The intertidal zone is characterized by persistent, tidally-driven fluctuations in both abiotic (e.g., temperature, [O2], salinity) and biotic (e.g., food availability, predation) conditions, which makes this a very physiologically challenging habitat for resident organisms. The magnitude and degree of variability of these environmental stressors differs between intertidal zones, with the most extreme physiological stress often being experienced by organisms in the high intertidal. Given that many of the fluctuating conditions in this environment are primary drivers of metabolic rate (e.g., temperature, [O2], food availability), we hypothesized that sessile conspecifics residing in different tidal zones would exhibit distinct ‘metabolic phenotypes,’ a term we use to collectively describe the organisms’ baseline metabolic performance and capacity. To investigate this hypothesis, we collected acorn barnacles (Balanus glandula) from low, mid, and high intertidal positions in San Luis Obispo Bay, CA and measured a suite of biochemical (whole-animal citrate synthase (CS) and lactate dehydrogenase (LDH) activity, aerial [lactate]), physiological (O2 consumption rates), morphological (body size), and behavioral (e.g., cirri beat frequency, % time operculum open) indices of metabolism. We found tidal zone-dependent differences in B. glandula metabolism that primarily related to anaerobic capacity, feeding behaviors and body size. Barnacles from the low intertidal tended to have a greater capacity for anaerobic metabolism (i.e., increased LDH activity), feed less when submerged, and be smaller in size compared to conspecifics in the high intertidal. We did not, however, see differences between barnacles from different tidal heights in whole-animal [lactate] following 24h of air exposure, which indicates that the enhanced capacity of low intertidal barnacles for anaerobic metabolism may have evolved to support metabolism during more prolonged episodes of emersion (>>24h) or during events other than emersion (e.g., coastal hypoxia, predation). There were also no significant differences in CS activity or baseline oxygen consumption rates (in air or seawater at 14˚C) across tidal heights, which implies that aerobic metabolic capacity may not be as sensitive to tidal position as anaerobic processes. Understanding how individuals occupying different shore heights differ in their metabolic capacity becomes increasingly interesting in the context of global climate change, given that the intertidal zone is predicted to experience even greater extremes in abiotic stress.

Adaptation to hypoxia is a well-known phenomenon increasing myocardial resistance to ischemia-reperfusion (I/R) injury as an appropriate physical exercise which improves the contractile function of the heart. Lactate is a major energy substrate for the heart muscle during physical activity and hypoxia. The metabolism of lactate was and still is associated with muscle fatigue, but in the last decades it has been considered its significant modulating function of metabolism during exercise at cellular level and whole organism level. It has been shown that its effects might be similar to the effects of hypoxia and its oxidized form, pyruvate, has the cardioprotective effects. The aim of this study was to compare the expression of LDHA and LDHB isoforms between left and right ventricle in the cardioprotective scheme of adaptation to hypoxia. Another objective/goal was to determine the left ventricular response to I/R insult in the perfused heart model adapted to hypoxia compared with the normoxic controls on/at the expression level of both LDH isoforms. Our results showed differences in the LDHA expression in the left and right ventricle and an increased response of the left ventricle to I/R insult in rats adapted to hypoxia which is reflected at the expression level of both isoforms. Key words: heart,...

Très appréciés des consommateurs du fait de leur qualité nutritionnelle, les produits de la mer jouent un rôle important dans l'alimentation humaine. Toutefois, la confiance du consommateur vis-à-vis de leur qualité est affectée par les riques associés à une exposition aux contaminants chimiques et à la fraîcheur des produits. Si le premier aspect résulte de la pollution des eaux marines sous l'effet d'une forte anthropisation des littoraux, le second est lié à la haute périssabilité de ces produits. Évaluer les niveaux de contamination chimique et la qualité-fraîcheur des produits de la mer est plus qu'un besoin : c'est aujourd'hui une nécéssité. C'est dans ce contexte que les travaux de cette thèse ont été menés. Deux objectifs principaux ont été visés dans la présente étude. Le premier objectif de ce travail était de faire une évaluation spatiale et saisonnière des niveaux de contamination par les polluants métalliques et organiques (HAPs et PCBs) des zones côtières du Sénégal en s'intéressant aux teneurs en contaminants dans les organismes marins. Nous avons étudié 7 espèces marines représentatives des différents maillons de la chaîne trophique (une macroalgue verte, un mollusque bivalve, un crustacé et 4 espèces de poisson) prélevées le long du littoral au niveau de 5 sites présentant des degrés d'anthropisation différents. Les résultats de cette étude montrent que les teneurs en contaminants chimiques des organismes marins sont variables selon les espèces et soulignent ainsi la nécéssité de l'approche multi-espèces pour l'étude de la contamination chimique du milieu. Des variations inter-sites de la teneur en contaminants chimiques dans les organismes ont été mis en évidence. Les sites les plus anthropisés comme Soubédioune et Rusfisque sont ceux qui présentent les teneurs les plus élevées. Les niveaux de contamination métalliques et organiques dans les organismes marins du littoral sénégalais sont inférieurs ou du même ordre de grandeur que ceux mesurés dans d'autres régions d'Afrique de l'Ouest ou dans d'autres régions du monde. L'évaluation des risques associés à l'ingestion des produits de la mer analysés montre que les teneurs en contaminants chimiques sont faibles et inférieures aux limites maximales admissibles pour la consommation humaine (norme EU). Seuls les sites de Rufisque et Soumbédioune présentent quelques dépassements chez certaines espèces (moules, sardinelles). Le deuxième objectif de cette étude était de développer des méthodes permettant d'évaluer la fraîcheur des filets de poisson et de différencier des filets frais des filets décongelés. Les méthodes retenues sont basées sur la mesure de l'augmentation de la perméabilité cellulaire du muscle de poisson. La conservation des filets de poisson à 4°C va conduire à une perméabilisation des cellules dans le temps qui peut être apréhendée par la mesure de la libération d'enzymes intracytoplasmiques ou par l'augmentation de la perméabilité des cellules à des colorants fluorescents. La mesure de l'activité LDH est intéressante à double titre : elle va permettre d'une part de mesure le niveau de lyse cellulaire, donc le niveau d'altération, des filets de poisson dans le temps. Elle va permettre d'autre part grâce à sa sensibilité à la congélation de mieux cerner les conditions qui permettraient à terme de faire la distinction entre les filets frais et des filets congelés/décongelés. / Much appreciated by consumers for its nutritional qualities, seafood plays an important role in human diet. Consumer confidence in the quality of these foodstuffs is nevertheless affected by concerns about risks associated with exposure to chemical contaminants and the freshness of these products. If the first of these is the result of pollution resulting from increasing human activities along coastlines, the second is linked to the highly perishable nature of these products. An evaluation of the levels of chemical contaminants in seafood and of its freshness is therefore a necessity. It is within this context that the work presented in this thesis was carried out. Two principal objectives were targeted in the present study. The first objective of this work was to evaluate the spatial and seasonal variability of seafood contamination by elements (including metals) and organic pollutants (PAHs and PCBs) along the Senegalese coast. We studied 7 marine species representative of different trophic level (a green macro algae, a bivalve mollusc, a crustacean and 4 species of fish) sampling them along the coastline at 5 sites representing different human activity pressur. The result of this study showed that contamination levels varied with species, underlining the importance of a multi-species approach to study contamination in the marine environment. Variations between sites were also observed. Sites with the greatest human activity, such as Soumbédioune and Rufisque, were also those where the highest levels of contaminants in seafood were found. The levels of contamination measured along the Senegalese coast, of both elements and organic pollutants, were inferior or of the same order of magnitude as those reported from other West African sites or from other regions of the globe. The risks associated with eating theseseafood products were low, with contaminant levels generally below the admissible limits (EU) for human consumption. Only a few samples of certain species (mussels, sardines) exceeded these limits at Soumbédioune and Rufisque. The second objective of this study was to develop methods to evaluate the freshness of fish fillets, and to distinguish fresh fillets from previously frozen ones. The methods developed were based upon a measure of cellular permeability within the fish muscle tissue. The conservation of fish fillets ar 4°C results in increased permeability of cells over time, measurable by studying the liberation of intra-cytoplasmic enzymes or the increasing permeability of cells to fluorescent colouring agents. The measurements of lactate dehydrogenase (LDH) is doubly interesting in this contex : on the one hand it enables a measure of cell lysis, and so the level of alteration of the fillets over time, to be established. It also, thanks to its sensitivity to freezing, to better distinguish fresh fillets from those that have been frozen then thawed.

