Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Previous studies indicated that cytosolic aldolase (ALDc) could be a rate limiting step in glycolysis and thus play a role in the regulation of carbon partitioning in sink tissues. In this study the role of ALDc in sugarcane was studied. Expression patterns of both ALDc transcript and protein were examined. In contrast to the leaves where ALDc expression is very low, the enzyme (transcript and protein) levels were high in all internodal tissues at all stages of maturity. In the leaves the plastidic isoform was prevalent as found previously in other C4 plants. The similar pattern of expression in transcript and protein abundance illustrate that there are no activators or inhibitors of ALDc activity present in sugarcane. The control on ALDc activity in sugarcane is therefore regulation of gene expression. To investigate the possibility that ALDc could be regulating carbon partitioning in sugarcane a series of transgenic sugarcane plants in the varieties NCo310 and N19 were produced. The presence and expression of the transgene and resultant effect on ALDc levels were determined for all the transgenic lines. The degree of ALDc reduction varied, with the biggest suppression of aldolase being 90% of that of the control plants. Alteration of ALDc activity caused no obvious phenotype. In both the varieties large decreases in ALDc tended to to lead to higher sucrose levels than that of the the control plants. 14C radiolabelling studies were conducted to investigate the effect of reduced ALDc levels on respiration and carbon partitioning. No differences in carbon metabolism could be found between the transgenic and control plants. Even in the line exhibiting a more than 90% decrease, the residual ALDc was sufficient for plants to grow normally under favourable glasshouse conditions. This would suggest that ALDc does not play a role in the regulation of flux through glycolysis, carbon partitioning and sucrose accumulation.

Les cancers agressifs se caractérisent par un métabolisme glycolytique exacerbé avec surexpression de protéines assurant le contrôle du pH intracellulaire par l’export efficace des déchets métaboliques acides (par CAIX, CAXII, MCT1 et MCT4 entre autres). Les cancers du sein dit "triple négatif" (sans expression des récepteurs à l'estrogène, progestérone et Her-2) présentent une consommation augmentée de glucose et un plus mauvais pronostic en comparaison avec les autres cancers du sein. L'analyse immunohistochimique de l'expression des protéines glycolytiques d'une cohorte de 159 patientes TNEG a montré que MCT4 était prédictif de la survenue de métastases et de décès. Le ciblage in vitro de MCT4 par la technique des Zinc Finger Nucléases (ZFN) a eu un effet anti-prolifératif. L'efficacité était toutefois maximale lors du ciblage combiné de MCT1 (inhibiteur pharmacologique), MCT4 et de la respiration mitochondriale par la phenformine dans la lignée de cancer TNEG Hs578t. Cette étude montre donc que le ciblage des protéines glycolytiques pourrait être une piste intéressante dans le traitement des cancers du sein TNEG. Un autre travail a permis d'exploiter le ciblage des protéines glycolytiques, notamment CAIX et CAXII, pour augmenter in vitro et in vivo la radiosensibilité de lignées de cancer colorectal tout en démontrant un mécanisme original de radiosensibilisation, via l'acidification intracellulaire. Enfin, nous avons mis en évidence l'importance de l'hypoxamiR miR210 dans la radiorésistance de lignées de cancer du poumon, avec une radiorésistance semblant dépasser l'effet oxygène. / Agressive cancers often harbor an exacerbated glycolytic metabolism with overexpression of proteins that maintain intracellular pH by extruding metabolic acid waste (via CAIX, CAXII, MCT1 and MCT4 among others). The "triple-negative" breast cancers (with no expression of estrogen, progesteron and Her-2 receptors) have an increased consumption of glucose and worse prognosis in comparison with other breast cancers. Immunohistochemical analysis of glycolytic proteins among 159 patients with TNEG breast cancer, showed that MCT4 was predictive for metastasis and death occurence. In vitro targeting of MCT4 by Zinc Finger Nuclease (ZFN) technique demonstrated an anti-proliferative effect. However, the maximal anti-proliferative effect was observed with the combination of MCT4/MCT1 (by pharmacological inhibition) and mitochondrial respiration by phenformine in the Hs578t TNEG cell line. This study demonstrated that targeting glycolytic protein could have a therapeutic effect in TNEG breast cancers. Another study also use targeting of glycolytic protein such as CAIX and CAXII to increase in vitro and in vivo radiosensitivity of colorectal cell lines while demonstrating an original mechanism of radiosensitization by increasing intracellular acidosis. Finally we demonstrated that the hypoxamiR miR210 was involved in the radioresistance of lung cancer cell line with a stronger impact than the oxygen effect.

O poli(tereftalato de etileno), PET, é um material polimérico importante, largamente utilizado na produção de garrafas para refrigerante e água mineral. Entretanto, a destinação das embalagens de PET pós-consumo vem criando sérias preocupações econômicas e ambientais. Uma das alternativas de destino desse material é a reciclagem química por glicólise, tendo como objetivo a produção do monômero tereftalato de bis-hidroxietila (BHET). Esta reação, embora referenciada, ainda apresenta problemas como rendimento de BHET e tempo de reação, entre outros, tendo como desafio o desenvolvimento de novos catalisadores eficientes e altamente seletivos. Dentro deste contexto, o presente trabalho busca estudar a atividade catalítica de um material nanoestruturado, os nanotubos de titanatos (TNT), na glicólise de PET (virgem e pós-consumo) comparado ao acetato de zinco (catalisador mais utilizado citado em literatura), a otimização de alguns parâmetros reacionais (granulometria de PET pós-consumo, razão Etilenoglicol:PET, porcentagem molar de TNT) e uma modificação dos nanotubos com zinco (ZnTNT) na despolimerização do PET Para a caracterização dos produtos as principais técnicas utilizadas foram a Calorimetria Exploratória Diferencial (DSC), Análise Termogravimétrica (TGA) e Ressonância Magnética Nuclear (RMN), além de resultados de rendimento, turnover number (TON) e turnover frequency (TOF). A despolimerização ocorreu por reação de glicólise utilizando PET virgem e pós-consumo e etilenoglicol para diferentes tempos a uma temperatura de 196°C. Os principais resultados mostram os TNT como catalisadores promissores, apresentando rendimentos em BHET de 83,9 e 76,7%, para PET virgem e pós-consumo, respectivamente, para 3 horas de reação. Esses valores são equiparáveis aos obtidos quando utilizado acetato de zinco, em que foi alcançado 79,4% (PET virgem) e 80,8% (PET pós-consumo). Após a modificação dos TNT com zinco, os resultados de rendimento em BHET alcançaram 87,1% para 3 horas de reação na menor granulometria estudada, demonstrando ser um catalisador ainda mais eficiente para essa reação. / Polyethylene terephthalate, PET, is an important polymer material, widely used in the production of bottles for soda and mineral water. However, the disposal of post-consumer PET packaging has created serious economic and environmental concerns. One of the alternatives for the disposal of this material is the chemical recycling by glycolysis, aiming the production of the monomer bis-(2hydroxyethyl) terephthalate (BHET). This reaction, although referenced, still presents problems such as BHET yield and reaction time, among others, having as challenge the development of new efficient and highly selective catalysts. In this context, the present work aims to study the catalytic activity of a nanostructured material, the titanate nanotubes (TNT) in PET glycolysis (virgin and post-consumer) compared to zinc acetate (the most used catalyst cited in literature), the study of some reaction parameters (post-consumer PET granulometry, Ethylene glycol:PET ratio and TNT molar percentage) and a modification of the catalyst with zinc (ZnTNT) in the depolymerization of PET For the characterization of the products, the main techniques used were Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) and Nuclear Magnetic Resonance (NMR), as well as yield, turnover number (TON) and turnover frequency (TOF) results. The depolymerization occurred by glycolysis reaction using virgin and post-consumer PET and ethylene glycol at different times at a temperature of 196°C. The main results show TNT as promising catalysts, with yields of BHET yields of 83.9 and 76.7%, for virgin and post-consumer PET, respectively, for 3 hours of reaction. These values are similar to those obtained when zinc acetate was used, in which 79.4% (virgin PET) and 80.8% (post-consumer PET) were reached. After the modification of the TNT with zinc, the yield results in BHET reached 87.1% for 3 hours of reaction at the smaller particle size studied, proving itself to be an even more efficient catalyst for this reaction.

