Orientador: Maria Célia Bertolini / Resumo: Glicogênio representa um dos principais carboidratos de reserva em muitos organismos e seu metabolismo está sob o controle de um complexo mecanismo envolvendo o balanço de nutrientes e sinais ambientais. A proteína glicogenina constitui a molécula iniciadora do processo de síntese de glicogênio, sendo as etapas seguintes efetuadas pelas enzimas glicogênio sintase e enzima ramificadora. Glicogênio sintase é a enzima limitante no processo e é regulada alosterismo e fosforilação reversível. Neste trabalho foi realizada uma caracterização do metabolismo de glicogênio no fungo N. crassa enfocando as enzimas glicogenina, glicogênio sintase e glicogênio sintase quinase-3. A proteína glicogenina (GNN) foi caracterizada do ponto de vista molecular, bioquímico e funcional. O cDNA codificando para esta proteína foi isolado e a seqüência polipeptídica deduzida mostrou uma proteína de 664 aminoácidos, uma das maiores proteínas glicogenina já isoladas. A inativação do gene gnn resultou em uma linhagem mutante incapaz de acumular glicogênio. A produção da proteína GNN em E. coli resultou em um polipeptídeo altamente susceptível à proteólise e formas truncadas da proteína mostraram ser mais estáveis e igualmente ativas nos processos de auto- e trans-glicosilação, além de servirem de substrato para ação da glicogênio sintase. Tais formas também foram capazes de complementar funcionalmente mutantes de S. cerevisiae. Além disso, a expressão do gene gnn foi mostrado ser regulado durante crescimento vegetativo e deprivação de carbono. Os resíduos Tyr196 e Tyr198 foram identificados como os sítios de glicosilação, os quais contribuem diferencialmente para este processo. Análise das interações entre GNN e a proteína glicogênio sintase de N. crassa (GSN) demonstrou que a região C-terminal da GNN é a mais importante para a interação. Entretanto, o...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glycogen represents one of the main reserve carbohydrates in many organisms and its metabolism is under control of a complex mechanism involving the balance of nutrients and environmental signals. The protein glycogenin is the initiator molecule in glycogen biogenesis and the subsequent steps are carried out by the enzymes glycogen synthase and branching enzyme. Glycogen synthase is the rate-limiting enzyme in the process and is regulated both by alosterism and reversible phosphorylation. In this work we performed a characterization of the glycogen metabolism in the filamentous fungus Neurospora crassa, focusing on the enzymes glycogenin, glycogen synthase and glycogen synthase kinase-3. The protein glycogenin (GNN) was characterized under the molecular, biochemical and functional aspects. The cDNA encoding for this protein was isolated and the deduced polypeptide sequence showed a protein with 664 residues, one of the largest glycogenins isolated so far. The inactivation of the gnn gene rendered a mutant strain that was no longer able to accumulate glycogen. The production of GNN protein in E. coli cells resulted in a polypeptide highly susceptible towards proteolysis and truncated forms were more stable and equally active, judged by their abilities to self- and trans-glucosylate, and to serve as substrate for glycogen synthase elongation. These proteins were also able to recover the glycogen deficiency phenotype in a S. cerevisae mutant strain. Moreover, the gnn gene expression was shown to be ...(Complete abstract, click electronic access below) / Doutor