INTRODUÇÃO: A resposta do osteossarcoma (OS) à quimioterapia (QT) préoperatória é atualmente o indicador mais sensível para o prognóstico de sobrevida dos pacientes diagnosticados com OS. Esta resposta é avaliada mediante a porcentagem de necrose encontrada pelo patologista após a extração da peça na cirurgia, utilizando- se o índice de necrose tumoral de Huvos, no qual a necrose é expressada percentualmente. Existem estudos correlacionando os valores da fosfatase alcalina (FA) e da desidrogenase lática (DHL) com a sobrevida do paciente. Neste trabalho foi pesquisada a relação que existe entre os valores sanguíneos, pré e pós QT, de FA e DHL, com a porcentagem de necrose tumoral encontrada na peça cirúrgica após a realização da QT pré-operatória. MÉTODOS: Foram estudados 647 prontuários de pacientes tratados pelo Grupo de Oncologia Ortopédica do Instituto de Ortopedia e Traumatologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, no período de 1990 até o inicio de 2013, com diagnóstico anatomopatológico de OS. Destes, 510 foram excluídos por não apresentarem dados completos para a análise posterior. Foram incluídos um total de 137 prontuários. Os valores da FA e da DHL dos pacientes incluídos foram obtidos da realização do estadiamento, antes da QT pré-operatória e dos valores reportados após a finalização da QT pré-operatória. Também foi coletado o grau de necrose tumoral de cada peça extraída na cirurgia de cada paciente. Classificamos os resultados da FA e DHL obtidos em dois grupos. No grupo I os pacientes com valores normais de FA e DHL, no grupo II, os pacientes com valores acima do limite de normalidade. A classificação utilizada para agrupar os valores de sorológicos em investigação foi adaptado do trabalho realizado por Bramer et. al. Foi reportada a porcentagem de necrose tumoral descrita pelos patologistas, obtida das peças extraídas nas cirurgias realizadas pós QT. Esta porcentagem foi classificada de acordo com a classificação de Huvos. Foram calculadas as correlações da necrose da peça cirúrgica (Huvos) com as enzimas pré, pós e alteração (pós-pré) QT e também entre as enzimas, com uso de correlações de Spearman para verificar a existência de correlação entre elas. RESULTADOS: Tanto a FA como a DHL diminuíram nos pacientes estudados, quando comparados os valores pré QT e pós QT. A média da diferença da FA obtida pré QT e pós QT foi de 795,12 U/L sendo o valor pré QT maior ao valor pós QT. A diferença entre os valores da DHL pré QT e pós QT foi em média de 437,40 U/L, sendo o valor pré QT maior ao valor pós QT. Não houve correlação estatisticamente significativa entre o índice de Huvos e os valores de FA e DHL. A ausência da relação foi observada tanto com os valores pré-quimioterapia, quanto com os valores pós-quimioterapia, não obtendo correlação com a alteração das enzimas após a quimioterapia (p < 0,05). CONCLUSÃO: A medição de FA e da DHL não são fatores preditivos sobre a resposta tumoral à quimioterapia préoperatória em pacientes portadores de osteossarcoma / BACKGROUND AND AIMS: The osteosarcoma´s (OS) response to pre surgical chemotherapy (CT) is actually the most sensible predictor for life prognosis in patients diagnosed with OS. This response is evaluated by the chemotherapy-induced necrosis percentage found by the pathologist after the removal of the surgical piece, using the Huvos tumor necrosis (TN) index, in which the necrosis is expressed in percentage. There are studies that correlate the alkaline phosphatase (AP) and the lactate deshydrogenase (LDH) with the patient\'s life prognosis. In this study we researched the relationship between the serum levels of pre and post CT of AP and LDH and the percentage of TN found in the surgical piece after the pre surgical CT. METHODS: This is a retrospective study in which we studied 647 medical history files with anatomopathologic diagnosis of OS, treated by the Orthopedic oncology group of the Orthopedic and Traumatology Institute of the Hospital das Clinicas of the Medical School of the University of São Paulo between 1990 and 2013. Out of these, 510 were excluded for not having complete data for posterior analyses. We included 137 files in the study. The AP and the LDH values were obtained before and after pre surgical CT. We also obtained the degree of chemotherapy-induces TN obtained from the surgical piece. We classified the AP and the LDH into two groups. In the first group the patients with normal AP and LDH values and in the second group those with values above normal limit. This classification was adapted from the study published by Bramer et al. We classified the percentage of TN obtained from the removed surgical pieces after CT, using the Huvos index. We calculated the correlation between the TN with the pre, post and pre-post difference CT enzymes, using the Spearman correlation. RESULTS: The AP and the LDH decreased when comparing the pre CT values to the post CT values. The mean of the difference was of 795.12 U/L, the pre CT value being higher than the post CT. The difference between the LDH values pre and post CT was of 437.40 U/L, being the pre CT higher than the post CT. There was no statistically significant correlation between the Huvos index and the AP and LDH values. This was observed with the pre CT as well as with the post CT values (p < 0,05). CONCLUSIONS: The values of AP and LDH are not predictors for the tumor\'s chemotherapy-induced necrosis to pre surgical CT in patients with osteosarcoma

Na lesão renal aguda, os mecanismos de defesa atuam como genes protetores, como a proteína heat shock 32 (HSP 32), também conhecida como heme oxigenase-1 (HO-1). A polimixina B (PmxB) é um antimicrobiano nefrotóxico. O objetivo deste estudo foi caracterizar a participação da enzima HO-1 na toxicidade da PmxB em células LLC-PK1. As células foram submetidas aos seguintes tratamentos: Controle (CTL- 0µM); Hemin (indutor de HO-1, 25µM); Hemin II (250M), Protoporfirina de zinco (ZnPP - inibidor de HO-1, 10M,); Nitro-L-arginina-metilester (L-NAME - inibidor de iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM de Hemin uma hora antes da PmxB); PmxB + ZnPP (10M de ZnPP uma hora antes da PmxB); PmxB + Hemin + L-NAME (25M de Hemin e 0,1mM de L-NAME uma hora antes da PmxB). Os grupos foram avaliados em 24 e 72 horas. Foram analisados os seguintes parâmetros: desidrogenase láctica (DHL), peroxidação lipídica (MDA), expressão gênica da HO-1 por RT-PCR, síntese protéica da HO-1 por imunofluorescência, óxido nítrico (NO) pelo método de Griess e expressão protéica da HO-1 e da iNOS por western blotting. Os resultados mostraram que a PmxB elevou o DHL com aumento dos níveis de MDA. O Hemin e a ZnPP elevaram as variáveis DHL, MDA e óxido nítrico (NO). O indutor de HO-1 incrementou a expressão protéica da HO-1 e da iNOS. A PmxB se confirmou como citotóxica e a HO-1 intensificou a lesão por mecanismos oxidativos. O efeito da HO-1 na lesão celular parece ser mediado pelo NO / In the acute kidney injury, the mechanisms of defense act as protector genes, as the protein heat shock 32 (HSP 32), also known as heme oxygenase-1 (HO-1). The polymyxin B (PmxB) is a nephrotoxic antimicrobial. The aim of this study was to distinguish the role of the HO-1 enzyme in the PmxB toxicity in LLC-PK1 cells. The cells were submitted to the following treatments: Control (CTL- 0µM); Hemin (inhibitor of HO-1, 25µM); Hemin II (250M), Zinc protoporphyrin (ZnPP - inhibitor of HO-1, 10M,); NG-nitro-L-arginine methyl ester (L-NAME - inhibitor of iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM of Hemin one hour before the PmxB); PmxB + ZnPP (10M of ZnPP one hour before the PmxB); PmxB + Hemin + L-NAME (25M of Hemin and 0,1mM of L-NAME one hour before the PmxB). All groups were evaluated in 24 and 72 hours. The following parameters were analysed: lactate dehydrogenase (LDH), lipid peroxidation (MDA), genic expression of HO-1 by RT-PCR, protein syntesis of HO-1 by immunofluorescence, nitric oxide (NO) by Griess method and protein expression of HO-1 and of iNOS by western blotting. The results showed that PmxB increased the LDH and the levels of MDA. Hemin and ZnPP also increased the LDH variables, MDA and nitric oxide (NO). The inducer of HO-1 improved the protein expression of HO-1 and of iNOS. The PmxB was confirmed as a cytotoxic and the HO-1 intensified the failure by oxidative mechanisms. The effect of HO-1 in the cell injury seemed to be mediated by NO

La prise de décision cellulaire se traduit par la capacité de toute cellule vivante à intégrer les différentes informations provenant de son environnement, et à les transformer en une réponse biologique cohérente. Il est aujourd'hui de plus en plus démontré que les populations cellulaires présentent une hétérogénéité quantitative et qualitative significative, qui pourrait jouer un rôle essentiel dans le fonctionnement des organismes vivants. La première partie de ma thèse a ainsi consisté à étudier la variabilité de l'expression génique au cours de la différenciation de progéniteurs érythrocytaires aviaires primaires, à l'échelle de la cellule unique. L'expression de 92 gènes a été analysée par RT-qPCR dans des cellules isolées à différents temps de différenciation. Les principaux résultats de cette étude ont montré que la variabilité de l'expression des gènes, mesurée par l'entropie de Shannon, atteint un niveau maximal à 8h-24h de différenciation, simultanément à une chute du nombre de gènes corrélés. Cette augmentation de la variabilité génique précède l'engagement irréversible des cellules dans le processus de différenciation érythrocytaire identifié entre 24 et 48h. Cette étude a également mis en lumière le gène LDHA (Lactate dehydrogenase A), codant pour une enzyme de la glycolyse anaérobie, dans les progéniteurs érythrocytaires en état d'auto-renouvellement et aux points critiques, 8h et 24h, de la différenciation. La deuxième partie de ma thèse a donc consisté à analyser le rôle précis de LDHA dans l'auto-renouvellement des progéniteurs érythrocytaires, ainsi que les variations du métabolisme du glucose au cours de la différenciation. Nos premiers résultats suggèrent que le processus de différenciation érythrocytaire s'accompagne d'un changement métabolique correspondant au passage de la glycolyse anaérobie dépendante de LDHA, vers une production d'énergie aérobie, reposant sur la phosphorylation oxydative / The meaning of cell decision making consists in the capacity of every living cell to integrate environmental information and to transform it in a coherent biological response. Nowadays it is increasingly demonstrated that cell populations present a significant quantitative and qualitative heterogeneity that could be involved in living organisms functions. Thus, the first part of my thesis consisted in studying gene expression variability at the single-cell level during the differentiation process of primary avian erythroid progenitor cells. The expression of 92 genes was analyzed using RT-qPCR in cells isolated at different differentiation time-points. The main results of this study showed that gene expression variability, as measured by Shannon entropy, reached a maximal level, simultaneously to a drop in the number of correlated genes, at 8-24h of differentiation. This increase of the gene expression variability preceded the irreversible commitment of cells into differentiation, identified between 24h and 48h. This analysis also highlighted the potential importance ofLDHA(Lactate dehydrogenase A) encoding a glycolytic enzyme, in erythroid progenitors self-renewal and at the critical differentiation time-point 8-24h. Therefore the second part of my thesis consisted in analyzing the role of LDHA in erythroid progenitors self-renewal and the variations of glucose metabolism during the differentiation process. Our first results suggested that erythroid differentiation might be accompanied with a metabolic change, corresponding to a switch from anaerobic glycolysisdepending upon LDHA, toward aerobic energy production, relying upon oxidative phosphorylation

Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done. Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59&plusmn / 2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.

Myc transcriptionally regulates genes involved in processes such as cell proliferation, metabolism, differentiation, and angiogenesis.  MYC expression is deregulated in many types of human cancer; therefore discovering the mechanisms behind MYCs role in tumorigenesis is essential.  In this dissertation, I have focused on several Myc target genes, Spermidine synthase (Srm); Lactate dehydrogenase (Ldh); 3-phosphoglycerate dehydrogenase (Phgdh); Serine hydroxymethyltransferase (SHMT) 1 and 2; and Pim-3 (a member of the Pim family of serine/threonine kinases).  These enzymes play a role in various functions: Spermidine synthase (polyamine synthesis); Lactate dehydrogenase (glycolysis); Phgdh and Shmt (serine metabolism); and Pim-3 (cell signaling).  In order to elucidate the impact Myc over-expression has on metabolism in tumorigenesis, we use human cell lines, and transgenic mice as well as cell lines and tissues derived from these mice.  The impact of inhibition of these target genes on Myc-driven tumorigenesis was done by genetically inhibiting the target gene (using RNAi or mouse models) or inhibiting the protein with a chemical inhibitor.  Investigating these Myc target genes will help determine if inhibition of Myc target genes is a viable approach for chemotherapeutics, and under what conditions this inhibition may be the most valuable.  In paper I, we examine SRM; a highly expressed enzyme in the polyamine synthesis pathway that converts putrescine to spermidine, and is important for actively growing cells.  Genetic inhibition via RNAi against Srm, or chemical inhibition of Srm, resulted in decreased proliferation of B-cell tumor lines from transgenic mice in vitro.  In vivo treatment of λ-Myc transgenic mice with a chemical SRM inhibitor exhibited a significant chemopreventative effect on tumor formation. These results support previous findings that inhibition of polyamine synthesis pathway enzymes has a place in cancer therapy.  Many Myc target genes have been suggested as attractive targets in battling Myc-driven tumorigenesis.  Surprisingly in paper II, when we analyzed the inhibition of other Myc target genes, such as Ldh, Shmt, and Phgdh, we found that inhibition of these genes did not inhibit Myc-driven tumorigenesis to any significant degree. However, inhibition of Ldh, Phgdh and Shmt2 had a notable effect on in vitro Ras-driven transformation.  These findings suggest that chemotherapeutic inhibition of metabolic genes such as Ldh, Phgdh and Shmt2 may be effective in genetically defined settings, keeping in mind the oncogenic lesion behind the tumor.  The Pim kinase family consists of three serine/threonine kinases, Pim1-3.  In paper III, we found that Pim-3 is a direct Myc target gene and that Pim-3 expression is high in Burkitt Lymphoma samples taken from human patients, as well as spontaneously arising lymphomas from Myc transgenic mice. We also found that inhibition of Pim-3 using a pan-Pim kinase inhibitor, Pimi, in these spontaneously arising Myc lymphomas resulted in caspase independent cell death.  These results indicate that Pim kinase inhibition may be a potential chemotherapeutic strategy in human lymphomas that rely on Pim-3 kinase expression.