O poli(tereftalato de etileno), PET, é um material polimérico importante, largamente utilizado na produção de garrafas para refrigerante e água mineral. Entretanto, a destinação das embalagens de PET pós-consumo vem criando sérias preocupações econômicas e ambientais. Uma das alternativas de destino desse material é a reciclagem química por glicólise, tendo como objetivo a produção do monômero tereftalato de bis-hidroxietila (BHET). Esta reação, embora referenciada, ainda apresenta problemas como rendimento de BHET e tempo de reação, entre outros, tendo como desafio o desenvolvimento de novos catalisadores eficientes e altamente seletivos. Dentro deste contexto, o presente trabalho busca estudar a atividade catalítica de um material nanoestruturado, os nanotubos de titanatos (TNT), na glicólise de PET (virgem e pós-consumo) comparado ao acetato de zinco (catalisador mais utilizado citado em literatura), a otimização de alguns parâmetros reacionais (granulometria de PET pós-consumo, razão Etilenoglicol:PET, porcentagem molar de TNT) e uma modificação dos nanotubos com zinco (ZnTNT) na despolimerização do PET Para a caracterização dos produtos as principais técnicas utilizadas foram a Calorimetria Exploratória Diferencial (DSC), Análise Termogravimétrica (TGA) e Ressonância Magnética Nuclear (RMN), além de resultados de rendimento, turnover number (TON) e turnover frequency (TOF). A despolimerização ocorreu por reação de glicólise utilizando PET virgem e pós-consumo e etilenoglicol para diferentes tempos a uma temperatura de 196°C. Os principais resultados mostram os TNT como catalisadores promissores, apresentando rendimentos em BHET de 83,9 e 76,7%, para PET virgem e pós-consumo, respectivamente, para 3 horas de reação. Esses valores são equiparáveis aos obtidos quando utilizado acetato de zinco, em que foi alcançado 79,4% (PET virgem) e 80,8% (PET pós-consumo). Após a modificação dos TNT com zinco, os resultados de rendimento em BHET alcançaram 87,1% para 3 horas de reação na menor granulometria estudada, demonstrando ser um catalisador ainda mais eficiente para essa reação. / Polyethylene terephthalate, PET, is an important polymer material, widely used in the production of bottles for soda and mineral water. However, the disposal of post-consumer PET packaging has created serious economic and environmental concerns. One of the alternatives for the disposal of this material is the chemical recycling by glycolysis, aiming the production of the monomer bis-(2hydroxyethyl) terephthalate (BHET). This reaction, although referenced, still presents problems such as BHET yield and reaction time, among others, having as challenge the development of new efficient and highly selective catalysts. In this context, the present work aims to study the catalytic activity of a nanostructured material, the titanate nanotubes (TNT) in PET glycolysis (virgin and post-consumer) compared to zinc acetate (the most used catalyst cited in literature), the study of some reaction parameters (post-consumer PET granulometry, Ethylene glycol:PET ratio and TNT molar percentage) and a modification of the catalyst with zinc (ZnTNT) in the depolymerization of PET For the characterization of the products, the main techniques used were Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) and Nuclear Magnetic Resonance (NMR), as well as yield, turnover number (TON) and turnover frequency (TOF) results. The depolymerization occurred by glycolysis reaction using virgin and post-consumer PET and ethylene glycol at different times at a temperature of 196°C. The main results show TNT as promising catalysts, with yields of BHET yields of 83.9 and 76.7%, for virgin and post-consumer PET, respectively, for 3 hours of reaction. These values are similar to those obtained when zinc acetate was used, in which 79.4% (virgin PET) and 80.8% (post-consumer PET) were reached. After the modification of the TNT with zinc, the yield results in BHET reached 87.1% for 3 hours of reaction at the smaller particle size studied, proving itself to be an even more efficient catalyst for this reaction.

