O metabolismo celular é substancialmente alterado durante a oncogênese, progressão tumoral e outros processos patológicos. Tem sido frequentemente analisado para compreensão dos processos que permitem o crescimento dos tecidos, reprodução, manutenção da homeostase e resposta a sinais extracelulares. Dentre os vários métodos para caracterização de alterações metabólicas, a espectrometria de massas tem contribuído significativamente para a identificação e quantificação de proteínas envolvidas no metabolismo, e também para a caracterização do metaboloma. A análise proteômica baseada em espectrometria de massas permite estudos qualitativos em grande escala, adequados para a busca e descoberta de analitos relevantes. A análise proteômica dirigida complementa esse caráter com qualidade quantitativa para proteínas alvo pré-selecionadas. Neste trabalho foram desenvolvidos métodos de proteômica dirigida para o monitoramento de alterações quantitativas nos níveis de proteínas envolvidas na via glicolítica do metabolismo da glicólise. Para tal peptídeos proteotípicos para cada proteína foram identificados e padronizados utilizando a estratégia de monitoramento de reações múltiplas. O painel foi aplicado para obter resultados das alterações que ocorrem em um modelo de progressão tumoral. Com esta estratégia empregada, foi possível selecionar e utilizar vários peptídeos representativos das enzimas da via glicolítica e também de algumas proteínas relevantes ao câncer. A utilização de peptídeos sintéticos facilitou substancialmente o processo de desenvolvimento do método. Por fim, com a metodologia desenvolvida, foi demonstrado para células MCF7, que o fator EGF alterou a expressão das proteínas da via glicolítica, aumento no fluxo para via das pentoses e capacidade aumentada da respiração celular. Este estudo, portanto, sugere uma nova disposição do metabolismo celular dado o conhecimento estabelecido em relação aos efeitos na respiração, como o efeito Warburg. / Cellular metabolism is altered during ontogenesis, cancer progression and several other pathological events. Because of that, the metabolism is constantly analyzed in order to provide further comprehension of processes that allow tissue growth, reproduction, homeostasis maintenance, and response to extracellular signals. Among the methods great diversity of methods used to characterize metabolic alterations, mass spectrometry has been contributing significantly to identify and quantify proteins involved in a diversity of metabolic pathways, but also to monitor the changes in metabolome. Proteomics based on mass spectrometry allows highthroughput in-depth qualitative resources for discovery phases stages, providing the new relevant candidates for further biochemical characterization. In a complementary way, targeted proteomics allows precise quantitative analyses of such selected protein targets. Here, it was developed a targeted proteomics method for multiplex monitoring glycolytic pathway enzymes and relevant cancer-related proteins. For that, proteotypic peptides representing proteins of interest were selected and studied in detail to be incorporated in a multiple reaction monitoring assay. The developed method was applied to monitor the alterations in the glycolytic pathway in a cancer progression model. Using targeted proteomics strategies, we selected and applied for quantification several proteotypic peptides representing glycolitic enzymes and cancerrelated proteins. The use of synthetic peptides allowed faster method development and more sensitive methods. The application of such methods, demonstrated alterations in glycolytic pathways and cancer-related proteins promoted in MCF7 cells treated with EGF. Also, an activation of pentose-phosphate pathway was suggested and increase in cellular oxygen consumption. This study, therefore, suggests changes in the cellular metabolism that differs from the classic Warburg effect observed during cancer development.