Made available in DSpace on 2016-08-17T18:39:34Z (GMT). No. of bitstreams: 1 3220.pdf: 7184046 bytes, checksum: d31437c1aa1937336c7b8cb91918b19b (MD5) Previous issue date: 2010-08-06 / Universidade Federal de Minas Gerais / Malaria disease, caused mainly by Plasmodium falciparum parasite, afflicts about 500 million people and causes nearly one million deaths every year. For the development of new drugs against this disease, one possible approach is to identify an enzyme that plays a key role in P. falciparum development and presents significantly different properties from the corresponding human one. These differences can be exploited in the design of specific inhibitors of the parasite s protein, thus, three different enzymes were selected as possible targets. As there are evidences suggesting that increasing oxidative stress can effectively inhibit the growth of the malarial parasite the enzyme Glutathione Reductase of P. falciparum (PfGR), responsible for the parasite s antioxidant defense, has become a potential target for the design and development of inhibitors. The second target was the P. falciparum Dihydrofolate Reductase-Thymidylate Synthase (PfDHFR-TS), and in this case blocking its action stops the dTMP production and DNA synthesis in the parasite. The third chosen target was the P. falciparum Lactate Dehydrogenase (PfLDH), whose inhibition interrupts the ATP formation and thus causing the death of the parasite. So that a family of arilloxazines compounds, together with chloroquine and methylene blue, were studied by means of docking simulations in the binding sites of these enzymes and also in the corresponding human enzymes for comparison. The three-dimensional structures of the enzymes and of chloroquine and methylene blue were obtained from the Protein Data Bank (PDB). The structures of the arilloxazines compounds, in turn, were obtained by molecular modeling with HyperChem 6.01 and MOPAC2009 programs, using as starting models similar crystallographic structures deposited in the Cambridge Structural Database. Docking simulations were performed using GOLD 4.0.1. The docking results showed that the enzymes PfGR and PfDHFR-TS are not the preferential targets of chloroquine. For the methylene blue it was possible to elucidate its binding mode in hGR and PfGR. Regarding the arilloxazines it was possible to show that they present their higher affinity for hGR, followed by PfGR, hDHFR, PfDHFR-TS, PfLDH and hLDH. In the case of GRs, the interface site was the preferred binding site. The results suggest that if arilloxazines compounds with higher affinity for PfGR are desirable then a pentafluorophenyl should be attached at the N10 position, as in the 2e compound. When searching for arilloxazines with higher affinity for PfLDH, it seems to be desirable a carboxymethyl group at the N3 position (as in 5b) and a pentafluorophenyl group at N10 (as in 2e). Finally, the results suggest that in general the studied arilloxazines probably will present a higher affinity for hDHFR than PfDHFR-TS. All these results are an important starting point for the design of new arilloxazines ligands so that they can be used as lead compounds in the search for new drugs against malaria. / A malária, causada principalmente pelo Plasmodium falciparum, atinge cerca de 500 milhões de pessoas e causa aproximadamente um milhão de mortes todos os anos. Para o desenvolvimento de novos fármacos contra esta doença, uma das abordagens possível é identificar uma enzima que desempenhe papel vital no desenvolvimento do P. falciparum e apresente propriedades significantemente diferentes das enzimas humanas correspondentes, de modo que tais diferenças possam ser exploradas no design de inibidores específicos à proteína do parasita. Existem evidências sugerindo que aumentar o estresse oxidativo pode inibir eficientemente o crescimento do parasita causador da malária e, portanto, a enzima Glutationa Redutase do P. falciparum (GRPf), responsável por sua defesa antioxidante, tornou-se um alvo em potencial para o desenvolvimento de inibidores. Também, o bloqueio da ação da Diidrofolato Redutase-Timidilato Sintase do P. falciparum (DHFR-TSPf) interrompe a produção de dTMP e a síntese de DNA no parasita. Ainda, espera-se que a inibição da Lactato Desidrogenase