A melhor compreensão dos mecanismos fisiopatológicos e farmacológicos aliados a métodos modernos de investigação tornaram possível a descoberta e o desenvolvimento de fármacos para diversas doenças e disfunções orgânicas em humanos. Os fármacos desenvolvidos atualmente são resultados de intensos esforços em pesquisa por equipes multidisciplinares, impactando diretamente na qualidade de vida das diversas populações no mundo. Nesse cenário, os grupos de pesquisas estabelecidos em Universidades com foco no planejamento de fármacos para doenças tropicais têm crescido. A Malária e a Doença de Chagas figuram com especial importância, a primeira pela expressiva mortalidade mundial, enquanto a segunda pela morbidade e seus impactos na população brasileira. O tratamento de ambas possui limitações que se agravam, seja pelo baixo número de opções terapêuticas, ou pelo desenvolvimento de cepas resistentes. As enzimas investigadas nesse doutoramento, enolase (PfEnolase) de Plasmodium falciparum e gliceraldeído3fosfato desidrogenase de Trypanosoma cruzi (TcGAPDH), são componentes da via glicolítica destes parasitas e são considerados alvos moleculares atrativos para o desenvolvimento de inibidores enzimáticos, dada a importância destas enzimas no processo de obtenção de energia do parasita. Os estudos fundamentamse na busca por modulação seletiva da atividade biológica dos alvos selecionados através do desenvolvimento de novas moléculas bioativas. O estabelecimento de protocolo de expressão e purificação para enzima Pfenolase permitiu sua obtenção em quantidade e pureza suficiente para condução de estudos cinéticos e de triagem biológica, com a identificação de cinco novas classes químicas bastante promissoras; além de ensaios de cristalização, que culminaram na determinação da enzima em diversos complexos cristalográficos. Os dados estruturais produzidos foram fundamentais para condução da abordagem computacional de triagem virtual, que permitiu a identificação de 31 moléculas candidatas a inibidoras de Pfenolase. Avanços significativos foram obtidos também com a enzima TcGAPDH, destacando-se as adaptações nos processos de obtenção da proteína recombinante e ensaio cinético, condução de ensaio de bioprospecção orientada com a identificação e caracterização da molécula isolada (tilirosídeo). Novas condições de cristalização foram identificadas e poderão ser empregadas no processo de obtenção de complexos cristalográficos futuros. Adicionalmente, desenvolveu-se uma ferramenta computacional, Kinecteasy, para processamento automatizado dos dados produzidos das etapas de triagem biológica. Os trabalhos integrados de biologia estrutural e química medicinal desenvolvidos contribuem significativamente para o avanço no processo de planejamento de novos inibidores para as enzimas selecionadas. / A better understanding of the pathophysiological and pharmacological mechanisms together with the modern research methods made possible the discovery and development of drugs for several humans´ diseases. The drugs currently developed are the result of intense efforts in research of multidisciplinary teams having as a direct consequence a remarkable impact on life quality of populations all over the world. In this scenario, research groups established at universities, with their focus on drug development for tropical diseases, are increasing. Malaria and Chagas disease deserve special attention, the former by the expressive world mortality, while the second by the morbidity and its impact on Brazilian population. Treatment for both has limitations, whether by the low number of therapeutic options, or by development of resistance. The target enzymes for this PhD project, enolase (PfEnolase) of Plasmodium falciparum and glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi (TcGAPDH), are essential components of glycolytic pathway and therefore related to the parasite energy production, thus, are considered attractive molecular targets for enzyme inhibitors development. Essentially, the proposed studies seek selective modulation of the target´s biological activity through the development of new bioactive molecules. The expression and purification protocols developed for Pfenolase have allowed us to obtain recombinant protein at suitable yield and purity for conducting screening assays, which has revealed five new chemical classes as Pfenolase inhibitors. Crystallization experiments were successfully conducted and 3D structure were determined for different complexes. Structural data was essential for performing the computational approach of virtual screening, which has allowed us to identify 31 inhibitor candidates for Pfenolase. Significant advances were obtained with TcGAPDH, highlighting the adaptations on recombinant protein protocol and kinetic assay. Assay-guided bioprospecting experiments were successfully performed with identification and characterization of isolated inhibitor (tiliroside). New crystallization conditions were identified and will be employed in future co-crystallization and soaking studies. Additionally, Kinecteasy, a computational tool, were developed for automated data processing of biological screening assays. The structure and medicinal chemistry studies presented here contribute significantly in the process of drug development for the selected enzymes.

