Made available in DSpace on 2016-06-02T20:36:19Z (GMT). No. of bitstreams: 1 2054.pdf: 3381379 bytes, checksum: b7e331b25f7899a9074fa9a68cff8f00 (MD5) Previous issue date: 2008-08-22 / Financiadora de Estudos e Projetos / This work describe the phytochemical study of bark and leaves of the specie E. paraensis (Rutaceae). This dissertation aims at contributing with the chemistry of the Rutaceae family and evaluation of the antichagasic activity of substances isolated. The phythochemical study allowed to isolate fifteen secondary metabolites belonging to different classes: two alkaloids: N-methylflindersine and skimmianine; the mixture of three steroids: β-sitosterol, stigmasterol e campestanol; the limonoid limonin; six coumarins, which are: marmesin, 8-methoximarmesin, isooxypeucedanin, prangol, isopimpinellin and xanthotoxin; and three glycosides flavonoids: quercetin-3-β-O-rhamnopyranoside, myricetin-3-β- O-rhamnopyranoside and hesperidin, while there is still no reports on the isolation of flavonoids in this specie. The isolated compounds were evaluated as the enzyme inhibitory activity in Ggapdh (glyceraldehyde-3-phosphate-dehydrogenase) of T. cruzi. However, a strategy was used for virtual screening based on physical and chemical properties of substances isolated by the computer program Volsurf 3.0. The ability of the inhibitory compounds pre-classified was tested by isothermal titration calorimetry (ITC), which has characterized the mechanism of action of active substances on the enzyme GAPDH. Through the standardized test the following compounds were tested front of GAPDH: Limonin, quercetin-3-α-O-rhamnopyranoside and prangol showed that the inhibition constant ranging between 91.8 (± 18.08) and 384.8 (145 ± , 05) μM; hesperidin and marmesin that have not submitted activity against the enzyme. / Este trabalho descreve o estudo fitoquímico do caule e das folhas da espécie E. paraensis (Rutaceae). Este estudo visa contribuir com a química da família Rutaceae e verificar a atividade tripanocida das substâncias isoladas desta espécie. O estudo fitoquímico resultou no isolamento de 15 metabólitos secundários, divididos em: dois alcalóides N-metil-flindersina e esquimianina; mistura de três esteróides β-sitosterol, estigmasterol e campesterol; o limonóide limonina; seis cumarinas: marmesina, 8-metoximarmesina, isooxipeucedanina, prangol, isopimpenilina e xantotoxina; e três flavonóides glicosilados, quercetina 3-β-O-raminosídeo, miricetina 3-β-O-raminosídeo e hesperidina, sendo esta última classe inédita nesta espécie. Os compostos isolados foram avaliados quanto a atividade enzimática inibitória de gGAPDH (Gliceraldeído-3-fosfatodesidrogenase) de T. cruzi. Contudo, foi utilizada uma estratégia de triagem virtual baseada nas propriedades físico-químicas das substâncias isoladas através do programa computacional Volsurf 3.0. A capacidade inibitória dos compostos pré-classificados foi testada através de uma Titulação Calorimétrica Isotérmica (ITC), que permitiu caracterizar o mecanismo de ação das subtâncias ativas sobre a enzima GAPDH. Através do ensaio padronizado foram testados os seguintes compostos frente à GAPDH: Limonina, quercetina-3-α-O-L-raminosídeo e prangol que apresentaram constantes de inibição variando entre 91,8 (±18,08) e 384,8 (±145,05) μM; hesperidina e marmesina não apresentaram atividade contra a enzima.