do P. falciparum (LDHPf) interrompa a produção de ATP no parasita e, consequentemente, cause sua morte. Portanto, estudou-se o comportamento de compostos da família das ariloxazinas, da cloroquina e do azul de metileno nos sítios de ligação destas enzimas, além das enzimas humanas correspondentes para fins de comparação, por meio de cálculos de docking. As estruturas tridimensionais das enzimas foram obtidas no Protein Data Bank (PDB). As estruturas dos inibidores da família das ariloxazinas, por sua vez, foram obtidas por meio de modelagem molecular, utilizando os programas HyperChem 6.01 e MOPAC2009, a partir de estruturas cristalográficas semelhantes obtidas no Cambridge Structural Database; já as estruturas da cloroquina e do azul de metileno foram obtidas também no PDB. Os cálculos de docking destes compostos nos sítios de ligação das enzimas estudadas foram realizados utilizando o programa GOLD 4.0.1. Com base nos resultados de docking, sugere-se que as enzimas GRPf e DHFR-TSPf não são alvos preferenciais da cloroquina. Também, pôde-se elucidar o possível modo de ligação do azul de metileno nas enzimas GRh e GRPf. No geral, foi possível sugerir ainda que as ariloxazinas devam apresentar maior afinidade pela GRh, seguida por GRPf, DHFRh, DHFR-TSPf, LDHPf e LDHh, nesta ordem. Nas GRs, o sítio da interface foi o sítio preferencial de ligação. Para se buscar inibidores da família das ariloxazinas com maior afinidade pela GRPf, sugere-se considerar um pentafluorfenil como substituinte na posição N10, como no composto 2e. Ainda, na busca por ariloxazinas com maior afinidade pela LDHPf, sugere-se considerar um carboximetil na posição N3 (como o de 5b) e um pentafluorfenil na posição N10 (como em 2e). Por fim, foi obtido que as ariloxazinas estudadas possivelmente apresentarão, em geral, uma maior afinidade pela DHFRh do que pela DHFR-TSPf. Estes dados podem ser tomados como ponto de partida para o design de novos compostos da família das ariloxazinas, a fim de que possam atuar como compostos líderes na busca por novos fármacos contra a malária.

Cancer cells are most clearly characterized by their abnormal and uncontrolled cell growth. One of the most notable theories that explains the vast proliferative capacity of tumorigenic cells is the Warburg effect, a significant shift in metabolism wherein cancer cells preferentially fuel cell division using aerobic glycolysis instead of aerobic respiration. This upregulation of glycolytic fermentation in aerobic environments is highly unusual - glycolysis is typically utilized in anaerobic conditions, but nonetheless dominates cancer metabolic activity in spite of the presence of oxygen. Since the discovery the Warburg effect in the 1920s, researchers have struggled to identify whether aerobic glycolysis is a cause or consequence of carcinogenesis. Interestingly, a new theory recently emerged that challenges this widely-accepted metabolic paradigm for cancer. Known as the reverse Warburg effect, this new mechanism shows that in carcinomas such as breast cancer, the Warburg effect occurs not in cancer cells, but rather in tumor-adjacent stromal fibroblasts. These cancer-associated fibroblasts (CAFs) in the greater tumor microenvironment produce lactate - a high-energy metabolite formed as a byproduct of aerobic glycolysis - to fuel aerobic respiration and rapid tumorigenesis in neighboring cancer cells. This emerging theory emphasizes the pivotal role of the tumor microenvironment in determining whether cancer cells undergo aerobic glycolysis or aerobic respiration. Central to this lactate-linked metabolic intersection are two critical enzymes that regulate a cell's metabolic commitment - lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHc). In order to clarify the mechanisms through which CAFs induce tumorigenesis in breast cancer, we plan to carry out two specific aims: (1) evaluate the enzymatic activity of LDH and PDHc, and (2) compare LDH and PDHc enzyme content. Using co-culture techniques to study the breast cancer tumor microenvironment in vitro, we will compare the enzymatic activity and enzyme content of both MCF7 breast cancer cells and CAFs to identify whether the reverse Warburg effect occurs due to post-translational enzyme activation or increased enzyme synthesis.