A presente pesquisa teve por objetivos principais: (1) estudar a bioacumulação do metal cromo (40-50 \'mü\'g/L) na Clorophyceae Pseudokirchneriella subcapitata (Korshikov) Hindak 1990 e dos metais cobre (2-10 \'mü\'g/L) e cromo (1-6 mg/L) na macrófita Pistia stratiotes L.; (2) avaliar os mecanismos de detoxificação, as estratégias de defesa e tolerância de Pistia stratiotes L., visando recomendar seu uso na fitorremediação; ambos através do uso do Delineamento Composto Central (DCC) e Metodologia de Superficie de Resposta (MSR), e (3) estudar a dinâmica de remoção de nutrientes em wetlands construídos, plantados e não plantados com as macrófitas Typha sp. e Phragmites sp., submetidos a diferentes regimes de fluxo e condições hidráulicas de operação. A bioacumulação de cromo em P. subcapitata e sua relação com o biovolume demonstraram uma possível estratégia de detoxificação. P. stratiotes desenvolve uma bioacumulação mais intensa nas raízes, resultados que são confirmados pela peroxidação de lipídios e a indução do estresse oxidativo causado pelo cromo. As enzimas catalase e glutationa redutase, induzidas pelo cobre em P. stratiotes, também apresentaram atividade mais intensa nas raízes. O teor de clorofila, em geral apresentou aumento nos tempos iniciais e decresceu no decorrer do tempo, em concentrações mais elevadas de cromo e cobre. Na análise da emissão de fluorescência da clorofila, o rendimento fotossintético e o índice de vitalidade foram os parâmetros mais sensíveis ao estresse causado por cromo em P. stratiotes. Os resultados obtidos na pesquisa com o DCC e a MSR permitem recomendar seu uso na ecotoxicologia aquática, pois podem gerar modelos preditivos de toxicidade; ampliar a compreensão dos mecanismos de detoxicaçao; reduzir o número de experimentos sem perder a confiabilidade dos dados e reduzir a geração de resíduos. Nos estudos realizados com os wetlands construídos, os parâmetros físico-químicos avaliados revelaram variação sazonal durante o período experimental (verão/2007, invemo/2008 e verão/2008). Typha sp. e Phragmites sp. estão entre as plantas mais comumente utilizadas nos wetlands construídos e sua presença amplia as condições de filtração do sistema, mas a eficiência da espécie na remoção dos nutrientes (amônia e fosfato) depende do regime de fluxo e das condições hidráulicas aplicadas. Os sistemas com fluxo subsuperficial com a superfície livre de água foram os wetlands que desempenharam melhor capacidade na remoção de nutrientes. Uma vez que a poluição dos corpos d\'água tem sido um problema constante na atualidade, estudos como estes oferecem subsídios para propostas futuras de preservação e recuperação ambiental, além de ampliar os conhecimentos sobre as macrófitas e sua aplicação na descontaminação ambiental em corpos d\'água e em sistemas de depuração de águas residuárias. / This research had as main objectives: (1) study the bioaccumulation of chromium metal (40- 50 \'mü\'g/L) in Clorophyceae Pseudokirchneriella subcapitata (Korshikov) Hindak 1990 and of copper (2-10 \'mü\'g/L) and chromium (1-6 mg/L) in the macrophyte Pistia stratiotes L. (2) study the mechanisms of detoxification, defense strategies and tolerance of Pistia stratiotes L. in order to recommend their use in phytoremediation, both through the use of Central Composite Design (DCC) and Response Surface Methodology (RSM), and (3) study the dynamics of nutrient removal in constructed wetlands, planted and unplanted with macrophytes: Typha sp. and Phragmites sp. subjected to different flow regimes and hydraulic conditions of operation. The bioaccumulation of chromium in P. subcapitata and its relation to biovolume shows a possible strategy for detoxification. P. stratiotes develops a more intense bioaccumulation in roots and these results are confirmed by lipid peroxidation and induction of oxidative stress caused by chromium. The enzymes catalase and glutathione reductase induced by copper in P. stratiotes, also showed the strongest activity in the roots. The chlorophyll content in general showed an increase in early and decreased over time, in higher concentrations of chromium and copper. In analyzing the fluorescence emission of chlorophyll, the photosynthetic yield and the index of vitality were the parameters most sensitive to stress caused by chromium in P. stratiotes. The results obtained in research with the DCC and MSR allowed to recommend their use in aquatic ecotoxicology, because they allow: to generate predictive models of toxicity, the simulation of such models expanding the understanding of the mechanisms of detoxification; reduce the number of experiments without losing the reliability of data and reducing waste generation. In studies with constructed wetlands, the physicochemical parameters evaluated showed seasonal variation observed during the experimental period (summer/2007, winter/2008, summer/2008). Typha sp. and Phragmites sp. are among the most commonly used plants in constructed wetlands, and its presence extends the conditions of filtration system, but the efficiency of the species in the removal of nutrients (ammonia and phosphate) depends on the flow regime and hydraulic conditions applied in the system. The systems with subsurface flow with free surface water wetlands that have been played better capacity in removing nutrients. Pollution of water bodies has been a constant problem at the moment, and studies like these provi de input for future proposals for the preservation and environmental restoration, in addition to expanding our knowledge on the macrophytes, and its application in environmental remediation in water bodies and systems purification of wastewater.