Orientador: Marco-Aurelio De Paoli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-16T00:53:24Z (GMT). No. of bitstreams: 1 Ribeiro_ElemCristinaCarlos_M.pdf: 2244998 bytes, checksum: d362a28fecd398558d6b535a95ecd592 (MD5) Previous issue date: 2010 / Resumo: Os poliuretanos representam uma variedade de produtos caracterizados pela presença de grupos carbamatos na cadeia principal, formados através da reação de poliadição dos compostos isocianatos com outros contendo hidrogênio ativo, assim como os alcoóis. São utilizados em diversas áreas e constituem um dos mais importantes grupos de polímeros devido à versatilidade em diferentes aplicações. Como conseqüência direta do sucesso comercial tem-se, proporcionalmente, uma grande quantidade de resíduos gerados, que muitas vezes são destinados ao descarte em aterros sanitários, constituindo um problema econômico e ambiental. Sendo assim, a reciclagem química dos poliuretanos torna-se oportuna quando outros processos usuais de reciclagem não podem ser aplicados, ou constitui uma opção mais viável ao invés do descarte nos aterros. O objetivo principal deste trabalho foi recuperar o poliol utilizado na produção da espuma de poliuretano através de reciclagem química. Utilizou-se uma reação de glicólise em duas fases com dietilenoglicol e dietanolamina, como solvente e catalisador, respectivamente. Os parâmetros da reação foram investigados e os produtos foram caracterizados, de forma a avaliar o poliol recuperado presente na fase superior, bem como os subprodutos presentes na fase inferior. A caracterização do poliol reciclado foi realizada através de Espectroscopia no Infravermelho com Transformada de Fourier, Cromatografia de Permeação em Gel, Ressonância Magnética Nuclear, titulação Karl Fischer e índice de acidez. O poliol recuperado foi utilizado na produção de espumas e as amostras comparadas à espuma submetida ao processo de reciclagem. A caracterização das espumas foi realizada por análise termogravimétrica, calorimetria diferencial de varredura, análise dinâmico-mecânica e microscopia eletrônica de varredura / Abstract: Polyurethanes are characterized by the presence of carbamate groups in the polymer chain, that are originated by polyaddition reaction of isocyanates with others compounds having an active hydrogen, like alcohols. Polyurethanes are applied in different areas of the industry and represent one of the most important polymer groups due to its versatile application. As direct consequence of the its successful applications, it has generates a lot of wastes of polyurethanes that sometimes are discarded in landfillings. Due to the environmental and economic issues, polyurethane chemical recycling became suitable when other recycling processes are not applicable to polyurethanes, being an alternative to landfillings. The main proposal of this work is to recover the constituent polyol from the polyurethane scrap, since it is a valuable raw material, through a chemical recycling process. Among the existing processes, it was considered the ¿splitphase¿ glycolysis using diethylene glycol (DEG), as a glycolysis agent, and diethanolamine (DEA), as a catalyst, in order to obtain better quality products by reactants activity. Reaction parameters were investigated and the products of the glycolysis reaction were characterized in order to evaluate the glycol recovered and present on the upper phase, while the by-products, like amides and urea; and excess of DEG and DEA excess composed the bottom lower phase. The characterization of recycled polyol was done by Gel Permeation Chromatography, Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, water content by Karl Fisher titration and acidity index. Recovered polyol was used to produce new foams and their characterizations were compared to the original one. The characterizations of the foams were done by Thermogravimetric Analysis, Differential Scanning Calorimetry, Dynamic Mechanic Analysis and Scanning Electron Microscopy / Mestrado / Quimica Inorganica / Mestre em Química

Os canais seletivos a ânions dependente de voltagem (VDACs) são um grupo de proteínas, primeiramente identificadas na membrana mitocondrial externa, capazes de formar estruturas de poros hidrofílicos em membranas. As VDACs são conhecidas pela sua função essencial no metabolismo celular e nos estágios recentes de apoptose. Em mamíferos, foram identificadas três isoformas de VDACs (VDAC1, 2 e 3). Uma pesquisa proteômica, consistindo de eletroforese bi-dimensional seguida por western blotting com anticorpos anti-VDAC 1, anti-VDAC 2 e anti-VDAC 3 e espectrometria de massas com fonte de ionização/desorção à laser assistido por matriz e tempo de vôo foi utilizada para estudar a expressão das isoformas de VDAC em mitocôndrias cerebrais de aves, ratos e bois. Foi estudada a possibilidade que diferenças na expressão relativa das isoformas de VDAC possam ser um fator determinante da proporção espécie-dependente dos sítios de ligação da hexoquinase tipo A: tipo B nas mitocôndrias cerebrais. Os spots foram caracterizados, e a intensidade de sinal foi comparada entre os spots. VDAC1 e VDAC2 foram divididas dentro de múltiplos spots. A VDAC1 foi dividida em dois spots nos géis bi-dimensionais realizados com amostras de cérebros de ratos e bois, e três spots para cérebros de aves. A VDAC2 foi separada em três, cinco e dois spots para cérebros de ratos, bois e aves, respectivamente. Os resultados reportam uma heterogeneidade de carga das VDACs 1 e 2 nos cérebros analisados. A VDAC1 foi a mais expressa das três isoformas. Além disso, a expressão da VDAC1 mais VDAC2 foi muito maior em cérebros de aves e bois do que em cérebros de ratos. Mitocôndrias de cérebro de aves mostraram uma maior expressão de VDAC1 e menor de VDAC2. As mitocôndrias de cérebro bovino apresentaram os níveis mais altos de VDAC2. A VDAC3 não foi detectada nos cérebros das espécies estudadas. / The voltage dependent anion selective channels (VDACs) are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. VDAC are known to play an essential role in cellular metabolism and in the early stages of apoptosis. In mammals, three VDACs isoforms (VDAC1, 2, 3) have been identified. A proteomic approach, consisting of two dimensional electrophoresis, followed by western blotting with anti-VDAC 1, anti-VDAC 2 and anti-VDAC 3 and by matrix assisted laser desorption/ionization time of flight mass spectrometry was used to study the expression of VDAC isoforms in rat, bovine and avian brain mitochondria. We were studying the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A: type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 and VDAC2 were divided into multiple spots. VDAC1 was divided in two spots in two dimensional gels of rat and bovine brains and three spots in avian brains. VDAC2 was separated into three, five and two spots in rat, bovine and avian brains, respectively. The results report charge heterogeneity of VDACs 1 and 2 in the analyzed brains. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was much higher in avian and bovine brains than in rat brains. Avian brain mitochondria showed the highest expression of VDAC1 and the lowest of VDAC2. Bovine brain mitochondria had the highest levels of VDAC2. No VDAC 3 was detected in studied species brains.

