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Thesis advisor: Evan R. Kantrowitz / The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of cellular regulation can be related in some manner to protein conformational changes such as the quaternary conformational changes of allosteric enzymes that alter enzyme activity to regulate metabolism. The control of metabolic pathways by allosteric enzymes is analogous to a molecular valve with "on" and "off" positions. In the "off" position, flow through the pathway is severely hindered, while in the "on" position the flow is normal. For a comprehensive understanding of allosteric regulation we must elucidate in molecular detail how the allosteric signal is transmitted to the active site to alter enzyme activity. In this work we use unnatural amino acid mutagenesis to introduce a fluorescent amino acid into the allosteric binding site of aspartate transcarbamoylase (ATCase), the enzyme responsible for regulation of pyrimidine nucleotide biosynthesis. The fluorescence from the amino acid is exquisitely sensitive to the binding of the allosteric effectors ATP, CTP, UTP, and GTP. In particular we show how the asymmetric nature of the allosteric sites of the enzyme are used to achieve regulatory sensitivity over a broad range of mixed heterotropic effector concentrations as is observed in the cell. Furthermore, employing the method of random sampling - high dimensional model representation (RS-HDMR) we derived a model for how ATCase is regulated when all four nucleotides are present at fluctuating concentrations, consistent with physiological conditions. We've discovered the fundamental requirements to induce the allosteric transition to the R state by showing that although ATCase can accept L-asparagine as an unnatural substrate, the transition to the R allosteric state requires the correct positioning of the alpha-carboxylate of its natural substrate L-aspartate. However, linking the functionalities of L-asparagine and carbamoyl phosphate into a single molecule is sufficient to correctly position the bi-substrate analog in the active site to induce the allosteric transition to the R-state. The cooperative nature of ATCase was further investigated through the isolation of a unique quaternary structure of ATCase consisting of two catalytic trimers linked covalently by disulfide bonds. By relieving the quaternary constraints imposed by the bridging regulatory subunits of the native holoenzyme, the flexibility of the c6 subunit significantly enhanced enzyme activity over the native holoenzyme. Unlike the native c3 catalytic subunit, the c6 species displays homotropic cooperativity for L-aspartate demonstrating that, when two catalytic trimers are linked, a binding event at one or more active sites can be transmitted through the molecule to the other active sites in the absence of regulatory subunits. The catalytic reaction of ATCase follows an ordered sequential mechanism that is complicated by the transition from the T state to the R state upon the binding of the second substrate L-aspartate. Acquiring X-ray crystal structures at each step along the pathway has advanced our understanding of the catalytic mechanism, yet R-state structures are difficult to obtain. Using a mutant version of ATCase locked in the R-allosteric state by disulfide bonds we captured crystallographic images of ATCase in the R state bound to the true substrates (CP and Asp), products (CA and Pi), and in the process of releasing the final product (Pi) prior to reversion of the molecule to the T state. These structures depict the steps in the catalytic cycle immediately before the catalytic reaction occurs, immediately after the reaction, and after the first product has been released from the active site. This work also focuses on developing allosteric inhibitors of the enzyme fructose-1,6-bisphosphatase (FBPase), one of the enzymes responsible for regulation of the gluconeogenesis pathway. Inhibitors of FBPase could serve as potential therapeutic agents against type-2 diabetes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Made available in DSpace on 2014-06-11T19:26:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-07Bitstream added on 2014-06-13T19:54:52Z : No. of bitstreams: 1 kohatsu_ds_me_botfca.pdf: 4312398 bytes, checksum: 91e1ff1dd9bac30e33d67d04a04522bd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho teve por objetivo avaliar o efeito de reguladores vegetais nas trocas gasosa durante o desenvolvimento da planta e na qualidade pós-colheita de frutos de melão rendilhado Galileu, armazenados em condições ambiente. A primeira etapa do experimento foi conduzida em estufa para cultivo protegido das plantas de melão, localizada na área experimental da Fazenda Experimental de São Manuel/UNESP, Botucatu, SP. A aplicação dos tratamentos foram realizadas no início da fase reprodutiva, 15 e 30 dias após a primeira aplicação. As medidas de trocas gasosas foram realizadas sempre no período das 8:00 às 11:00 horas da manhã, nas plantas controle e tratadas com os reguladores vegetais, no dia anterior e posterior à aplicação. Ci, A, gs e E foram feitas com um Infra Red Gas Analyser IRGA, modelo LI-6400 da LI-COR. As plantas receberam os tratamentos no campo (T1 controle, T2 GA3 (ácido giberélico) a 100 mg L-1, T3 IBA (ácido indolilbutírico) a 100 mg L-1, T4 cinetina a 100 mg L-1, T5 mistura de GA3 + IBA + cinetina a 5%), os frutos foram colhidos (51 dias após a primeira aplicação) e transportados ao Laboratório de Frutas e Hortaliças, pertencente ao Departamento de Gestão e Tecnologia Agroindustrial/UNESP, Botucatu, SP. Os frutos foram avaliados a cada 5 dias, durante 20 dias de armazenamento. As alterações pós-colheita foram detectadas por meio de análises de perda de massa fresca, firmeza, acidez titulável, pH, sólidos solúveis, relação SS/AT, açúcares totais, redutores e sacarose, atividade das enzimas peroxidase (POD), pectinametilesterase (PME) e poligalacturonase (PG). Os macronutrientes e micronutrientes foram analisados na colheita. O delineamento estatístico utilizado foi em blocos ao acaso com o controle e mais quatro tratamentos e quatro repetições que correspondem aos blocos. Nas condições em que o experimento... / This work's objective was to evaluate the effects of plant growth regulators on gas exchange during plant development and on post harvest quality of Galileu cantaloupes stored at conditions atmosphere. The first stage of the assay was conducted in a greenhouse (protected cultivation of cantaloupe plants), in the experimental area of São Manuel's Experimental Farm/UNESP, Botucatu, SP. The treatments were applied in the beginning of the reproductive stage, and 15 and 30 days after the first application. Gas exchange measurements were performed from 0800 to 1100 a.m, both in control plants and plants treated with plant growth regulators, on the previous and subsequent day from application. Ci, A, gs, and E were evaluated with a LI-COR model LI-6400 Infra Red Gas Analyzer - IRGA. The cantaloupe plants were treated in the field (T1 controls, T2 GA3 (gibberellic acid) 100 mg L-1, T3 IBA (indolebutyric acid) 100 mg L-1, T4- kinetin 100 mg L-1, T5- GA3 + IBA + kinetin mixture at 5%), and fruits were harvest (51 days after the first application) and transported to the Fruits and Vegetables Laboratory of Departamento de Gestão e Tecnologia Agroindustrial/UNESP, Botucatu, SP. The fruits were evaluated every 5 days during a 20-day storage period. Alterations in post harvest quality were detected through analyses comprising fresh weight loss, firmness, titratable acidity, pH, soluble solids, SS/TA ratio, total sugars, reduced sugars and sucrose, and activity of the enzymes peroxidase (POD), pectin methylesterase (PME), and polygalacturonase (PG). Macronutrients and micronutrients were analyzed at harvest time. The statistical design consisted of randomized blocks including a control, in addition to four treatments and four replicates that corresponded to blocks. Under the conditions in which the experiment was conducted, the results allow us to... (Complete abstract click electronic access below)

Orientador: Elizabeth Orika Ono / Banca: Regina Marta Evangelista / Banca: José Maria Monteiro Sigrist / Resumo: O presente trabalho teve por objetivo avaliar o efeito de reguladores vegetais nas trocas gasosa durante o desenvolvimento da planta e na qualidade pós-colheita de frutos de melão rendilhado Galileu, armazenados em condições ambiente. A primeira etapa do experimento foi conduzida em estufa para cultivo protegido das plantas de melão, localizada na área experimental da Fazenda Experimental de São Manuel/UNESP, Botucatu, SP. A aplicação dos tratamentos foram realizadas no início da fase reprodutiva, 15 e 30 dias após a primeira aplicação. As medidas de trocas gasosas foram realizadas sempre no período das 8:00 às 11:00 horas da manhã, nas plantas controle e tratadas com os reguladores vegetais, no dia anterior e posterior à aplicação. Ci, A, gs e E foram feitas com um Infra Red Gas Analyser IRGA, modelo LI-6400 da LI-COR. As plantas receberam os tratamentos no campo (T1 controle, T2 GA3 (ácido giberélico) a 100 mg L-1, T3 IBA (ácido indolilbutírico) a 100 mg L-1, T4 cinetina a 100 mg L-1, T5 mistura de GA3 + IBA + cinetina a 5%), os frutos foram colhidos (51 dias após a primeira aplicação) e transportados ao Laboratório de Frutas e Hortaliças, pertencente