Le superoxyde O2●- est une espèce réactive de l’oxygène produite par de nombreux métabolismes chez les espèces vivant en condition aérobie. Ses propriétés radicalaires font de O2●- une espèce très réactive capable d’endommager les macromolécules présentes dans les cellules, conduisant entre autre au stress oxydant et à de possibles troubles neurodégénératifs. Pour se protéger, la Nature utilise des enzymes, appelées superoxydes dismutases (SOD), qui sont chargées de catalyser la dismutation du superoxyde en oxygène et peroxyde d’hydrogène. La dernière qui fut découverte est la NiSOD qui comporte un ion nickel dans son site actif. Contrairement aux autres types de SOD, celle-ci n’est pas présente chez l’homme et ne l’est que chez certaines bactéries comme Chlamydiae. Ainsi, cibler la NiSOD est une méthode prometteuse pour le développement de nouveaux antibiotiques. De même, le développement de nouveaux complexes biomimétiques des SOD peut conduire à de nouveaux agents thérapeutiques contre les maladies liées au stress oxydant. Notre projet consiste à développer de nouveaux modèles actifs de la NiSOD, avec comme stratégie l’utilisation de ligands peptidiques. Les deux principaux objectifs sont (1) de développer des catalyseurs de type SOD, actifs dans l’eau, mais aussi (2) d’acquérir des informations supplémentaires sur le mécanisme mis en jeu lors de la catalyse par la NiSOD, afin de mettre en évidence les intermédiaires clés et les différences majeures entre la NiSOD et les autres SODs présentes chez l’homme. Nos résultats montrent qu’une bonne activité catalytique peut être obtenue même avec une sphère de coordination différente de celle de l’enzyme, et mettent en évidence des facteurs clés pour l’activité. De plus, des études mécanistiques tendent à montrer un mécanisme en sphère interne pour la réduction du superoxyde. / The superoxide radical anion, O2●-, is generated by many life processes. Its radical properties make it a highly reactive species able to damage all macromolecules contributing to the pathogenesis of many diseases including neurodegenerative disorders. In order to protect cells against O2●-, Nature uses superoxide dismutases (SODs) which catalyze the dismutation of O2●- into hydrogen peroxide and oxygen. The last discovered SOD contains a nickel cofactor. Importantly the NiSOD is found in several pathogenic bacteria but not in humans. Therefore targeting the NiSOD is a promising approach to develop antibiotics. Secondly, the development of novel SOD mimics may have potential uses as therapeutic agents in oxidative stress-related diseases. Our project aims at developing innovative active NiSOD mimics, based on the use of peptide-based ligands with two main objectives: (i) to develop efficient SOD like catalysts, active in water, displaying antioxidant properties for potential therapeutic applications and (ii) to contribute to the full understanding of the catalytic mechanism of the NiSOD to highlight the specific key elements that differentiate NiSOD from the human MnSOD for the design of potential antibiotics. Our results show that, even with a coordination sphere different from the one in the enzyme, a good catalytic activity can be obtained. Key elements for the activity are also determined. Moreover, mechanistic studies indicates an inner sphere mechanism for superoxide reduction.

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1983, härtill 5 uppsatser</p> / digitalisering@umu

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

La superoxyde dismutase à Manganèse (SOD Mn ou SOD2) est une enzyme importante dans la défense antioxydante, qui semble jouer un rôle mal défini dans le développement des tumeurs selon l’expression constitutive de son gène. Cependant, les mécanismes de régulation de cette expression constitutive sont mal connus, en particulier dans les cellules tumorales mammaires. Ce travail a reposé sur la mise en évidence préalable d’une protéine, appelée la Damaged DNA Binding 2 (DDB2) protein, se fixant spécifiquement sur la région promotrice du gène SOD2. La DDB2 est connue pour sa participation dans la réparation de l’ADN par excision de nucléotides. Dans un 1er temps, nous avons caractérisé la séquence d’ADN spécifiquement reconnue dans la région proximale du promoteur du gène SOD2, sur laquelle la DDB2 s’y fixe sous la forme d’un monomère, pour réguler négativement la transcription constitutive de la SOD Mn dans les cellules tumorales mammaires non métastatiques de type MCF-7. Par ailleurs, la DDB2 n’est pas impliquée dans le mécanisme d’induction du gène SOD2, lorsque les cellules MCF-7 sont exposées à des substances inductrices. En revanche, nous avons montré que l’absence de la protéine DDB2, associée à celle du facteur de transcription AP-2?, déjà connu comme répresseur du gène SOD2, entraîne une expression constitutive élevée de la SOD Mn dans les cellules tumorales mammaires métastatiques n’exprimant pas le récepteur aux œstrogènes (ER-). De plus, cette expression constitutive élevée est principalement dépendante du facteur de transcription Sp1. Dans un 2ème temps, nous avons évalué la signification biologique de la régulation de l’expression constitutive de la SOD Mn par la DDB2 dans les cellules tumorales mammaires. Nos résultats montrent que la DDB2 active la prolifération des cellules tumorales mammaires ER+, en exerçant sa régulation négative sur l’expression de la SOD Mn. Dans un 3ème temps, nous avons cherché à montrer les conséquences sur la croissance des cellules tumorales mammaires ER-, qui surexpriment naturellement la SOD Mn. Nos résultats révèlent que l’enzyme antioxydante joue un rôle important dans les mécanismes moléculaires impliqués dans le pouvoir invasif des cellules tumorales mammaires ER-. La surexpression de la SOD Mn, associée à un taux faible des enzymes éliminant l’H2O2, entraînent une augmentation du pouvoir invasif déjà élevé des cellules tumorales mammaires ER-, associée une augmentation de l’activité de la métalloprotéinase 9. L’élimination, en présence d’antioxydants, de l’H2O2 libéré par l’activité de la SOD Mn surexprimée, entraîne une inhibition à la fois de la croissance et des capacités invasives des cellules tumorales mammaires ER-. L’ensemble de ce travail contribue à mieux comprendre l’importance de la SOD Mn et du mécanisme de régulation de son gène dans la croissance et l’invasion tumorales. Ainsi ce travail révèle également la SOD Mn et la DDB2 comme de potentiels facteurs prédictifs de la progression tumorale mammaire. Enfin, la découverte de la nouvelle activité biologique de la DDB2 ouvre un vaste champ de perspectives intéressantes en cancérologie mammaire. / Manganese superoxide dismutase (Mn SOD or SOD2) is an important enzyme in the antioxidizing defence, which seems to play an unclear role in the cancer development, according to the constitutive expression of its gene. However, the regulation of this constitutive expression is not totally known, particularly in the breast cancer cells. This work is based on a preliminary revealing that a protein, called Damaged DNA Binding 2 (DDB2), specifically binds the SOD2 gene promoter. The DDB2 is known for its involvement in the nucleotide excision repair. At first step, we characterized the specific DNA sequence recognized in the proximal area of the SOD2 gene promoter, on which a DDB2 monomer binds, in order to regulate negatively the Mn SOD transcription in the MCF-7 non metastatic breast cancer cells. Besides, DDB2 is not involved in the mechanism of SOD2 gene induction, when MCF-7 cells are exposed to induced substances. However, we showed that the lack of the DDB2 protein, associated with the lack of the AP-2a transcription factor, already known as a repressor of the SOD2 gene, lead to a high Mn SOD constitutive expression in the metastatic breast cancer cells. Furthermore, this high constitutive expression is mainly dependent of the Sp1 transcription factor. Secondly, we estimated the biological meaning of the regulation of the Mn SOD constitutive expression by the DDB2 in the breast cancer cells. Our results show that the DDB2 activates the positive ER breast cancer cell proliferation, by exercising its negative regulation on the Mn SOD expression. Thirdly, we tried to show the consequences on the negative ER breast cancer cell growth, which naturally and highly express the Mn SOD. Our results reveal that the antioxidizing enzyme plays an important role in the molecular mechanisms involved in the invasive capacities of the negative ER breast cancer cells. The high Mn SOD expression, associated in a decrease of the H2O2 detoxifying enzymes expression, enhance the negative ER breast cancer cell invasion and an increase of the matrix metallopeptidase-9 activity. The H2O2 elimination, with specific antioxidants, decreases both negative ER breast cancer cell growth and invasive capacities. This whole work contributes to better understand the Mn SOD importance and the mechanism of its gene regulation, in the tumoral growth and invasion. This work also reveals the Mn SOD and DDB2 as potential predictive factors of the breast cancer progress. Finally, the discovery of this new DDB2 biological activity opens a huge field of interesting perspectives in breast cancer research.