Under normal conditions, non-transformed cells rely on glycolysis followed by oxidative phosphorylation to generate ATPs. When oxygen is scarce or when cells are actively proliferating, cellular ATPs come mainly from glycolysis. Pyruvate is converted into lactate to allow glycolysis to continue. Interestingly, cancer cells have adapted to favor lactate production even at normal oxygen tensions, exhibiting a metabolic shift known as the Warburg effect. However, the metabolic state of other cellular constituents within the tumor remains mostly unknown. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells. They aid tumor growth and metastasis by providing growth factors, cytokine, ECM remodeling proteins and interacting with other tumor stromal cells. Here I show that the Warburg effect also operates in stromal fibroblasts of the tumor microenvironment. Using mass spectrometry, genetic mouse models, gene expression and methylation studies, I demonstrate that CAFs from human and mouse mammary tumors exhibit hyperactive glycolysis and a metabolic shift towards lactate production. Furthermore, this phenotype may be sustained through epigenetic modifications of endogenous hypoxia-inducible factor 1α, key regulatory enzymes fructose-bisphosphatase 1 and pyruvate kinase M2. Depletion of stromal fibroblasts or suppression of lactate production specifically in these cells alters the metabolic profile of not only the tumors but also the cancer cells and results in impeded tumor growth. These results collectively suggest that tumor growth is dependent on metabolic state and metabolic support of stromal fibroblasts, highlighting these cells as attractive therapeutic targets in controlling cancer progression.

This thesis investigates the effects of alanine on rainbow trout’s glucose metabolism at the organismal and molecular levels. Rainbow trout is an important aquaculture species that belongs to the salmonid family. As a carnivorous fish, the requirement of protein/amino acids in trout’s diet is high. In contrast, rainbow trout are poor utilizers of carbohydrates. One prevalent hypothesis suggests that high levels of dietary amino acids could indeed contribute to the poor utilization of carbohydrates in this species. In mammals, there is evidence supporting the importance of alanine as a gluconeogenic precursor. However, a recent study found that alanine stimulates hepatic AMP-activated protein kinase (AMPK) to lower circulating glucose levels in mice. Alanine levels are high in all tissues in rainbow trout. The role of alanine in gluconeogenesis is less clear in trout and there is no evidence, to our knowledge, regarding its effects on glucose kinetics. Therefore, the main goal of the study was to investigate the impact of the continuous infusion of exogenous alanine for 4h on glucose fluxes and to identify potential mechanisms in tissues that could interpret the observed changes in glucose fluxes in vivo. Glucose turnover, appearance and disposal, Rt, Ra and Rd, respectively, were measured to determine the impact of alanine on glucose fluxes. The expression and/or activity of key genes in glucose transport, utilization and gluconeogenesis were assessed in liver and muscle. An additional goal was to assess whether alanine activates AMPK in trout. The levels of phosphorylated AMPK and other signaling proteins known to interact with the latter were quantified. Results show that alanine reduced plasma glucose levels and inhibited Ra and Rd glucose, consistent with previously observed effects of insulin in rainbow trout. The reduction in the expression of a paralogue of glut4, a key gene in glucose transport, and the activity of hexokinase (HK), a key enzyme in glucose utilization, in muscle can partially explain the observed reduction in Rd glucose. Together, these results suggest that glucose was not a preferred substrate under conditions of increased alanine availability and that alanine was probably oxidized to provide energy. Alanine failed to activate AMPK in trout, contrary to mammalian findings. However, it increased AKT (also known as protein kinase B) phosphorylation in muscle, similar to the effect of insulin in trout. In conclusion, my results suggest that alanine mediated at least some of the observed effects by stimulating insulin secretion given the similarities between the effects of exogenous alanine and insulin in rainbow trout as discussed above. Future studies are warranted to investigate the hypothesis that alanine is an insulin secretagogue in rainbow trout.

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

Serum lactate levels are traditionally interpreted as a marker of tissue hypoxia and often used clinically as an indicator of severity and outcome of sepsis/septic shock. Interestingly, recent studies involving the effects of tumor-derived lactate suggest that lactate itself may have an immunosuppressive effect in its local environment. This finding adds to the recent advances in immunometabolism that shed light on the importance of metabolism and metabolic intermediates in the regulation of innate immune and inflammatory responses in sepsis. In this article, we summarize recent studies, showing that the activation of immune cells requires aerobic glycolytic metabolism and that lactate produced by aerobic glycolysis may play an immunosuppressive role in sepsis.

Background. Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy. Methods. Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-d-glucose [2-DG], 0.25-2 g/kg, n = 6-8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. Results. Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG-treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1β as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium. Conclusions. Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第17893号 / 工博第3802号 / 新制||工||1581(附属図書館) / 30713 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17977号 / 医博第3841号 / 新制||医||1001(附属図書館) / 80821 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 稲垣 暢也, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM