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.

Le complexe de remodelage de la chromatine NuRD, composé des sous-unités catalytiques CHD3 et CHD4, est un régulateur épigénétique de l’expression génique. Nos résultats montrent que NuRD s’associe avec les facteurs de transcription essentiels du mélanome que sont MITF et SOX10. Cependant, malgré une association physique et une co-localisation génomique, CHD4/NuRD ne semble pas agir comme un cofacteur important pour MITF ou SOX10. Néanmoins, la répression de CHD4 conduit à un ralentissement de la prolifération et déréprime l’expression des enzymes PADI1 et PADI3 dans les cellules de mélanome ainsi que dans de nombreux types de cellules cancéreuses. Ainsi, l’induction de ces enzymes, responsables de la conversion des arginines en citrullines, entraîne la citrullination spécifique de PKM2, une enzyme glycolytique essentielle, diminuant ainsi sa sensibilité aux inhibiteurs allostériques, et donc altérant l’équilibre physiologique entre activateurs et inhibiteurs de l’enzyme. L’ensemble de ce travail de thèse a permis de mettre en évidence une nouvelle voie reliant, d’une part la régulation épigénétique de l’expression de PADI1 et PADI3 par CHD4/NuRD ainsi que la reprogrammation de la régulation allostérique de PKM2 via la citrullination d’arginines, au flux glycolytique et au contrôle de la prolifération des cellules cancéreuses d’autre part. / The Nucleosome Remodelling and Deacetylation (NuRD) complex is an epigenetic regulator of gene expression that includes two mutually exclusive ATPase subunits CHD3 and CHD4. Our results show that NuRD associates with essential melanoma cell transcription factors namely MITF and SOX10. However, despite their physical association and genomic co-localization, CHD4-NuRD does not appear to act as a cofactor for MITF or SOX10 regulated gene expression. Nevertheless, CHD4 silencing leads to a slow growth phenotype and de-represses the expression of PADI1 (Protein Arginine DeIminase 1) and PADI3, two enzymes involved in converting arginines to citrullines in melanoma and multiple types of cancer cells. Increased expression of PADI1 and PADI3 enhances citrullination of arginines within the key glycolytic regulatory enzyme PKM2 then promoting excessive glycolysis, lowering ATP levels and slowing down proliferation. PKM2 citrullination lowers its sensitivity to allosteric inhibitors thus shifting equilibrium towards allosteric activators thereby bypassing the normal physiological regulation of glycolysis. Overall, our results lead to describe a novel pathway linking, epigenetic regulation of PADI1 and PADI3 expression by CHD4/NuRD and reprogramming of PKM2 allosteric regulation through arginines citrullination, to glycolytic flux and cancer cell proliferation.

Das Glioblastoma multiforme (GBM) ist der am häufigsten vorkommende maligne Hirntumor mit äußerst ungünstiger Prognose für die betroffenen Patienten. Typisch für die Tumore ist eine hohe Aktivität der Glykolyse zur Generierung von ATP und zur Bereitstellung von Makromolekülen für die Zellproliferation, während die oxidative Phosphorylierung auch in Gegenwart von Sauerstoff praktisch keine Bedeutung für die Generation von ATP hat, was auch als Warburg Effekt bekannt ist. Das natürlich vorkommende Carnosin (β-Alanyl-LHistidin) wirkt sich antiproliferativ auf Tumorzellen aus, was mit einer Inhibition der glykolytischen ATP Produktion einhergeht. Der Mechanismus der Inhibition ist weitgehend unverstanden und ist Gegenstand der vorliegenden Arbeit. Im Rahmen der durchgeführten Arbeit wurde der Einfluss von Carnosin auf die mRNA Expressionen von für die Glykolyse relevanten Genen untersucht, wobei eine starke Induktion der Pyruvatdehydrogenase Kinase (PDK) 4 in drei GBM Zelllinien beobachtet wurde. Weiterhin konnte gezeigt werden, dass L-Histidin den gleichen Effekt wie Carnosin zeigt, nicht jedoch β-Alanin, L-Alanin oder L-Alanyl-L-Histidin. Da Tumorzellen die intrazelluläre Gewebscarnosinase aber kaum die extrazelluläre Serumcarnosinase exprimieren, liegt die Vermutung nahe, dass die antineoplastische Wirkung des Carnosins auf die enzymatische Spaltung von Carnosin und die daraus resultierende Freisetzung von L-Histidin zurückzuführen ist. In weiteren Untersuchungen wurden Hinweise erbracht, dass Carnosin durch eine Beeinflussung von Histon-Deacetylasen, die endogene PDK4 mRNA Expression steigern könnte. Zusätzlich wurden die Proteinexpressionen der PDK1 und 4 unter dem Einfluss von Carnosin untersucht.

Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Human immunodeficiency virus (HIV) primarily infects T helper (Th) cells. Decrease in the number of Th cells is the hallmark of HIV infection. Latent reservoirs of human immunodeficiency virus (HIV) are the leading barrier towards eradication of HIV infection. T Follicular helper (Tfh) cells are a subset of Th cells that function to provide aid to B cells for their maturation, affinity selection and antibody class switch. Several studies have shown that Tfh cells are a major reservoir of latent as well as productive hiv infection. But in contrast to the fate of other Th cell subsets, the frequency of Tfh cells was shown to have increased during HIV infection which could not be attributed to their reduced susceptibility to HIV infection. The hypothesis was that Tfh cells possess a unique metabolic phenotype that protects them from HIV induced cell death. Transcriptome analysis of Th subsets from human donors and showed that Tfh cells rely less on glycolysis for their energetic requirements and instead have increased transcription of fatty acid synthesis genes. This finding was corroborated by seahorse extracellular flux assay. The results shoId that glycolysis was not essential for Tfh cell differentiation in-vitro. The observed increase in Tfh cell frequency could not be attributed to increased Tfh differentiation upon HIV infection since HIV infection inhibited the differentiation of both non-Tfh and Tfh cells. The results found that bypassing the glycolytic pathway by providing Tfh cells with Galactose in the medium protected ex-vivo infected primary tonsillar cells from HIV induced cell death. This protection could be partly explained by the induction of Baculovirus IAP repeat containing 5 (BIRC5) when the cells utilized Galactose instead of Glucose. The studies together show that Tfh cells have an oxidative metabolic phenotype which protects them from HIV induced cell death in part by induction of BIRC5 expression.

No description available.

The brain is one of the most energetically demanding organs within the human body and is cognitively susceptible to energetic deficits such that the rise in obesity, insulin resistance, and Alzheimer’s disease in recent decades pose a substantial threat to cognitive longevity. The therapeutic efficacy of ketones are well-established in epilepsy and are currently being applied to other disease states. Alzheimer’s disease is characterized by impairments in brain glucose uptake and metabolism in regions relevant to learning, memory, and cognition that progress with the disease. While brain glucose uptake is impaired, ketone uptake is unaltered, potentially enabling ketones to fuel the glucose-deficient brain. Using RNA-seq data acquired from multiple publicly available AD databases, we assessed glycolytic and ketolytic gene expression in post-mortem AD and cognitively normal control brains. Gene expression was normalized to brain region – parietal lobe, cerebellum, temporal cortex, frontal lobe, inferior frontal gyrus, parahippocampal gyrus, superior temporal gyrus – and cell type – neurons, astrocytes, oligodendrocytes, and microglia. We report impairments in glycolytic gene expression in regions of the brain relevant to memory and cognition in neurons and oligodendrocytes, but not ketolytic gene expression in neurons. The data are consistent with previous work and support clinical ketone intervention. The cognitive effects of ketogenic diets remain controversial, especially in healthy adults. To elucidate the effects of a ketogenic diet in healthy mice, C57BL6 mice were placed on a ketone-supplemented ketogenic diet for eight weeks. Recognition memory was assessed in a novel object recognition test and hippocampal bioenergetics were measured using high-resolution respirometry, western blot, and biochemical assays. The diet significantly improved recognition memory and enhanced hippocampal mitochondrial efficiency, measured by ATP production per unit of oxygen consumed, suggesting cognitive validity of the diet in middle-age. Long-term potentiation (LTP), the activity-dependent strengthening of synapses, within the hippocampus, is one of the molecular mechanisms of learning and memory formation. LTP of hippocampal Schaffer-collaterals was quantified in young adult C57BL/6 mice with field electrophysiology following ex vivo brain slice incubation with a β-hydroxybutyrate-rich ACSF. Mice were then placed on the ketone-supplemented diet for four weeks. Behavioral spatial memory was measured in the Morris water maze and Schaffer-collateral LTP was assessed with field electrophysiology. No meaningful changes in LTP and behavioral memory were observed with ketone treatment, suggesting ketogenic interventions may be more applicable in aging and pathologies that display cognitive deficits, rather than in healthy young adults. Together, these studies support the exploration of ketogenic interventions as a potential restorative measure in Alzheimer’s disease and preventative measure in aging, which may be impactful facing the rise of obesity and insulin resistance.