ao Departamento de Gestão e Tecnologia Agroindustrial/UNESP, Botucatu, SP. Os frutos foram avaliados a cada 5 dias, durante 20 dias de armazenamento. As alterações pós-colheita foram detectadas por meio de análises de perda de massa fresca, firmeza, acidez titulável, pH, sólidos solúveis, relação SS/AT, açúcares totais, redutores e sacarose, atividade das enzimas peroxidase (POD), pectinametilesterase (PME) e poligalacturonase (PG). Os macronutrientes e micronutrientes foram analisados na colheita. O delineamento estatístico utilizado foi em blocos ao acaso com o controle e mais quatro tratamentos e quatro repetições que correspondem aos blocos. Nas condições em que o experimento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work's objective was to evaluate the effects of plant growth regulators on gas exchange during plant development and on post harvest quality of Galileu cantaloupes stored at conditions atmosphere. The first stage of the assay was conducted in a greenhouse (protected cultivation of cantaloupe plants), in the experimental area of São Manuel's Experimental Farm/UNESP, Botucatu, SP. The treatments were applied in the beginning of the reproductive stage, and 15 and 30 days after the first application. Gas exchange measurements were performed from 0800 to 1100 a.m, both in control plants and plants treated with plant growth regulators, on the previous and subsequent day from application. Ci, A, gs, and E were evaluated with a LI-COR model LI-6400 "Infra Red Gas Analyzer - IRGA". The cantaloupe plants were treated in the field (T1 controls, T2 GA3 (gibberellic acid) 100 mg L-1, T3 IBA (indolebutyric acid) 100 mg L-1, T4- kinetin 100 mg L-1, T5- GA3 + IBA + kinetin mixture at 5%), and fruits were harvest (51 days after the first application) and transported to the Fruits and Vegetables Laboratory of Departamento de Gestão e Tecnologia Agroindustrial/UNESP, Botucatu, SP. The fruits were evaluated every 5 days during a 20-day storage period. Alterations in post harvest quality were detected through analyses comprising fresh weight loss, firmness, titratable acidity, pH, soluble solids, SS/TA ratio, total sugars, reduced sugars and sucrose, and activity of the enzymes peroxidase (POD), pectin methylesterase (PME), and polygalacturonase (PG). Macronutrients and micronutrients were analyzed at harvest time. The statistical design consisted of randomized blocks including a control, in addition to four treatments and four replicates that corresponded to blocks. Under the conditions in which the experiment was conducted, the results allow us to... (Complete abstract click electronic access below) / Mestre

Orientador: Giuseppina Pace Pereira Lima / Banca: João Domingos Rodrigues / Banca: Denise Laschi / Banca: Terezinha Rangel Câmara / Banca: José Luis Mosca / Abstract: Beehive ginger inflorescences have yellow bracts when they are young, which are ornamental and are specially used in gardening projects and as cut flowers. However, there are crop management and postharvest factors that affect the expansion of the species. So, the objective of this work was to study some physiological postharvest aspect of beehive ginger inflorescences grown in the lower middle São Francisco river basin. Flower stems just harvested were submitted to different treatments (distilled water; 75 mg L-1 of silver nitrate - AgNO3; 1000 mg L-1 of cobalt chloride - CoCl2; 5 mg L-1 de GA3 - Progibb® and 10 mg L-1 of 6-benzylamino purine - BAP), in an environment with controlled temperature and humidity, for 15 days. The longevity was monitored from non-destructive analysis (grading scale, fresh weight, consumption and pH of the preservative solution) as well as destructive ones (peroxidase and polyphenol oxidase activity, total soluble and reducing carbohydrates, phenols, putrescine, spermine and spermidine content). The non-destructive analysis showed that the beehive ginger stems treated with gibberelin and silver nitrate presented a better visual aspect according to the grading scale, the ones treated with AgNO3 absorbed a greater volume of the solution during the experimental period, while the ones treated with silver nitrate and BAP had a greater fresh weight. The smallest variation of the preservative solution pH took place with the treatments containing the plant regulators. The destructive analysis revealed that the inflorescences maintained in preservative solutions with gibberelin and distilled water kept their stocks of total soluble sugars for 3 days longer than the stems submitted to the other treatments. The contents of reducing sugars increased 7 considerably in inflorescences treated with cytokinin. The BAP promoted alterations in the activity of the... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:32:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-03-16Bitstream added on 2014-06-13T19:43:14Z : No. of bitstreams: 1 santos_mhlc_dr_botfca.pdf: 1509424 bytes, checksum: 0e38e7b79ee2e60018b38f884c2d8420 (MD5) / Beehive ginger inflorescences have yellow bracts when they are young, which are ornamental and are specially used in gardening projects and as cut flowers. However, there are crop management and postharvest factors that affect the expansion of the species. So, the objective of this work was to study some physiological postharvest aspect of beehive ginger inflorescences grown in the lower middle São Francisco river basin. Flower stems just harvested were submitted to different treatments (distilled water; 75 mg L-1 of silver nitrate - AgNO3; 1000 mg L-1 of cobalt chloride - CoCl2; 5 mg L-1 de GA3 - Progibb® and 10 mg L-1 of 6-benzylamino purine - BAP), in an environment with controlled temperature and humidity, for 15 days. The longevity was monitored from non-destructive analysis (grading scale, fresh weight, consumption and pH of the preservative solution) as well as destructive ones (peroxidase and polyphenol oxidase activity, total soluble and reducing carbohydrates, phenols, putrescine, spermine and spermidine content). The non-destructive analysis showed that the beehive ginger stems treated with gibberelin and silver nitrate presented a better visual aspect according to the grading scale, the ones treated with AgNO3 absorbed a greater volume of the solution during the experimental period, while the ones treated with silver nitrate and BAP had a greater fresh weight. The smallest variation of the preservative solution pH took place with the treatments containing the plant regulators. The destructive analysis revealed that the inflorescences maintained in preservative solutions with gibberelin and distilled water kept their stocks of total soluble sugars for 3 days longer than the stems submitted to the other treatments. The contents of reducing sugars increased 7 considerably in inflorescences treated with cytokinin. The BAP promoted alterations in the activity of the... (Complete abstract click electronic access below)

Die Chorismatmutase (CM, EC 5.4.99.5), kodiert durch ARO7, katalysiert die Claisen-Umlagerung von Chorismat zu Prephenat in der Biosynthese von Tyrosin und Phenylalanin. Das relativ kleine, dimere Enzym der Hefe Saccharomyces cerevisiae wird allosterisch durch Tryptophan aktiviert und allosterisch durch Tyrosin inhibiert. In der vorliegenden Arbeit wurde die Theorie widerlegt, dass die Chorismatemutase an der Osmoregulation und Vakuolenentstehung beteiligt ist. Die Analyse einiger Stämme mit punktmutiertem oder deletiertem ARO7-Gen zeigte ausschließlich eine Funktion in der Aminosäure-Biosynthese. Die Fusion an das grün-fluoreszierende Protein ermöglichte die Lokalisierung der CM in Cytoplasma und Kern der Hefezelle.Auf Proteinebene wurde der intramolekulare Signalübertragungsweg von den allosterischen zu den aktiven Zentren näher untersucht. Es wurden Chimären-Enzyme hergestellt, in denen das molekulare Scharnier L220s zwischen der katalytischen und allosterischen Domäne ausgetauscht wurde gegen den entsprechenden Bestandteil homologer Pilzenzyme. Die kinetische Analyse zeigte, dass dieser Proteinteil essentiell ist für die Unterscheidung zwischen dem Signal Aktivierung bzw. Inhibierung. Diese Region ist auch für die Dimerisierung der CM von Bedeutung. Durch Austausch hydrophober Aminosäuren gegen geladene Reste in und in der Nähe dieses Scharniers wurde eine stabile, monomere Enzymvariante hergestellt. Diese CM zeigte reduzierte Aktivität und keine Regulation, aber das kodierende Gen komplementierte die Tyrosin- und Phenylalanin-Auxotrophie der Zellen. Diese Ergebnisse unterstützen die Theorie, dass das Hefeenzym durch gleichzeitige Evolution von Regulations- und Stabilisierungsmechanismen aus einem monomeren, unregulierten Vorläuferprotein entstanden ist, welches dem der Escherichia coli CM ähnlich war. Um weitere Erkenntnisse über die Prinzipien der Proteinstabilisierung zu erhalten, wurde auch die Chorismatmutase on Thermus thermophilus charakterisiert, nachdem das kodierende Gen kloniert war. Dieses Enzym ist ähnlich zu der strukturell einzigartigen Chorismatmutae aus Bacillus subtilis, wird aber, im Gegensatz zu letzterem durch Tyrosin in seiner Aktivität gehemmt. Modellierungsstudien zeigten, dass wie auch bei anderen Proteinen verstärkte Hydrophilität von Oberflächen, erhöhte Hydrophobizität innerhalb der Struktur wie auch die Versteifung von Loops in der Nähe des aktiven Zentrums zur Stabilisierung dieser Proteinfaltung beitragen.