Orientadores: Eduardo Galembeck, Denise Vaz de Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T04:47:20Z (GMT). No. of bitstreams: 1 Sarraipa_MarioFerreira_M.pdf: 5351663 bytes, checksum: 54171c332f541db2d4c1eda62a4f5ad7 (MD5) Previous issue date: 2009 / Resumo: O estudo de vias metabólicas é fundamental em todos os cursos das áreas biológicas. O metabolismo de carboidratos, lipídios e proteínas é usado como modelo para estudo de vias metabólicas na maioria das disciplinas de Bioquímica básica e o seu conhecimento é importante para a compreensão dos processos de obtenção e utilização de energia pelos seres vivos. O presente trabalho utilizou-se de uma nova abordagem para o estudo de vias metabólicas baseado na montagem, passo a passo, de uma via metabólica, sendo escolhida a glicólise anaeróbica como modelo. Foi desenvolvido um programa de computador para montagem das reações da via metabólica de maneira que os estudantes manuseassem independentemente com orientações localizadas em um tutorial e com uma seqüência de instruções dos componentes de cada reação Essa metodologia foi aplicada nos cursos de Educação Física, Enfermagem, Medicina e Biologia da Universidade Estadual de Campinas - UNICAMP e no curso de Educação Física do Instituto Adventista de São Paulo - IASP, nas disciplinas de bioquímica básica e fisiologia respectivamente. Como instrumento de coleta, foi estruturado e montado um banco de dados, de modo que, ao utilizar o programa, todas as informações de utilização do mesmo foram armazenadas. Analisando estatisticamente os dados, pudemos observar que durante a montagem da via metabólica os usuários apresentaram uma tendência à diminuição do número de tentativas ao longo das onze reações. Dentre as onze reações, os estudantes apresentaram maior dificuldade na primeira e na sexta, conforme demonstrado pelo percentual de usuários (21% para a 1ª reação e 14% para a 2ª reação) que não conseguiram concluir a reação até a vigésima tentativa. Ainda foi analisado o tipo de erro por reação, possibilitando a discussão individual de cada reação e dos possíveis motivos que ocasionaram este erro. Como instrumento didático, o programa foi muito bem aceito pelos estudantes, que tiveram uma alternativa em relação às "aulas tradicionais sobre glicólise anaeróbica", possibilitando uma abordagem diferenciada do conteúdo. Além disso, o programa permitiu aos estudantes construir as vias metabólicas prestando atenção em cada uma de suas reações e identificando pontos de maior dificuldade Para os professores, o programa serviu como uma ferramenta didática adicional, proporcionando uma apresentação diferenciada do assunto e o conhecimento das etapas de maior dificuldade para os estudantes, permitindo a retomada dos conceitos necessários para a montagem da via glicolítica. / Abstract: The study of metabolic pathways is fundamental for all Biology-related courses. Carbohydrates, lipids, and proteins metabolism is used as a model for the study of metabolic pathways in most Basic Biochemistry subjects, and having such knowledge is important for the understanding of processes such as energy attainment and use up by living beings. This project has used a new approach to study metabolic pathways based on their mounting step-by-step, choosing anaerobic glycolysis as a model. A computer program has been developed for mounting the metabolic pathways reactions so that students could handle it by themselves with support available at a tutorial, and also a sequence of instructions for each reaction components. This methodology was applied to the following courses: Physical Education, Nursing, Medicine, and Biology at Universidade Estadual de Campinas - UNICAMP, and Physical Education at Instituto Adventista de São Paulo - IASP, to the subjects of Basic Biochemistry and Physiology. To collect data, a database has been developed so that, whenever the software was used, every use information was stored. By analyzing data statistically, it was noted that, during metabolic pathways mounting, users were likely to decrease the number of attempts along the eleven reactions. Amongst them, students found it harder on first and sixth reactions, as shown by the percentage of users who were not able to complete the reaction by the twentieth attempt (21% on the first reaction, 14% on the second). Additionally, the type of mistake by reaction has also been analyzed, allowing discussion of each reaction individually, and of possible reasons for such mistakes. As a teaching tool, the program has been very well accepted by students, who had an alternative in relation to the "anaerobic glycolysis traditional classes", making it possible to have a different approach of contents. Furthermore, the program enabled students to build metabolic pathways paying attention to each one of their reactions, and also identifying the most difficult points. For teachers, the software was an additional tool, providing a different presentation of the topic, and also knowledge on the most difficult stages for students, allowing the resumption of the concepts necessary to assemble the glycolytic pathway. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Pour développer des biomarqueurs permettant d'estimer l'état de santé des animaux et de prévoir les effets des contaminants sur les niveaux d'organisation biologique supérieurs, le métabolisme énergétique apparaît comme un candidat de choix. En effet, il participe à la mise en place de grandes fonctions comme par exemple la reproduction ou la croissance. L'altération des processus d'allocation énergétique par la contamination peut avoir des conséquences sur le devenir des individus et de leurs populations. L'objectif de ce travail est d'analyser en laboratoire les impacts potentiels de xénobiotiques (cuivre et éthofumésate) sur le métabolisme énergétique d'un cyprinidé, le gardon Rutilus rutilus, à travers l'étude des processus participant à la formation d'énergie cellulaire(ATP). L'effet des xénobiotiques sur l'état de santé général des juvéniles de gardons a d'abord été appréhendé, et peu d'impacts ont pu être observés sur les indices généraux mesurés. L'effet des contaminants sur la formation aérobie et anaérobie de l'énergie (glycolyse, ses substrats et produits) a ensuite été évalué. Si des différences d'effet des deux contaminants ont été observées au niveau moléculaire, ils ont tous deux induit une augmentation du métabolisme anaérobie au niveau biochimique. Enfin, l'impact des contaminants sur les fonctions mitochondriales a été appréhendé.Des atteintes ont été observées au niveau de la chaîne respiratoire et des ultra-structures des mitochondries, impliquant un effet sur l'énergie cellulaire disponible. Ce travail constitue la base du développement de nouveaux marqueurs précoces de troubles physiologiques des organismes utilisables en biosurveillance. / The energy metabolism constitutes an appropriate approach for the development of biomarkers allowing to estimate the health status of animals and to predict the effects of contaminants on higher levels of biological organization. Indeed, it participates in the establishment of key functions such as reproduction or growth. Alterations of energy allocation process by the contamination can affect the fate of individuals and populations. The aim of this study was to determine in laboratory the potentials effects of chemicals (copper and ethofumaste) on energy metabolism of a cyprinid species, the roach Rutilus rutilus, through the study of processes involved in the synthesis of cellular energy (ATP). The effect of chemicals was first performed on general health status of juvenile roach, and few impacts were found on general indexes measured. Secondly, the study of aerobic and anaerobic energy production (glycolysis, substrates and products) was performed. Significant differences were observed at the molecular regulation level, depending on chemicals. However, at the biochemical level, an increase in anaerobic metabolism was observed with both contaminants. Finally, the effects of contaminants on mitochondrial functions were assessed. Mitochondrial alterations were observed either in the respiratory chain and in the ultra-structure of mitochondria;these results involved an effect on the availability of cellular energy. This work constitutes the basis for the development of new early markers of physiological disorders in organisms used in biomonitoring studies.

An approach to investigating cancer that has recently seen resurgence of interest is the “Warburg effect”. Otto Warburg originally described the altered metabolism of cancer cells and identified that they exhibit an increase in glucose uptake and lactate production. This up-regulation of glycolytic flux and glucose transport is now associated with 90% of cancers. In order to improve the overall understanding of the “Warburg effect” two forms of systems biology have been implemented - comparative in vitro analysis of kinetic activities and dynamic modelling. In this analysis, human breast cancer cell lines MCF-7, MDA-MB-231 and T47D and a non transformed breast cell line MCF-10A were used to identify key similarities and differences in kinetic activities across the glycolytic pathway. Additionally, activities of key glycolytic enzymes hexokinase, pyruvate kinase and lactate dehydrogenase were compared under hypoxic conditions to further understand regulation of cancer cells. The most prominent feature that arose from comparing the kinetic activities of the three malignant and one non-malignant cell line is that each cell line has its own specific set of activities for glycolysis. This indicates that there are differences in regulation across the glycolytic pathway for each of these cell lines. This is of specific interest in the search for therapeutic targets. Further, we determined that despite the prominence of oncogenic HIF signalling activities of hexokinase, pyruvate kinase and lactate dehydrogenase were further modulated by growth under hypoxic conditions. Despite the lack of obvious distinct kinetic differences between the non-cancerous and cancerous cells lines some discernible differences are apparent when modelled in silico.

