Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against an anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the substrate specificity of Cp3-O-GT. An initial quickscreening assay using radioactive UDP-glucose as a sugar donor suggested there may have been expansion of substrate acceptance. Confirming time course assays did not support this. Additionally, results of these assays show that D344P protein has decreased activity with flavonols as compared to wild type Cp3-O-GT. with no expansion of substrate specificity. Models suggest that a change in protein conformation has resulted in decreased activity.

Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases are the enzymes that mediate glucosylation by transferring glucose from a high energy sugar donor to the acceptor substrates. Our study focuses on the structural and functional analysis of a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) clone from Citrus paradisi that has been characterized previously in our lab. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. Cp-3-O-GT was modeled with a flavonoid 3-O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT- P145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-OGT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at 7-OH position forming prunin. Product identification with other substrates is in progress. Results are being used to revisit and refine the structure model. Structural and functional analysis of flavonoid GTs may contribute to custom design of GTs for the synthesis of novel glucosides by changing glucosylation patterns.

Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. This process can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and anthocyanidins. Homology modeling using the crystallized structure of the V. vinifera GT predicted sites of amino acids that could influence substrate binding site. The 382 position was of particular interest with arginine in C. paradisi and tryptophan in V. vinifera. This change is hypothesized to cause a shift in substrate specificity of the Cp3OGT to accept anthocyanidins as well as flavonols. Site-directed mutagenesis was performed to form the R382W mutant Cp3OGT and transformed into yeast for expression. Western blot determined the optimal protein induction period for the cells, after which the cells were broken to extract the recombinant mutant protein. Purification of the R382W 3GT allowed for enzyme analysis to be performed by measuring the incorporation of radioactive glucose into the reaction product. HPLC will be used to identify reaction products. An enzyme kinetics study will show the extent of any biochemical change in function as a result of this mutation; results will then be incorporated into a refined protein model.

Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity as well as regiospecificity. Several F3GT's characterized from other plants also had the ability to glucosylate anthocyanidins, however the grapefruit F3GT did not. This research is designed to test the hypothesis that specific amino acid residues impart the substrate specificity and regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modelling. Enzyme activity of the mutant F3-GT proteins will be tested with flavonols for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant F3-GT enzyme to test for change in substrate specificity. The result from this study will add to our knowledge of GTs.

Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.

Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against a anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the regio and/or steriospecificity of Cp3-OGT. An initial screening assay has been performed using radioactive UDP- glucose as a sugar donor. Early results indicated that the mutant D344P has particular affinity for flavonols and for diosometin, a flavone. Kinetic assays are being performed to confirm these results. Studies of time course, enzyme concentration, HPLC product analysis, pH optimum and reaction kinetics will be performed to further complete D344P protein characterization.

Made available in DSpace on 2014-06-11T19:31:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-03-02Bitstream added on 2014-06-13T19:21:13Z : No. of bitstreams: 1 wiezzel_acs_dr_mar.pdf: 734223 bytes, checksum: f5797ae72c1b9d765bbc46528a643179 (MD5) / Esta tese resulta de uma investigação que tem por foco o estudo acerca de quanto e como os Programas de Pós-Graduação stricto sensu em Educação das Universidades Estaduais Paulistas contribuem na formação de seus alunos enquanto professores universitários. Os dados coletados são oriundos de análise documental, questionários e entrevistas informais com alunos dos três Programas de Pós-Graduação em Educação em questão. Nesta coleta de dados foram priorizados três aspectos do problema: as necessidades de formação dos pós-graduandos enquanto professores universitários, a atuação dos Programas quanto a esta formação e a opinião dos pós-graduandos sobre as contribuições do Programa em sua formação docente. O trabalho demonstra a ocorrência de esforços dos Programas quanto à formação docente universitária e aponta algumas sugestões que possam vir a desencadear pesquisas futuras, bem como auxiliar na discussão a respeito da formação do professor universitário nos cursos de Pós-Graduação em Educação, e nas Pós-Graduações de forma mais geral. / This thesis is the result of an investigation that focus on how and how much the Post-Graduate Programs on Education of USP, UNICAMP and UNESP, the three State Universities in São Paulo State, contribute in the training of their students as University Professors. The techniques that were used to collect the data were documental analysis, questionnaries and intervieus with students of the three Programs. Three aspects of the problem were stressed: the necessities of the training of the post-graduate students as University Professors, from the Programs act on this training, and the opinion of the students about the contributions of the Program about his/her training. This work demonstrates the occurrence of efforts by the Programs on the training of University Professors and points out some suggestions that can trigger future researches, as well as contribute in the question of the training of Professors in the Post-Graduate Programs on Education and in other Post-Graduate Programs.

A presente pesquisa caracteriza-se como um estudo de caso de caráter exploratório e teve como objetivo mapear a influência do estágio pós-doutoral sobre a produção dos docentes em programas de pós-graduação da Universidade de São Paulo. O estágio pós-doutoral é um item extra na carreira acadêmica que atua como processo de atualização e incremento de conhecimento, por meio de socialização e contato com mainstream da ciência de uma determinada área. O presente estudo trabalhou basicamente utilizando como fonte de dados o sistema Lattes, onde a construção de um índice permitiu uma pesquisa exaustiva e objetiva trabalhando em duas vertentes, uma se pautou no levantamento da produção docente antes e depois da realização do pós-doutorado e outra se pautou na comparação do desempenho acadêmico coletivo entre os pesquisadores que optaram por realizar estágio pós-doutoral e os que optaram por não realizar estágio pós-doutoral. A pesquisa encontra-se circunscrita nas grandes áreas de Biológicas, Engenharias, Exatas e da Terra e Saúde. Com metodologia bifurcada, no panorama ex-ante e ex-post a pesquisa trabalhou com a população de 86 docentes e na comparação entre doutores com e sem realização de estágio pós-doutoral, com uma amostra de 198 docentes. Os resultados da pesquisa apontam que o pós-doutorado possui uma leve influência sobre o aumento de publicações em revistas internacionais Qualis A, possui influência mais visível sobre a produção quando realizado no Exterior e possui impactos positivos diante de um maior intervalo entre a conclusão do doutorado e a realização do pós-doutorado, entre os docentes com e sem pós-doutorado existe uma tendência de acréscimo de produção para os pesquisadores com pós-doutorado, mas essa tendência não é tão forte e incisiva, mesmo após um maior tempo de conclusão do doutorado, diversos casos de pesquisadores com produção e desempenhos similares são vistos, independentemente da realização ou não de pós-doutorado. / This research is an exploratory case study and has as it as objective to map the influence of the post-doctorate stage about production of the professors in graduate studies of the University of São Paulo. The post-doctorate stage is an item extra in academic career and it acts as process of actualization and increment of knowledge, through of socialization and contact with mainstream of the science of a determinate area. The present study worked using as source of data the system Lattes, where construction of an index it allowed an ample and objective research working in two approaches, an as guideline of teacher production before and after of the realization of the post doctor’s degree and other guideline in the comparison of collective academic performance between the researchers that had opted to realize post-doctorate stage and the that had opted not to realize post-doctorate stage. The research is circumscribed in the areas of Biological, Engineering, Exactas and Geosciences and Health. With two methodologies, in the context ex-ante and ex-post the research worked with the population of 86 teachers and in comparison between doctors with and without realization of post-doctorate stage, with a sample of 198 teachers. The results of research indicated that an post doctor’s degree it possess a small tendency of influence in the increase of papers in internationals periodicals Qualis A, possess bigger influence in the production when for the made in out Brazil and possess impacts positives with an greater interval between the conclusion of the doctor’s degree and the realization of post doctor’s degree. Between the teachers with and without post doctor’s degree it exists a tendency of increase of production for the researchers with post doctor’s degree, but this tendency not is so strong, with after a bigger time of conclusion of doctor’s degree, diverse cases of researchers with productions and performances similar are found independent of the realization or not realization of post doctor’s degree.