Orientador: Maria Célia Bertolini / Banca: Marcia Aparecida Silva Graminha / Banca: Marcos Roberto Mattos Fontes / Resumo: O fungo filamentoso Neurospora crassa é um organismo modelo muito utilizado para estudos de diversos aspectos da biologia dos eucariotos. Nosso laboratório tem utilizado este fungo para o estudo dos mecanismos moleculares e bioquímicos da regulação do metabolismo de glicogênio. A presente proposta de trabalho é uma consequência de experimentos anteriores realizados com o objetivo de identificar proteínas (fatores de transcrição ou não) que se ligam à região promotora do gene gsn, o qual codifica a enzima glicogênio sintase, regulatória do processo de síntese do carboidrato. Esses estudos combinaram experimentos de ensaios de retardamento em gel utilizando fragmentos do promotor gsn e proteínas do extrato total do fungo, acoplados à análise proteômica e identificação das proteínas por espectrometria de massas. Os experimentos resultaram na identificação de algumas proteínas do fungo, as quais podem ou não estar envolvidas na regulação da expressão do gene. Alguns estudos preliminares com estas proteínas foram anteriormente realizados no laboratório e apontaram um provável papel das mesmas na regulação do metabolismo do carboidrato em N. crassa. Duas dessas proteínas, as codificadas pelas ORFs NCU3482 e NCU06679 foram objeto de estudo neste trabalho. Portanto, o objetivo deste trabalho foi realizar a caracterização das linhagens mutantes nestas ORFs, além da produção e purificação das proteínas na forma recombinante. Foram realizadas análises morfológicas da linhagem mutante na ORF NCU06679, tais como: crescimento colonial e radial, crescimento linear e análise microscópica das extremidades das hifas. Esses experimentos foram realizados em comparação com a linhagem selvagem do fungo e, mostraram esta proteína está envolvida no processo de desenvolvimento do... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The fungus Neurospora crassa has been widely used as a model organism for fundamental aspects of eukaryotic biology. We have been studying the biochemical and molecular mechanisms involved in glycogen metabolism regulation in this fungus. The present work is a consequence of previous experiments performed in the laboratory to identify proteins that bind to the promoter region of the gsn gene which encodes glycogen synthase, the regulatory enzyme in glycogen synthesis. Previous studies were performed by using a combination of DNA gel shift assay coupled to proteomic analysis, followed by identification of proteins by mass spectrometry. The assays resulted in the identification of proteins likely involved in the regulation of gene expression. Preliminary studies with these proteins have previously been carried out and suggested that they might have a role in the regulation of glycogen metabolism in N. crassa. Two of them, the ORFs NCU3482 and NCU06679 gene products were object of study in this work. The main objective was to characterize the mutant strains in both proteins and to produce and purify the recombinant proteins. Morphological analyzes were performed in the ORF NCU06679 mutant strain such as colony and radial linear growth and microscopic examination of the ends of the hyphae. These experiments showed that this protein is involved in the fungus development since growth and ability to conidiate were deficient when compared to the wild-type strain. The expression of gsn and gpn (encodes glycogen phosphorylase, the regulatory enzyme in glycogen degradation) genes were analyzed by qPCR and the results showed differences in gene expression of both genes during vegetative growth of the NCU06679 mutant strain when compared to the wild-type strain. The protein encoded by ORF NCU06679 was produced as a recombinant protein and the purification... (Complete abstract click electronic access below) / Mestre

Typescript (photocopy)

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.

Six men were studied to compare the effects of pre-exercise carbohydrate feedings on endurance performance and muscle glycogen utilization during exhaustive exercise. Trials consisted of a cycling ride to exhaustion at 757. of the subjects' maximal oxygen uptake preceded by the ingestion of either fructose (FRU), glucose (GLU), or sweet placebo (CON). No differences were observed between trials for oxygen uptake, respiratory exchange ratio, heart rate, or exercise time to exhaustion. Blood glucose was elevated (P<0.05) as a result of the glucose feeding, but fell rapidly with the onset of exercise, reaching a nadir of 4.02 + O.-'4 mmcl: i (mean + SE) at 15 min of exercise (P<0.05). Serum insulin also increased (P<0.05) following the glucose feeding: by 30 min of exercise, however, insulin had returned to pre-drink levels. No differences in blood glucose and insulin were observed between FRU and CON. Muscle glycogen utilization during the first 30 min of exercise (CON = 46.3 ± 82 mmol/kg w.w., FRU = 56.3 + 3, GLU = 50.0 + 4.9) and total glycogen use (CON = 93.4 + 11.1, FRU = 118.8 + 10.9, GLU = 99.5 + 4.3) was similar between trials (P>O.05). It was concluded that despite more stable blood glucose and insulin levels in FRU and CON, compared with GLU, this provided no advantage to endurance performance or muscle glycogen utilization.

Thesis--Auburn University, 2005. / Abstract. Vita. Includes bibliographical references (leaves 76-90).

Thesis (Ph. D.)--University of Michigan, 1933. / Caption title: Pentose metabolism III. The rate of absorption of l-rhamnose and the formation of glycogen in the organism of the white rat after oral administration of l-rhamnose, by Alfred K. Silberman and Howard B. Lewis. "Reprinted from the Journal of Biological Chemistry, vol. 101, no. 3 ... August, 1933." Bibliography: p. 750-751.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 51-53).

Thesis (PH. D.)--University of Michigan, 1933. / Caption title: Pentose metabolism III. The rate of absorption of l-rhamnose and the formation of glycogen in the organism of the white rat after oral administration of l-rhamnose, by Alfred K. Silberman and Howard B. Lewis. "Reprinted from the Journal of Biological Chemistry, vol. 101, no. 3 ... August, 1933." Bibliography: p. 750-751.