Não Consta Resumo na Publicação / Abstracts Not Available

Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus

Não Consta Resumo na Publicação / Abstracts Not Available

A importância na geração de oxaloacetato foi investigada através da determinação da atividade da piruvato carboxilase nos músculos estriados e da suplementação de seus precursores (aspartato e asparagina) na dieta de ratos. A atividade da piruvato carboxilase eleva-se durante o exercício físico e, portanto, deve fornecer mais oxaloacetato para a etapa inicial do ciclo de Krebs. A suplementação crônica (5 semanas) de aspartato e asparagina promove aumento da resistência ao esforço em ratos treinados em natação durante 1 hora diária por 5 semanas. Este efeito foi acompanhado de elevação no número e tamanho das mitocôndrias e alteração no metabolismo de glicose dos músculos esqueléticos (elevação do conteúdo de glicogênio e de sua síntese e diminuição da glicólise). Esses resultados sugerem que a geração de oxaloacetato desempenha papel fundamental na manutenção do esforço prolongado. A suplementação de aspartato e asparagina na dieta melhora a performance nessas condições, porém causa lesões na ultraestrutura muscular (mitocôndrias, linha \"Z\" e miofibrilas). / The importance of oxaloacetate formation was investigated by measuring pyruvate carboxylase activity in muscles and by given its precursors (aspartate, asparagine) in the diet of rats. The activity of pyruvate carboxylase markedly raised during physical effort and so might provide oxaloacetate for Krebs cycle functioning. The supplementation of aspartate and aspagine for a prolonged period of time (5 weeks) promotes increment in the resistance to exercise in rats trained to swimming during 1 hour daily for 5 weeks. This effect is accompanied by an increase in the size and number of mitochondria and also changes in glucose metabolism; elevation in glycogen synthesis and content and reduction in the rate of glycolysis. These results suggest that the production of oxaloacetate plays a role to maintain the moderate exercise during a prolonged period of time. Nevertheless, the aspartate and asparagine supplemented in the diet, despite improving the perfomance to moderate and prolonged exercise, provokes muscle ultraestructure lesions of mitochondria, \"Z\" line and miofibrils.

Doctor Pharmaceuticae - DPharm / Natural products have been an important source of drugs and novel lead compounds in drug discovery. Their unique scaffolds have led to the synthesis of derivatives that continue to give rise to medicinally relevant agents. Thus, natural product-inspired drugs represent a significant proportion of drugs in the market and with several more in development. Cancer is among the leading public health problems and a prominent cause of death globally. Chemotherapy has been important in the management of this disease even though side effects that arise due to lack of selectivity is still an issue.

The diploma thesis deals with the Influence of the Enzyme L-asparagine and the Inorganic salts (NaCl, CaCl2, NaHCO3 and NH4HCO3) on the elimination of the acrylamide in food-stuffs and a simulated model cereal matrix. The acrylamide belongs to the probable carcinogenic compounds which is incipient in the course of thermal processing of food, which are rich in the reducing sugars and amino acids as L-asparagine. Because of L-asparagine is the natural component of cereals and simultaneously is dominant antecedent incipient acrylamide, the way of the elimination by enzyme L-asparaginase (or the combination of L-asparaginase and salt) leads to the reduced level of acrylamide in a final product. The L- asparagine and salts were used on food-stuffs and a simulated model cereal matrix. It was found that individual used substances (except for NH4HCO3) cause the reduction of acrylamide production even about 90 % without change in the sensory properties of final product.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23608号 / 医博第4795号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 中島 貴子, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20239号 / 医博第4198号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Cancer cells display increased metabolic demands to support their proliferation and biosynthetic needs. It has been extensively shown in cancers, that amino acids have functions beyond the role of mRNA translation. The breadth of functions makes amino acid restriction an effective strategy for cancer therapy; hence an important line of research involves targeting amino acid acquisition and metabolism therapeutically. Currently, asparagine depletion via L-Asparaginase in acute lymphoblastic leukemia (ALL) remains the only clinically approved therapy to date. In the first project, we showed that ALL cells are auxotrophic for asparagine and rely on exogenous sources for this non-essential amino acid. However, sensitivity to L-Asparaginase therapy is mitigated by the expression of the enzyme asparagine synthetase (ASNS), involved in de novo asparagine biosynthesis. We showed that this adaptive response requires two essential steps; demethylation of the ASNS promoter and recruitment of activating transcription factor 4 (ATF4) to the promoter to drive ASNS transcription. Our follow-up study in ALL cells showed that asparagine bioavailability (through de novo biosynthesis or exogenous sources) is essential to maintain the expression of the critical oncogene c-MYC. c-MYC is a potent transcription factor and is dysregulated in over 60% of cancers, including hematopoietic malignancies. We showed that this regulation by asparagine is primarily at the translation level and c-MYC expression is rescued only when exogenous asparagine is available or when cells can undertake de novo biosynthesis. At the biochemical level, asparagine depletion also causes an induction of ATF4 mediated stress response and suppression of global translation mediated by decreased mammalian target of rapamycin complex 1 (mTORC1) activity. However, we found that neither inhibition of the stress response or rescuing global translation rescued c-MYC protein expression. We also extended this observation to c-MYC-driven lymphomas using cell lines and orthotopic in vivo models. We showed that genetic inhibition of ASNS or pharmacological inhibition of asparagine production can significantly limit c-MYC protein and tumor growth when environmental asparagine is limiting. Overall, our work shows an essential role for asparagine in lymphoid cancers and has expanded on the usage of L-Asparaginase to resistant leukemias and lymphomas.