Orientador: Roger Frigério Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T14:26:13Z (GMT). No. of bitstreams: 1 Ruas_JulianaSilveira_M.pdf: 1871713 bytes, checksum: a20e3cd08d0b770aed3b059541e382e3 (MD5) Previous issue date: 2015 / Resumo: A maioria das células tumorais depende da glicólise para a ressíntese de ATP durante um processo de rápida proliferação, mesmo que haja disponibilidade de oxigênio para a transdução de energia mitocondrial (Efeito Warburg). O objetivo do presente estudo foi avaliar o papel do metabolismo oxidativo mitocondrial na proliferação de células de glioblastoma humano U-87MG e T98G. Quando as células foram cultivadas na presença de oligomicina (um inibidor da ATP sintase) ou antimicina A (um inibidor do complexo III da cadeia transportadora de elétrons), observou-se apenas uma inibição parcial da proliferação das células. Notadamente, a incubação dessas células com ambos os inibidores causou uma inibição quase completa na proliferação celular. Resultados semelhantes foram observados em cultura primária de astrócitos, havendo uma queda na proliferação celular somente quando ambos os inibidores mitocondriais estavam presentes. Medidas de consumo de oxigênio indicaram que células de glioblastoma utilizam parcialmente a fosforilação oxidativa para a ressíntese de ATP e apresentam uma respiração bem acoplada. Quando se inibiu, nestas células, a fosforilação oxidativa do ADP com oligomicina ou antimicina A, houve um pequeno aumento no consumo de glicose e na produção de lactato. No entanto, o tratamento com ambos os inibidores mitocondriais promoveu um menor consumo de glicose e produção de lactato, em comparação com os efeitos que a antimicina A promoveu. Isso indica que a cadeia transportadora de elétrons quando inibida pela presença de antimicina A, promove um funcionamento inverso da ATP sintase, promovendo a hidrólise de ATP para que haja um bombeamento de prótons para o espaço intermembranar mitocodrial. De acordo com os resultados acima descritos, uma queda quase completa do potencial de membrana mitocondrial foi observada apenas quando as células de glioblastoma foram incubadas na presença de ambos os inibidores mitocondriais, oligomicina e antimicina A. Quando a análise do ciclo celular foi realizada, observou-se uma diminuição da percentagem das células em G0-G1 e um aumento nas fases S e G2-M quando tratadas com oligomicina. Quando as células foram tratadas com antimicina A e oligomicina mais antimicina A foi constatado uma diminuição significativa nas fases G0-G1 e G2-M, e um aumento na fase S. Em conclusão, estes resultados indicam que a rápida proliferação de células de glioblastoma depende da existência do potencial de membrana mitocondrial, mas não da fosforilação oxidativa ou do transporte de elétrons na cadeia respiratória / Abstract: Most tumor cells rely on glycolysis for ATP resynthesis during rapid proliferation, despite the availability saturating levels of oxygen for mitochondrial energy transduction (Warburg effect). The aim of the present study was to evaluate the role of mitochondrial oxidative metabolism on proliferation of human glioblastoma cells U-87MG and T98G. When cells were cultured in the presence of oligomycin (ATP synthase inhibitor) or antimycin A (inhibitor of complex III of the electron transport chain), we observed only a partial inhibition of cell proliferation. Remarkably, incubation of cells with both inhibitors caused an almost complete inhibition of cell proliferation. Similar results were observed in primary culture of astrocytes, with a decrease in cell proliferation only when both mitochondrial inhibitors were present. Oxygen consumption measurements indicated that glioma cells partially rely on oxidative phosphorylation for ATP turnover and exhibit a well-coupled respiration. In fact, shutting down mitochondrial ADP phosphorylation in these glioma cells with either oligomycin or antimycin inhibitors slightly increased glucose consumption and lactate release. However, the treatment with both mitochondrial inhibitors promoted lower glucose consumption and lactate release as compared with the effects of antimycin alone, which indicates that ATP synthase is operating reversely and thus hydrolyzing ATP and pumping H+ out when the respiratory chain is inhibited by antimycin. In agreement, an almost complete collapse of mitochondrial membrane potential was only observed when the glioma cells were incubated in the presence of both antimycin and oligomycin, but not of only antimycin. When cell cycle analyses were performed in oligomycin-treated cells, a decrease in the percentage of cells in G0-G1 phase and an increase in S and G2-M phases were observed. When cells were treated with antimycin A or oligomycin plus antimycin A, it was observed a significant decrease in G0-G1 and G2-M cell phases and an increase in S phase. Overall, our results suggest that the rapid proliferation of glioblastoma cells is dependent on the mitochondrial membrane potential, but not on oxidative phosphorylation or electron transport in the respiratory chain / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Ciências