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-09T23:35:51Z No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-15T22:34:21Z (GMT) No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) / Made available in DSpace on 2016-08-15T22:34:21Z (GMT). No. of bitstreams: 1 VirginiaPenellopeMacedoESilva_TESE.pdf: 2028754 bytes, checksum: 8e6e495b2056248d59d55bf24abbf089 (MD5) Previous issue date: 2015-04-23 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Nas Am?ricas, a infec??o por Leishmania tem como principal agente etiol?gico Leishmania infantum. Nos ?ltimos 30 anos o padr?o de distribui??o das leishmanioses tem mudado substancialmente e a doen?a tem apresentado um perfil emergente na periferia dos grandes centros urbanos. A infec??o por Leishmania pode evoluir com um amplo espectro cl?nico desde o acometimento da pele, mucosas e v?sceras. Dos indiv?duos infectados por L. infantum apenas 10% desenvolvem a doen?a, sabe-se que 90% da infec??o humana ? assintom?tica e diversos fatores est?o envolvidos no curso da infec??o. Os principais fatores envolvidos no desenvolvimento da doen?a s?o a resposta imune do hospedeiro, a esp?cie e o conte?do g?nico do parasita. O sequenciamento dos isolados de Leishmania poderia aumentar a compreens?o acerca da sintomatologia dos indiv?duos. Dessa forma, o objetivo desse estudo foi avaliar a diversidade gen?tica de cepas de Leishmania circulantes entre humanos, sintom?ticos e assintom?ticos e c?es de ?reas end?micas do Rio Grande do Norte. A variabilidade gen?tica de um grupo de amostras de humanos e caninos com a doen?a visceral, doen?a cut?nea e infec??o assintom?tica foi avaliado com os marcadores moleculares hsp70 e ITS1. O genoma completo de 20 isolados de Leishmania oriundos de humanos, sintom?ticos e assintom?ticos, e c?es foram sequenciados para avaliar a diversidade dessas amostras. Os fragmentos amplificados de hsp70 e ITS1 das amostras e foram analisados e montadas utilizando o pacote Phred/Phrap. Os dendogramas foram constru?dos aplicando o m?todo neighbor joining com 500 bootstraps, seguido das infer?ncias sobre as rela??es entre as variantes de Leishmania. As sequ?ncias dos 20 isolados brasileiros foram mapeadas com o genoma de refer?ncia Leishmania infantum JPCM5, usando o programa Bowtie2, com identifica??o de 36 contigs. As informa??es dos SNPs v?lidos foram utilizadas na PCA. Os SNPs foram visualizados pelo Geneious 7.1 e IGV. As anota??es do genoma foram ent?o transferidas para seus respectivos cromossomos e visualizadas utilizando o Geneious. As sequ?ncias consenso de todos os cromossomos (com m?nimo de 75% das reads com a mesma base) foram alinhadas usando Mauve. As ?rvores filogen?ticas foram calculadas de acordo com c?lculos de m?xima verossimilhan?a e modelos JTT. Como resultados obtivemos que hsp70 e ITS1 n?o foram capazes de definir variabilidade gen?tica entre os isolados de humanos e c?es; nem para os isolados de cultura e de sangue perif?rico, oriundos de um mesmo paciente.O sequenciamento gen?mico dos 20 isolados brasileiros revelou uma forte rela??o entre as cepas de Leishmania circulantes em no Rio Grande do Norte. Os isolados da Grande Natal de humanos e caninos permaneceram agrupados em todas as an?lises, sugerindo que existe proximidade genot?pica e geogr?fica entre os isolados. As amostras isoladas na d?cada de 1990 apresentaram uma maior diversidade genot?pica quando comparadas as amostras recentemente isoladas. De forma geral, n?o encontramos correla??o entre as formas cl?nicas sintom?ticas e assintom?ticas e o conte?do g?nico dos isolados brasileiros de Leishmania. / Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector?s species and the parasite?s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

No description available.

Cancer therapy traditionally seeks to achieve complete tumor eradication via induction of cancer cell death by chemotherapeutic agents or radiation. An alternative strategy is to induce cytostasis, i.e. to arrest proliferation of cancer cells, perhaps in parallel with conventional chemotherapy. Such an alternative strategy could provide prolonged survival with less severe consequences of cytotoxic agents. To be truly effective, a chemotherapeutic drug should exert its action on biochemical targets specific for neoplastic cells while leaving the normal cells unaffected. Therefore, the knowledge of tumor cell-specific biochemical and signaling pathways is a pre-requisite for development of new, prospective anticancer drugs. In this study, we concentrated on the energy metabolism which is remarkably different in tumor and healthy cells. Cancer cells generate ATP mainly through the glycolytic pathway, and depend far less on oxidative phosphorylation (the Warburg effect). The way cancer cells generate energy reflects their need for energy as well as building blocks required for fast biosynthesis. Glycolysis, in contrast to oxidative phosphorylation, enhances biosynthetic pathways thus accelerating progression of tumor cells through the cell cycle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) occupies a central position in the glycolytic pathway thus playing a critical role in the energy metabolism of cancer cells. Along with its enzymatic activity, GAPDH is a multifunctional protein which acts as a signaling and regulatory molecule in several cellular mechanisms. Based on the fact that glycolysis plays a pivotal role in survival of cancer cells, we hypothesized that down-regulation of GAPDH protein would alter the cancer cell proliferation, and cellular sensitivity of cancer cells to chemotherapy. The goal of this study was to evaluate GAPDH as a potential molecular target for treatment of cancer. In this project, our aims were: 1) To determine the effect of GAPDH level on cell proliferation and cell cycle progression of human carcinoma cells; 2) To elucidate the molecular mechanism(s) causing proliferation arrest in GAPDH-depleted cells; 3) To identify the chemotherapeutic agents exhibiting cytotoxic effect against non-dividing, senescent cells; 4) To analyze molecular dynamics of nuclear GAPDH and its mutant variants in the context of chemotherapy-induced stress. Towards these aims, we developed an experimental model where the level of GAPDH in human carcinoma cells was modulated by RNA interference (RNAi) technology. In vitro experiments were performed in this model system to evaluate the energy status, and signaling pathways in cancer cells after GAPDH depletion. Human carcinoma isogenic cell lines with different levels of GAPDH protein were analyzed for the sensitivity to various chemotherapeutic agents. Using site-mutagenesis, we prepared mutated variants of GAPDH and estimated their enzymatic activity. We also prepared constructs where GAPDH cDNA was fused with green fluorescent protein (EGFP) cDNA, and transiently expressed them in human cancer cells, to assess GAPDH localization and biological effects. We analyzed intranuclear localization and dynamic characteristics of GAPDH and its variants in the live cells using image confocal technologies (e.g. FRAP). In our study, we demonstrated that GAPDH is a molecular target with clinical potential for senescence-based tumor suppression. Our experiments revealed that depletion of GAPDH induces energy crisis and proliferation arrest in human carcinoma cells. We elucidated the molecular mechanisms initiated by GAPDH depletion, and demonstrated that GAPDH-depleted cells acquire the accelerated senescence phenotype. Moreover, we found chemotherapeutic agents cytotoxic to the senescent cells, a finding that opens a way to combination chemotherapy with therapy-induced senescence agents. Our results on dynamic characteristics of intranuclear GAPDH and its mutant forms indicate that in the nucleus, GAPDH interacts with biomolecules yet to be identified. The results of this study suggest a novel, prospective molecular target for pharmacotherapeutic intervention in cancer management. / Pharmaceutical Sciences

Baker's yeast (Saccharomyces cerevisiae) undergoes major biochemical and structural rearrangements in order to survive cycles of desiccation and rehydration, yet a firm understanding of the response is lacking. The purpose of this study was to examine the response of S. cerevisiae to desiccation and rehydration at both the physiological and molecular levels. Transmission electron microscopy was used to show that loss of vacuolar structure, enlarged nuclear boundaries, as well as cell wall thickening were all associated with the desiccation response. Molecular analysis focused on glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.13), a multifunctional protein that is involved in several cellular processes other than glycolysis, including nuclear translocation under stress and intracellular sensing of oxidative stress during apoptosis. Here, GAPDH was studied primarily to determine its potential role in mediating the changes in cell wall physiology identified through our structural studies. GAPDH appears to be shuttled between the cell wall and the cytoplasm during the desiccation/rehydration process. Western analyses in combination with the use of inhibitors of translation (cycloheximide) suggest that the shuttling process does not require de novo protein synthesis. Western analyses also identified an immuno-reactive peptide in the cell wall and cytoplasmic fractions of lower molecular mass than native GAPDH (27 KDa vs. 37 KDa). This lower molecular weight peptide exhibited the translocation process similar to that of the full length GAPDH. Studies with GAPDH deletion strains suggested that the 27 kDa fragment is encoded by tdh3. The importance of this lower molecular weight form is yet to be determined. / Master of Science in Life Sciences

La diatomée d'eau douce A. formosa peut produire des lipides neutres en plus ou moins grandes quantités en fonction des conditions de culture. Ainsi, nous avons montré par exemple qu'une carence en silice stimule la production de triacylglycérols (TAGs) mais génère une diminution de la biomasse. En revanche, nous avons montré que l'addition de bicarbonate et de phytohormones augmente à la fois la biomasse et la production de TAGs. L'ajout de phytohormones dans les milieux de culture de cette diatomée résulte en une augmentation de l'activité d'enzymes dans les extraits et notamment celles du cycle de Benson-Calvin. Parmi ces enzymes, la GAPDH est une enzyme dont l'activité augmente significativement. Nous avons montré que chez A. formosa, cette enzyme forme un complexe ternaire avec la CP12 et la Férrédoxine NADP Réductase (FNR) et non pas avec la CP12 et la phosphoribulokinase comme chez la plupart des organismes photosynthétiques. La régulation de cette enzyme en est de fait modifiée. La phytohormone, 24-épibrassinolide conduit à une augmentation d'activité de la GAPDH qui résulte de la dissociation du complexe GAPDH-CP12 et la GAPDH n'est plus redox régulée. La GAPDH chez les diatomées est donc régulée par des interactions protéineprotéine. / A. formosa, a freshwater diatom, can produce different amounts of neutral lipids such as triacylglycerols (TAGs) under different growth conditions. We showed that as it is well-known for diatoms, starvation for silica increased the production of TAGs but decreased biomass. However, the addition of bicarbonate or phytohormones into the growth medium increased both biomass and TAGs. Addition of phytohormones increased the activities of enzymes in particular those of the Benson-Calvin cycle. Among the target enzymes of the Benson-Calvin cycle, GAPDH was strongly affected. We purified this enzyme and demonstrated that, in the diatom A. formosa, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Calvin cycle, forms a complex with the small chloroplast protein CP12 and Ferredoxin NADP Reductase (FNR), which is involved in the photochemical phase of photosynthesis. In cells treated with the phytohormone, 24-epibrassinolide, GAPDH was "free", not redox-regulated and not associated anymore with CP12. Therefore GAPDH from this diatom is regulated by protein-protein interaction but the GAPDH/CP12/FNR complex replaces the one formed between GAPDH, CP12 and phosphoribulokinase found in most photoautotrophs.

Streptococcus pneumoniae est un pathogène humain majeur causant des pneumonies, méningites et septicémies. Pour assurer sa survie et sa dissémination le pneumocoque déploie un ensemble de facteurs de virulence favorisant l'invasion des tissus et l'évasion du système immunitaire. Une classe particulière de protéines dites « moonlighting », ne sont associées à aucun système d'export connu et pourtant se trouvent localisées en surface du pneumocoque. Les protéines « moonlighting » sont des protéines cytoplasmiques conservées, localisées dans divers compartiments cellulaires et présentant des fonctions additionnelles. La glycéraldéhyde-3-phosphate déshydrogénase (GAPDH) est retrouvée en surface de nombreuses cellules et exerce divers rôles dans les processus de virulence d'organismes pathogènes. La GAPDH de surface du pneumocoque agit comme un facteur de virulence en recrutant le plasminogène / la plasmine de l'hôte, ce qui facilite l'invasion bactérienne à travers la matrice extracellulaire et les barrières endothéliales et épithéliales. Cependant, les mécanismes permettant l'export et la fixation de la GAPDH à la surface de la bactérie n'avaient pas encore été découverts. Ce travail démontre que la GAPDH est relarguée par lyse cellulaire et s'associe au peptidoglycane. C1q, un composant clé de la voie classique du complément, est un acteur majeur dans la réponse aux infections microbiennes et peut également détecter des éléments nocifs du soi-altéré comme les cellules apoptotiques. L'usage d'approches expérimentales complémentaires a permis d'identifier la GAPDH comme un partenaire de C1q quand elle est exposée en surface de S. pneumoniae et de cellules apoptotiques humaines. Néanmoins et de manière plutôt inattendue, seule la GAPDH du pneumocoque active la cascade du complément à la différence de la protéine homologue humaine. Ces résultats encouragent la poursuite d'études afin de comprendre comment la reconnaissance par C1q de deux protéines très proches peut conduire à de telles différences sur ses propriétés d'activation du complément. / Streptococcus pneumoniae is a major human pathogen, which causes pneumonia, meningitis and septicemia. To insure its survival and dissemination, the pneumococcus deploys an array of virulence factors promoting invasion of tissues and evasion from the immune system. A particular class of proteins not associated with any known export system, the moonlighting proteins, is found at the pneumococcal surface. Moonlighting proteins are conserved cytoplasmic metabolic enzymes or molecular chaperones localized in various cellular compartments and exhibiting additional functions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is found at the surface of numerous eukaryotic and prokaryotic cells, and display diverse roles in the virulence processes of pathogenic organisms. The pneumococcal surface GAPDH acts as a virulence factor by binding to host plasminogen/plasmin, which facilitates the bacterial invasion through the extracellular matrix and the endothelial and epithelial cell barriers. However, the mechanisms leading to the GAPDH export and binding to the bacterial surface had not been deciphered yet. This work demonstrates that the GAPDH is released upon cell lysis and associates with the peptidoglycan. C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. The use of complementary experimental approaches allowed the identification of the GAPDH as a C1q partner when exposed at the surface of S. pneumoniae and human apoptotic cells. However, and rather unexpectedly, the pneumococcal GAPDH activates the complement cascade unlike the human one. Those results encourage further studies in order to understand how C1q recognition of two closely related proteins can lead to such striking differences on its complement activation properties.