Neste trabalho o estágio pós-doutoral é visto como um item de treinamento, desenvolvimento e educação próprio da carreira acadêmica, que atua como instrumento de atualização, reciclagem docente e incremento de conhecimento, por meio da socialização e contato entre pesquisadores. A presente pesquisa é exploratória, de abrangência nacional e tem como objetivo principal analisar qual o impacto do estágio pós-doutoral no exterior sobre o desempenho de docentes e programas de pós-graduação brasileiros. Para esse intento o estudo trabalhou em duas vertentes e que produziram resultados convergentes; uma vertente se pautou no levantamento da produção docente antes e depois da realização do pós-doutorado, basicamente utilizando como fonte de dados secundários o sistema Lattes, onde a construção de um Índice de Desempenho - ID, permitiu uma pesquisa exaustiva e objetiva acerca da produção científica e da produção técnica dos participantes, e outra vertente, pautou-se na coleta de dados primários por meio da aplicação de questionário estruturado com escalas psicométricas para mensurar a percepção e o julgamento dos docentes sobre o pós-doutorado e o seu impacto. Foram convidados a participar do survey todos os 2.565 docentes egressos do Programa de Estágio Pós-doutoral no Exterior, atuantes na pós-graduação e que tiveram bolsa da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, no período de 1996 a 2008 (1996, ano de criação do programa, a 2008 quando este foi desmembrado em dois subprogramas); a taxa de retorno dos questionários enviados foi de 38,1% e configurou uma amostra de 978 professores participantes. O presente levantamento cobre todas as regiões geográficas do país e abrange todas as grandes áreas do conhecimento. Com o método de coleta bifurcado, no panorama ex ante e ex post do desempenho acadêmico, a pesquisa encontrou como resultado um impacto moderado do pós-doutorado sobre as publicações e pesquisas e um impacto quase nulo sobre o desempenho dos docentes em atividades técnico-científicas. Em relação ao questionário aplicado, as escalas utilizadas mostraram bons índices de confiabilidade e o resultado acerca das visões e das percepções dos docentes, também não foi entusiasta, registrou-se a percepção de impacto moderado e positivo sobre o trabalho docente e prevaleceu uma tendência de registro do julgamento por parte dos participantes de que o pós-doutorado no exterior possui impacto moderado e positivo sobre as atividades de pesquisa, um impacto menor sobre atividades de ensino-pesquisa e um impacto praticamente inexistente e pouco expressivo sobre as atividades de extensão universitária. Os resultados, no entanto são muito ricos e vão muito além do que os aqui expressos e sintetizados neste resumo, assim convidamos o leitor que se interesse pelo tema a passear por essa tese em sua íntegra, para acessar com maior amplitude às questões levantadas acerca do pós-doutorado e o seu efeito percebido pelos docentes no nível individual e o seu efeito julgado no nível do coletivo, bem como, em especial a influência do pós-doutorado sobre o desempenho docente no que diz respeito ao aumento da produção acadêmica e o cenário desafiador do contexto das produções docentes amplas que vão além das publicações em revistas especializadas e produções científicas no sistema de pós-graduação. / This research consider the post-doctoral stage as an item of training, development and education typical of the academic career, which operates as professor recycling, upgrade and knowledge increase instrument, by means of the socialization and the join contact of researchers professors. The main goal of the present research exploratory is to investigate what is the impact of the post-doctoral stage abroad on the professor\'s performance and programs of Brazilian Graduate Studies. For this purpose the study worked in two fronts and that produced convergent outputs, a front was based in weighing of the professor production before and after of post-doctoral realization basically using as a secondary data source the Lattes system, where the construction of a performance index allowed an ample and objective research of analysis of scientific production and technical production of the participants, and another front structured questionnaires were used for primary data collection via psychometric scales to measure the perception and the judgment of professors about the post-doctoral and its impact. Were invited to join the survey, all the 2.565 egresses professors of the program of post-doctoral stage abroad, active in graduate studies and that have scholarship of the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), in the period 1996-2008 (1996, year of program creation, to 2008 when it was divided into two subprograms); the return rate of the questionnaires sent out was 38.1% which set up a sample of 978 professors participants. This survey covers all geographical regions of Brazil and covers all areas of knowledge. With the collection method bifurcated, in the perspective of academic performance ex- ante and ex-post, the research found a result of moderate impact on the publications and researches and an impact almost null on the professor\'s performance in technical-scientific activities. In relation the questionnaire applied, the scales used presented good levels of reliability and the result about the visions and professors perceptions, also was not enthusiastic, was registered the perception of moderate and positive impact on the professor work and prevailed a tendency de register of judgment that the postdoctoral abroad has moderate and positive impact on the researches activities, an impact less on the teaching-research and an impact practically nonexistent and little expressive in university extension activities. The results nonetheless are very rich and goes beyond the here expressed an synthesized this abstract, thus invite the reader, who has familiarity with Portuguese and that interest in the theme to walk this thesis in its entirety, to access with higher amplitude the questions raised about the postdoctoral studies and its effect judged in level of the collective, as well as in special the influence postdoctoral on the professor performance regarding the increase of scientific production and the defiant scenario of ample professor productions scenario which go beyond of publications in specialized journals and scientific productions in the graduate studies system.