Aconitase is an enzyme of the Krebs cycle that catalyzes the isomerization of citrate to isocitrate. In addition to its enzymatic activity, Aco1 has been reported to bind to mitochondrial DNA (mtDNA) and mediate its maintenance in the budding yeast S. cerevisiae. In the absence of Aco1, cells rapidly lose mtDNA and become “petite” mutants. The purpose of this study is to uncover the mechanism behind mtDNA loss due to an aco1 deletion mutation. We found that an aco1 mutation activates the mitochondria-to-nucleus retrograde (RTG) signaling pathway, resulting in increased expression of citrate synthases (CIT) through the activation of two transcription factors Rtg1 and Rtg3. Increased activity of CIT leads to increased iron accumulation in cells, which is known to raise reactive oxygen species (ROS). By deleting RTG1, RTG3, genes encoding citrate synthases, orMRS3 and MRS4, encoding two irontransporters in the mitochondrial inner membranes, mtDNA loss can be prevented in aco1 deletion mutant cells. We further show that the loss of SOD1, encoding the cytoplasmic isoform of superoxide dismutase, but not SOD2, encoding the mitochondrial isoform of superoxide dismutase, prevents mtDNA loss in aco1 mutant cells. Altogether, our data suggest that mtDNA loss in aco1 mutant cells is caused by the activation of the RTG pathway and subsequent iron accumulation and toxicity in the mitochondria.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur.  Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.

Este trabalho teve como objetivo analisar os efeitos fisiológicos do herbicida Paraquat, indutor de estresse oxidativo, em plantas de cana-de-açúcar (Saccharum officinarum L.). A escolha desta espécie como modelo fisiológico deste estudo deveu-se a sua importância econômica para o país, em especial para o Estado de São Paulo. O Paraquat (PQ) é um herbicida de contato que atua especificamente no Fotossistema II bloqueando o fluxo de elétrons e impedindo a redução do NADPH, aceptor final de elétrons. Como consequência, há a formação de radicais instáveis, reativos com a molécula de oxigênio (EROs), os quais causam danos em membranas, proteínas e ácidos nucleicos. A hipótese central foi a de que a atividade de peroxidase de ascorbato é mais sensível ao PQ do que a atividade de superóxido dismutase, nas mesmas condições. O experimento principal consistiu da aplicação, via pulverização foliar, de diferentes doses de PQ (0, 2, 4, 6 e 8 mM de Metil Violágeno, agente ativo), que permaneceu em contato por 24 e 48 horas. Após 18 horas de exposição, observou-se sintomas visuais de toxocidade e uma redução significativa da atividade fotossintética, medida através de parâmetros relacionados com a fluorescência da clorofila a. O nível de peroxidação de lipídeos, indicado pela concentração de TBARS, foi aumentado, indicando danos nas membranas causados pela geração de EROs. Um resultado importante foi o aumento da atividade específica de SOD (Superóxido Dismutase), primeira enzima de defesa contra EROs. Esse aumento foi gradativo com o aumento da dose e do tempo de exposição ao PQ. A isoforma mais evidente foi a SOD Cu/Zn, visualizada por atividade em gel nativo. Esta isoforma está presente preferencialmente em cloroplastos, organela alvo da ação do PQ. É importante ressaltar que mesmo em doses quase letais, a atividade desta enzima manteve-se em níveis semelhantes ao controle. Entretanto, a atividade de APX (Ascorbato Peroxidase), enzima que faz a remoção de radicais H2O2, gerados pela SOD, foi reduzida abruptamnete a partir de 4 mM PQ, apesar de ter sido observado um aumento na dose inicial (2 mM). Este fato sugere que os danos oxidativos em cana-de-açúcar tratadas com PQ podem ser causados pelo excesso de radicais H2O2. Diferente da SOD, a APX é considerada bastante susceptível a certos tipos de estresse ambiental, logo perdendo sua atividade. Analisou-se também a expressão de proteínas por SDS-PAGE e eletroforese 2-D. Em ambos resultados, foram observados diminuição na expressão de algumas proteínas ocasionada pelo tratamento com PQ. Contudo, não foi possível detectar claramente a indução de peptídeos nesse caso, provavelmente devido ao alto grau de degradação das estruturas celulares. O aumento da atividade de SOD em cloroplastos de folhas de cana-de-açúcar tratadas com PQ, deve ser mais estudado, pois esta pode ser uma enzima alvo para programas de melhoramento genético visando obtenção de plantas com resistência ao PQ e a outros herbicidas com modo de ação no sistema fotossintético. / This work had the objective to analyze physiological effects of Paraquat herbicide, an oxidative stress inductor, in sugarcane (Saccharum officinarum L.) plants. The choice