L’orge (Hordeum vulgare L.) est l'une des céréales les plus importantes du monde et l’une des premières cultures domestiquées. Elle a été utilisée pendant des siècles pour l'alimentation humaine. Comme toutes les autres plantes, l'orge est dépendante de l'azote inorganique. L’efficacité de remobilisation de l'azote est donc très importante pour le remplissage des grains et pour la teneur en protéines du grain. L'objectif de ce travail est de donner une image du métabolisme des feuilles sénescence chez l'orge lorsque les plantes sont cultivées dans des conditions limitantes ou non en nitrates. Les analyses biochimiques, physiologiques et moléculaires de la sénescence des feuilles d'orge ont été réalisées. La gestion de l'azote pendant la sénescence des feuilles a été suivie par l'évolution des différents composés azotés au cours du vieillissement de la feuille. Une étude de profilage métabolique a été effectuée afin de déterminer les caractéristiques métaboliques de la sénescence des feuilles dans l'orge. En parallèle, les enzymes impliquées dans la remobilisation de l'azote ont été étudiées. Leurs activités et les niveaux de leurs transcripts ont été mesurés. Une attention particulière a été portée aux glutamine synthétases et asparagine synthétases et aux protéines de la machinerie de l'autophagie, processus connus pour jouer un rôle dans la remobilisation de l'azote pendant la sénescence des feuilles. A partir de toutes les données de séquences disponibles, ADNc, EST et séquences génomiques, cinq gènes codant pour les isoformes de glutamine synthétase cytosoliques (GS1), cinq gènes codant pour les isoformes d’asparagine synthétase (AS) isoformes et 19 gènes codant pour des protéines de la machinerie de l'autophagie ont été identifiés. Les expressions de tous les gènes identifiés ont été suivies au cours de la sénescence des feuilles et en fonction de l'alimentation en nitrates. La plupart de ces gènes sont sur-exprimés dans les feuilles sénescentes et de façon différentielle en fonction des conditions de nutrition. Toutes les données de séquences fournies par ce travail seront utiles à d'autres études translationelles et d'association génétique. / Barley (Hordeum vulgare L.) is one of the most important cereals in the world. It was one of the first domesticated crops and was used for centuries for human food. As all plants, barley has a fundamental dependence of inorganic nitrogen and nitrogen remobilization efficiency is very important for grain filling and grain protein content. The aim of this work was then to give a picture of the leaf-senescence metabolism in barley leaves when plants are grown under low or high nitrate conditions. Biochemical, physiological and molecular analyses of barley leaf senescence were performed. Nitrogen management during leaf senescence was monitored measuring changes in the different nitrogen pools during leaf ageing. In addition a large metabolite profiling study was performed in order to determine the metabolic hallmarks of leaf senescence in barley. In parallel enzymes involved in nitrogen remobilization were studied measuring their activity and the transcript levels of their coding genes. There was a special focus on glutamine synthetase and asparagine synthetase enzymes and for autophagy machinery that are known to play a role in nitrogen remobilisation during leaf senescence.From all the sequences data available, cDNA, EST and genomic sequences, we could identified five genes coding for cytosolic glutamine synthetase (GS1), five genes coding for asparagine synthetase (AS) and 19 genes coding for autophagy machinery proteins. Transcript levels of all the genes identified were monitored during leaf senescence and depending on nitrate nutrition. Most of these genes were over-expressed in senescing leaves and differentially expressed depending on nitrate conditions. In addition to the characterization of autophagy, GS1 and ASN genes, phylogenic and gene structures were analysed. All the sequences data provided by this work will be helpful to further translational and genetic association studies.