularidade e diferenciação social ao grupo e referenciam as disputas internas. Também foi dada ênfase à criação e à consolidação de instituições de governo que desempenharam um papel ativo na estruturação da pós-graduação, tendo em vista que as mesmas expressam, dentro de lógicas burocráticas e administrativas, os arranjos e relações de força empreendidas pelos setores governamentais e a universidade. Neste sentido, tomamos o 4º Plano Nacional de Pós-graduação como uma expressão e referência fundamental dos valores hegemônicos que orientam as políticas e, simultaneamente, como plano de ação formalizado, que delimita o curso da ação governamental. Considerando que as políticas de apoio à ciência têm como um de seus princípios de formação e gestão a “participação” dos agentes ligados à universidade (ou seja, o próprio “público alvo” das políticas), a análise recai nas concepções e representações sociais do papel dos cientistas e pesquisadores nos processos decisórios, nos critérios e nas condições de acesso dos mesmos às arenas de influência e espaços de poder e de controle burocrático da universidade. / The main focus of this work is on the relations between segments active in and connected to science and Brazilian universities and the field of "politics", as well as the resulting government policy. Understanding these relations as broader interconnected social processes, such as the constitution of scientific work as a legitimate social and professional practice in possession of a minimum rule set, mutual recognition and a certain degree of consensus among those practicing it regarding its values and interests, which at the same time confers singularity and social differentiation to the group and serves as a reference for its internal disputes. Emphasis has also been given to the creation and consolidation of government institutions that have played an active role in structuring graduate studies, considering that these express, within bureaucratic and administrative logic, the arrangements and power relations undertaken by governmental sectors and the universities. In this sense, we understand the 4º Plano Nacional de Pós-graduação (4th National Post-Graduate Studies Plan) as an expression of and fundamental reference to the hegemonic values that guide the policies, and simultaneously as a formal plan of action delimiting governmental course of action. Considering that policies in support of science hold as one of their principles of education and management the “participation” of agents tied to the university (in other words, the “target”of the policies), the analysis falls back on social conceptions and representations regarding the role of scientists and researchers in decision making processes, criteria and their conditions of access to the arenas of influence and spaces of power and bureaucratic control of the university.

Ce travail prend pour thème général les relations entre, d’un côté, les segments actifs et liés à la science et à l’université au Brésil et, de l’autre, le champ de la « politique » ainsi que les politiques publiques qui en proviennent. Pour comprendre ces rapports, il faut mettre en relation des processus sociaux plus amples, comme la constitution du travail scientifique en tant que pratique sociale et professionnelle légitime, dotée d’un règlement minimum, de la reconnaissance mutuelle et d’un certain degré de consensus entre ses participants pour ce qui est des valeurs et intérêts qui confèrent à la fois une singularité et une différenciation sociale à ce groupe et servent de référence aux disputes internes. La création et la consolidation d’institutions gouvernementales ayant joué un rôle actif dans la structuration des masters/doctorats ont également été mises en relief du fait qu’elles expriment, dans des logiques bureaucratiques et administratives, les arrangements et les rapports de force mis en oeuvre par les secteurs gouvernementaux et l’université. En ce sens, nous avons pris le 4e Plano Nacional de Pós-graduação (Plan national de master/doctorat) comme une expression et une référence fondamentale des valeurs hégémoniques orientant les politiques et, en même temps, comme plan d’action formalisé, délimitant le cours de l’action gouvernementale. Sachant que l’un des principes de formation et de gestion des politiques de financement de la science est la « participation » des agents liés à l’université (soit, le « public cible » des politiques), l’analyse s’est concentrée sur les conceptions et représentations sociales du rôle des scientifiques et chercheurs dans les processus de décision, sur leurs critères et conditions d’accès dans les arènes d’influence et les espaces de pouvoir et de contrôle bureaucratique de l / O presente trabalho tem como foco geral as relações entre os segmentos atuantes e ligados à ciência e à universidade no Brasil e o campo da “política”, bem como as políticas públicas que daí advêm. Na compreensão destas relações estão interligados processos sociais mais amplos, como a constituição do trabalho científico como prática social e profissional legítima, dotada de um regramento mínimo, do reconhecimento mútuo e de algum grau de consenso entre os seus praticantes acerca dos valores e interesses que, ao mesmo tempo, conferem singularidade e diferenciação social ao grupo e referenciam as disputas internas. Também foi dada ênfase à criação e à consolidação de instituições de governo que desempenharam um papel ativo na estruturação da pós-graduação, tendo em vista que as mesmas expressam, dentro de lógicas burocráticas e administrativas, os arranjos e relações de força empreendidas pelos setores governamentais e a universidade. Neste sentido, tomamos o 4º Plano Nacional de Pós-graduação como uma expressão e referência fundamental dos valores hegemônicos que orientam as políticas e, simultaneamente, como plano de ação formalizado, que delimita o curso da ação governamental. Considerando que as políticas de apoio à ciência têm como um de seus princípios de formação e gestão a “participação” dos agentes ligados à universidade (ou seja, o próprio “público alvo” das políticas), a análise recai nas concepções e representações sociais do papel dos cientistas e pesquisadores nos processos decisórios, nos critérios e nas condições de acesso dos mesmos às arenas de influência e espaços de poder e de controle burocrático da universidade. / The main focus of this work is on the relations between segments active in and connected to science and Brazilian universities and the field of "politics", as well as the resulting government policy. Understanding these relations as broader interconnected social processes, such as the constitution of scientific work as a legitimate social and professional practice in possession of a minimum rule set, mutual recognition and a certain degree of consensus among those practicing it regarding its values and interests, which at the same time confers singularity and social differentiation to the group and serves as a reference for its internal disputes. Emphasis has also been given to the creation and consolidation of government institutions that have played an active role in structuring graduate studies, considering that these express, within bureaucratic and administrative logic, the arrangements and power relations undertaken by governmental sectors and the universities. In this sense, we understand the 4º Plano Nacional de Pós-graduação (4th National Post-Graduate Studies Plan) as an expression of and fundamental reference to the hegemonic values that guide the policies, and simultaneously as a formal plan of action delimiting governmental course of action. Considering that policies in support of science hold as one of their principles of education and management the “participation” of agents tied to the university (in other words, the “target”of the policies), the analysis falls back on social conceptions and representations regarding the role of scientists and researchers in decision making processes, criteria and their conditions of access to the arenas of influence and spaces of power and bureaucratic control of the university.