Objective: The aim was to discriminate among several hypotheses of preconditioning of isolated rabbit cardiomyocytes and to determine if ischaemic preincubation would evoke a protective response. Methods: Isolated myocytes were subjected to 5 min of preincubation, in the presence or absence of glucose, and incubated in the presence of 1 mM iodoacetic acid during the final sustained ischaemic period. In a second series, the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA), ingenol 3, 20-dibenzoate, and thymeleatoxin were added during preincubation. In a third series, preincubation periods were substituted by brief ischaemic pelleting of cells. Final prolonged ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury was determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 5 min glucose-free preincubation. Addition of iodoacetic acid into the final ischaemic pellet increased the rates of rigor contracture and injury, but did not abolish the protective response. Direct protein kinase C activation with PMA, a non-selective phorbol ester, and ingenol, an ε, δ-PKC isozyme selective activator, protected cells, but thymeleatoxin, an α,β,γ-PKC isozyme selective activator, did not. A 10 min ischaemic preincubation preconditioned, but the protection was not enhanced when ischaemia was extended to 30 min, or when PMA was included during the initial ischaemic preincubation. Adenosine partially inhibited the response. Conclusions: (1) Preconditioning of isolated myocytes is not dependent on glycolysis or glucose transport. (2) Preconditioning appears dependent on activation of the ε-PKC isoformn. (3) Ischaemia is capable of preconditioning isolated myocytes in vitro, and initiation of this effect is modified by simultaneous additional of adenosine but not by direct protein kinase C activation with PMA. Induction of protection by PMA and ingenol shows that protection requires protein kinase C activation, but direct potassium channel activation by regulatory G proteins is not critical.Cardiovascular Research 1994;28:1700-1706.

Objective: The aim was to determine if isolated rat cardiomycytes could be protected from ischaemic cell death by preincubation with adenosine or adenosine agonists. Methods: Cardiomyocytes isolated from rat hearts were preincubated in the presence of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), or carbachol prior to concentration into an ischaemic slurry. Effects of glycolysis and of isoprenaline were determined by addition of iodoacetic acid or isoprenaline to the ischaemic incubates and by exclusion of glucose from all media. Rates of ischaemic contracture were determined and survival of the myocytes versus paired control preparations was determined after various times of ischaemia, following resuspension of the cells in isotonic or hypotonic media. Results: Adenosine and CCPA produced only a small reduction of the rates of contracture and death of isolated myocytes. Carbachol gave no significant protection. Neither the degree of injury of control cells nor the amount of protection by CCPA was altered in the presence of added isoprenaline. Protection was abolished by the A1 receptor blocker sulphophenyl theophylline, iodoacetic acid, and exclusion of glucose. Conclusions: Adenosine and adenosine agonists afford a minimal degree of protection to ischaemic isolated myocytes by a glucose dependent mechanism. This protection does not appear to account for the larger degree of protection seen in intact hearts, following similar preconditioning protocols. The failure of adenosine to protect may be related to the quiescent state of isolated cardiomyocytes, or be species specific in that adenosine may not be the trigger for preconditioning in rats.Cardiovascular Research 1993;27:1670-1676.

Glucose 1,6-bisphosphate is an important small molecule involved in the regulation of glycolysis. Four enzymes synthesize this compound. One enzyme is known to degrade it, glucose 1,6-bisphosphatase. Other groups have produced work that indicates that there are two isoforms of this enzyme, one predominant in the brain and one in the muscle. This thesis contains the work performed in attempts to isolate and characterize the muscle isoform of glucose 1,6-bisphosphatase. While this enzyme was not isolated, much was learned about it and the results from this work may help in the future identification of this enzyme.