Das Ziel dieser Arbeit war es einen geeigneten internen Standard für die linksventrikuläre mRNA-Quantifizierung bei kardialer Hypertrophie und Herzinsuffizienz in der Ratte zu finden. Die mRNA-Expression von GAPDH, 18SrRNA, Cyclophilin and Porphobilinogen-Desaminase (PBGD) wurde vier Wochen nach Induktion von Hypertrophie (kleiner aortokavaler Shunt) und Herzinsuffizienz (großer aortokavaler Shunt bzw. Myokardinfarkt) mit Hilfe des Ribonuklease Protektion Assay (RPA) und der TaqMan PCR bestimmt. Die linksventrikuläre ANP-mRNA-Expression war in allen untersuchten Modellen unabhängig von der angewendeten Detektionsmethode erhöht. Die mRNA-Expression der Housekeeping Gene mit Hilfe des RPA bestimmt, war in allen untersuchten Modellen im Vergleich zu den Kontrollen unverändert (GAPDH: kleiner Shunt: 105.1+-7.4, großer Shunt: 105.2+-6.8, MI: 88.4+-3.7; 18SrRNA: kleiner Shunt: 110.7+-8.2, großer Shunt: 104.4+-8.9, MI: 107.5+-12.0; Cyclophilin: kleiner Shunt: 96.4+-7.9, großer Shunt: 112.9+-4.9, MI: 95.7+-13.8; PBGD: kleiner Shunt: 81.9+-6.3, großer Shunt: 83.7+-4.7, MI: 79.8+-9.7; % Kontrolle). In der sehr sensitiven TaqMan PCR zeigte sich eine veränderte mRNA-Expression von GAPDH, PBGD und Cyclophilin, lediglich 18S wurde unverändert exprimiert (GAPDH: kleiner Shunt: 114.5+-18.7, großer Shunt: 133.6+-19.1, MI: 64.2+-6.2, p / The purpose of this study was to identify an appropriate left ventricular mRNA as internal standard in gene expression analysis in cardiac hypertrophy and heart failure in the rat. Expression levels of GAPDH, 18SrRNA, Cyclophilin and porphobilinogen desaminase (PBGD) were measured four weeks after induction of either cardiac hypertrophy (small aortocaval shunt) or heart failure (large aortocaval shunt or myocardial infarction) using Ribonuclease protection assay (RPA) and TaqMan PCR. The left ventricular expression of ANP mRNA was increased in all these experimental models independently of the used method. Using RPA the mRNA expression of all studied housekeeping genes was unchanged in all experimental models compared to controls (GAPDH: small shunt: 105.1+-7.4, large shunt: 105.2+-6.8, MI: 88.4+-3.7; 18SrRNA: small shunt: 110.7+-8.2, large shunt: 104.4+-8.9, MI: 107.5+-12.0; Cyclophilin: small shunt: 96.4+-7.9, large shunt: 112.9+-4.9, MI: 95.7+-13.8; PBGD: small shunt: 81.9+-6.3, large shunt: 83.7+-4.7, MI: 79.8+-9.7; % control). Using the TaqMan PCR as a much more sensitive method only 18SrRNA levels were unchanged whereas GAPDH, PBGD and Cyclophilin mRNA expression was regulated (GAPDH: small shunt: 114.5+-18.7, large shunt: 133.6+-19.1, MI: 64.2+-6.2, p

O T. cruzi, é o agente causador da doença de Chagas. O estado redox NAD+/NADH intracelular é fundamental na manutenção do metabolismo celular. A GAPDH apresenta a função de proteção do telômero em mamíferos contra degradação, isto por causa de ligar se ao telômero. Aqui, mostramos que a GAPDH recombinante de T. cruzi (rTcGAPDH) interage com o DNA telomérico. A rTcGAPDH liga ao DNA de simples fita. Mostramos que a GAPDH liga ao DNA telomérico in vivo em células epimastigotas, onde a [NADH] é maior que [NAD+], mas a adição de NAD+ exógeno bloqueia esta interação. Corroborando a hipótese de que o equilíbrio NAD+/NADH determina a interação GAPDH-telômero, vimos que o tripomastigota tem maior [NAD+] intracelular que a [NADH] e a GAPDH não é capaz de ligar se ao DNA telomérico. Além disso, o NADH exógeno resgata a interação GAPDH-telómero nesta fase. É importante o equilíbrio NAD+/NADH desta interação em tripanosomas, sugerindo que a proteção do telômero do