Ce travail prend pour thème général les relations entre, d’un côté, les segments actifs et liés à la science et à l’université au Brésil et, de l’autre, le champ de la « politique » ainsi que les politiques publiques qui en proviennent. Pour comprendre ces rapports, il faut mettre en relation des processus sociaux plus amples, comme la constitution du travail scientifique en tant que pratique sociale et professionnelle légitime, dotée d’un règlement minimum, de la reconnaissance mutuelle et d’un certain degré de consensus entre ses participants pour ce qui est des valeurs et intérêts qui confèrent à la fois une singularité et une différenciation sociale à ce groupe et servent de référence aux disputes internes. La création et la consolidation d’institutions gouvernementales ayant joué un rôle actif dans la structuration des masters/doctorats ont également été mises en relief du fait qu’elles expriment, dans des logiques bureaucratiques et administratives, les arrangements et les rapports de force mis en oeuvre par les secteurs gouvernementaux et l’université. En ce sens, nous avons pris le 4e Plano Nacional de Pós-graduação (Plan national de master/doctorat) comme une expression et une référence fondamentale des valeurs hégémoniques orientant les politiques et, en même temps, comme plan d’action formalisé, délimitant le cours de l’action gouvernementale. Sachant que l’un des principes de formation et de gestion des politiques de financement de la science est la « participation » des agents liés à l’université (soit, le « public cible » des politiques), l’analyse s’est concentrée sur les conceptions et représentations sociales du rôle des scientifiques et chercheurs dans les processus de décision, sur leurs critères et conditions d’accès dans les arènes d’influence et les espaces de pouvoir et de contrôle bureaucratique de l / O presente trabalho tem como foco geral as relações entre os segmentos atuantes e ligados à ciência e à universidade no Brasil e o campo da “política”, bem como as políticas públicas que daí advêm. Na compreensão destas relações estão interligados processos sociais mais amplos, como a constituição do trabalho científico como prática social e profissional legítima, dotada de um regramento mínimo, do reconhecimento mútuo e de algum grau de consenso entre os seus praticantes acerca dos valores e interesses que, ao mesmo tempo, conferem singularidade e diferenciação social ao grupo e referenciam as disputas internas. Também foi dada ênfase à criação e à consolidação de instituições de governo que desempenharam um papel ativo na estruturação da pós-graduação, tendo em vista que as mesmas expressam, dentro de lógicas burocráticas e administrativas, os arranjos e relações de força empreendidas pelos setores governamentais e a universidade. Neste sentido, tomamos o 4º Plano Nacional de Pós-graduação como uma expressão e referência fundamental dos valores hegemônicos que orientam as políticas e, simultaneamente, como plano de ação formalizado, que delimita o curso da ação governamental. Considerando que as políticas de apoio à ciência têm como um de seus princípios de formação e gestão a “participação” dos agentes ligados à universidade (ou seja, o próprio “público alvo” das políticas), a análise recai nas concepções e representações sociais do papel dos cientistas e pesquisadores nos processos decisórios, nos critérios e nas condições de acesso dos mesmos às arenas de influência e espaços de poder e de controle burocrático da universidade. / The main focus of this work is on the relations between segments active in and connected to science and Brazilian universities and the field of "politics", as well as the resulting government policy. Understanding these relations as broader interconnected social processes, such as the constitution of scientific work as a legitimate social and professional practice in possession of a minimum rule set, mutual recognition and a certain degree of consensus among those practicing it regarding its values and interests, which at the same time confers singularity and social differentiation to the group and serves as a reference for its internal disputes. Emphasis has also been given to the creation and consolidation of government institutions that have played an active role in structuring graduate studies, considering that these express, within bureaucratic and administrative logic, the arrangements and power relations undertaken by governmental sectors and the universities. In this sense, we understand the 4º Plano Nacional de Pós-graduação (4th National Post-Graduate Studies Plan) as an expression of and fundamental reference to the hegemonic values that guide the policies, and simultaneously as a formal plan of action delimiting governmental course of action. Considering that policies in support of science hold as one of their principles of education and management the “participation” of agents tied to the university (in other words, the “target”of the policies), the analysis falls back on social conceptions and representations regarding the role of scientists and researchers in decision making processes, criteria and their conditions of access to the arenas of influence and spaces of power and bureaucratic control of the university.

This study examines the educational persistence of women of African descent (WOAD) in pursuit of a doctorate degree at universities in the southeastern United States. WOAD are women of African ancestry born outside the African continent. These women are heirs to an inner dogged determination and spirit to survive despite all odds (Pulliam, 2003, p. 337).This study used Ellis’s (1997) Three Stages for Graduate Student Development as the conceptual framework to examine the persistent strategies used by these women to persist to the completion of their studies.