Orientador: Nora Rut Krawczyk / Dissertação (mestrado) ¿ Universidade Estadual de Campinas, Faculdade de Educação. / Made available in DSpace on 2018-08-20T16:38:26Z (GMT). No. of bitstreams: 1 Santos_LucianaAparecidados_M.pdf: 1373220 bytes, checksum: 411c305395f72b7fb8927b69ed3229bd (MD5) Previous issue date: 2012 / Resumo: A defesa do modelo gerencial de universidades corporativas apóia-se na emergência de uma organização não hierárquica, enxuta e flexível, no advento da economia do conhecimento, na redução do prazo de validade do conhecimento, no foco em capacidade de empregabilidade para toda a vida e em uma mudança no mercado de educação. As universidades corporativas geralmente são apresentadas como uma reorganização dos centros de treinamento e desenvolvimento dos departamentos de recursos humanos das grandes empresas. Para alguns autores, as universidades corporativas extrapolam seu foco de atendimento para um público externo, atuando como um ¿guarda-chuva estratégico¿ que abrange diversos públicos e diversas modalidades de cursos. As universidades setoriais funcionam como uma adequação do modelo de universidade corporativa para diferentes setores. Geralmente organizadas e gerenciadas por associações patronais do setor industrial, do setor de transporte, do setor imobiliário, do setor de papel e celulose, etc., oferecem formação, qualificação e treinamento por meio de parcerias com fundações, universidades públicas e privadas. A partir desse quadro de referência, optou-se por uma investigação de natureza qualitativa, que envolve: revisão e análise bibliográfica e, trabalho de campo, por meio de análise documental e entrevistas. Para as entrevistas, identificou-se uma universidade setorial representativa no cenário nacional, nesse caso do setor sucroenergético, com intuito de enriquecer a realização do objetivo da pesquisa. / Abstract: The defense of the managerial model of corporate universities rests in the emergence of a non-hierarchical, leaner and flexible, organization, in the advent of a knowledge-based economy, and within the reduction of the period of validity of knowledge, and in the focus on employability skills for life and in a change in the education market. Corporate universities are generally presented as a reorganization of the training centres and the development of human resources departments of large companies. For some authors, corporate universities extrapolate their focus of care for an external audience, acting as a "strategic umbrella" covering diverse audiences and various forms of courses. Sectoral universities function as an adequacy of corporate University model for different industries. Usually organized and managed by employers of the industrial sector, the transport sector, the real estate industry, the pulp and paper sector, etc., they offer training, qualification and training through partnerships with foundations, public and private universities. From this frame of reference, we opted for a qualitative research involving: bibliographic review and analysis and field work, through documentary analysis and interviews. For interviews, it has been identified a sectoral university representative in the national scenario, in this case with sugar and ethanol sector, to enhance the achievement of the purpose of this search. / Mestrado / Ciencias Sociais na Educação / Mestre em Educação