parasita pode ser regulada pelo estado metabólico das células. / The T. cruzi, is the causative agent of Chagas disease. The redox state of NAD+/NADH intracellular is critical in the maintenance of cellular metabolism. The GAPDH has the protection function of the telomere in mammals against degradation, because it is connecting to the telomere. Here we show the recombinant GAPDH of T. cruzi (rTcGAPDH) interacts with telomeric DNA. The rTcGAPDH binds to single-stranded DNA. We show GAPDH to bind to telomeric DNA in vivo epimastigotes cells, where [NADH] is greater than [NAD+], but the addition of exogenous NAD+ blocks this interaction. Corroborating the hypothesis that the NAD+/NADH balance determines the GAPDH-telomere interaction, we saw that the trypomastigote has higher [NAD+] that intracellular [NADH] and GAPDH is not able to connect to telomeric DNA. In addition, the exogenous NADH recovers the GAPDH-telomere interaction at this stage. It is important the NAD+/NADH balance this interaction in trypanosomes, suggesting that the protection of the telomere of the parasite can be regulated by the metabolic state of the cells.

Les objectifs de ce travail étaient d’approfondir les connaissances sur les capacités d’adaptation des diatomées à différentes concentrations de CO2 et plus précisément sur l’implication des CCM dans l’assimilation du carbone minéral dissous. Des études sur la physiologie, les enzymes de différentes voies métaboliques et des analyses de transcriptomique et de protéomique ont été réalisées. Nous avons observé que les espèces de diatomées étudiées étaient capables de réguler leurs systèmes de concentration du carbone minéral suivant les conditions environnementales. Certaines semblent utiliser préférentiellement le CO2 ou le bicarbonate tandis que d’autres espèces utilisent les deux. L’activité des anhydrases carbonique est fortement induite en faible concentration de CO2. Nos travaux montrent cependant, que l’activité de cette enzyme est variable d’une espèce à l’autre. Nous avons aussi observé que dans la majorité des diatomées que nous avons étudiées, la photosynthèse de type C3 et non de type C4 est présente, apportant ainsi un éclairage à une véritable controverse sur le métabolisme du carbone chez les diatomées. Nos travaux soulignent de plus, une grande diversité de stratégies de CCM chez les diatomées. Chez T. pseudonana, une nouvelle protéine, LCIP63, a été observée en conditions de faible concentration de CO2. Son rôle physiologique est actuellement inconnu ouvrant de nouvelles perspectives de recherche. / The objectives of this work were to improve the knowledge on ability of diatoms to scope with different CO2 concentrations and to study their carbon concentrating mechanisms (CCM). Studies of their physiology, their metabolic enzymes, and analyses at transcriptomic and proteomic levels were performed. In all studied diatoms, the CCMs can be regulated according to CO2 availability in the environment. Some diatoms seem to use preferentially CO2, others, bicarbonate and some can use both. The carbonic anhydrase (CA) activity is strongly induced when cells were grown at low vs high CO2. However, our work shows that CA activity is highly variable among the different diatoms. Most of the studied diatoms perform a C3 photosynthesis and not C4 photosynthesis. In diatoms, there is a huge diversity in the CCM strategy. A new protein, LCIP63, was observed when T. pseudonana was grown under low CO2. The physiological role of this protein is yet unknown and this finding opens new research perspectives.