to use this species as a physiological model for this study was due its recognized economical importance for our country, in special to the State of Sao Paulo. Paraquat (PQ) is a contact herbicide that acts specifically on Photosystem II by blocking the normal electron flux and the reduction reaction of NADPH, the electron acceptor molecule. As consequence, there is formation of instable radicals and highly reactive oxygen species (ROS), which cause damage to membranes, proteins and nucleic acids. The central hypothesis was that ascorbate peroxidase activity is more sensitive to PQ than the activity of superoxide dismutase in the same conditions. The main experiment consisted on the sprayed of different concentrations of PQ (0, 2, 4, 6 e 8 mM of Metil Violagen) that stayed in leaf contact for 24 and 46 hours. After 18 hours, it was observed visible symptoms of toxicity and the photosynthetic activity decreased, measured by chlorophyll a fluorescence. Also, the level of lipids peroxidation measured by the increase concentration of TBARS formation, indicate damage to cell membrane caused by the generation of ROS. An important result was the increase of SOD specific activity (superoxide dismutase) first enzyme related to defense against ROS. This gradation increase was correspondent to the exhibition period to PQ. The most evident SOD isoform by native PAGE was the SOD Cu/Zn. This isoform is located preferentially in chloroplasts, organelle target to the action of PQ. It is important to remark that even in almost lethal concentrations of PQ the chloroplast SOD activity still remains expressed beilng responsible for the removal of H2O2 generated by SOD, which was reduced abruptly after 4 mM treatment. Although, it has been observed increase with the initial dose (2 mM). This fact suggests that the oxidative damages in sugarcane treated with PQ can be caused by excess of H2O2. Differently of SOD, this enzyme is considered to be fragile to certain kinds of environmental adversities, loosing its activity Also the protein expression was analyzed by SDSPAGE and 2-D electrophoresis. In both analyses, it was observed decrease of some protein expression caused by PQ. However, it was not possible to clearly detect peptides induction in this case, probably due to the elevated level of degradation of cell structures. The increase of chloroplast SOD activity in sugarcane leaves treated with PQ should be detailed investigated since it can be a molecular target for breeding programs with the goal to obtain plants with an acquired resistance to PQ or others herbicides with model action in the photochemical system.

La protéine Damaged DNA-Binding 2 (DDB2) est connue pour son rôle dans la réparation de l'ADN lésé par les UV. Cependant, le laboratoire a montré que cette protéine est surexprimée naturellement dans les cellules tumorales mammaires non métastatiques et active leur prolifération, en favorisant leur entrée en phase de transition G1/S du cycle cellulaire. Il a été montré que cette nouvelle activité biologique de DDB2 dépend de sa capacité à intervenir dans la transcription de gènes cibles, comme celui codant l'enzyme anti-oxydante, la superoxyde dismutase à manganèse (SOD Mn). Sur la base que DDB2 est peu ou pas exprimée dans les cellules tumorales mammaires métastatiques, ce travail a consisté à étudier le rôle de cette protéine dans les capacités invasives de ces cellules. Dans un 1er temps, nous avons montré que les cellules tumorales mammaires hautement métastatiques (MDA-MB231 et SKBR3), lorsqu'elles surexpriment DDB2 après introduction de son gène, ont des capacités migratrices et invasives in vitro, ainsi que des propriétés in vivo à développer des métastases pulmonaires, fortement réduites, en association avec une diminution importante de l'expression de la métalloprotéase matricielle 9 (MMP-9). De même, lors d'une analyse rétrospective sur 92 échantillons cliniques provenant de patientes, une corrélation inverse entre l'expression de DDB2 et le haut grade (SBR>ou =3) des tumeurs mammaires est observée. Dans un 2ème temps, nous avons identifié le mécanisme moléculaire par lequel DDB2 agit négativement sur les capacités invasives des cellules tumorales mammaires. Nous avons montré que DDB2 intervient positivement sur l'expression du gène codant I kappa B alpha (IkBa), en se fixant sur une séquence d'ADN localisée dans la région proximale du promoteur, qui entraîne en conséquence une forte diminution de l'activité du facteur de transcription NF-kB. Ce dernier est connu pour son rôle dans les capacités invasives et migratrices des cellules tumorales mammaires métastatiques, en régulant de nombreux gènes cibles comme celui codant la MMP-9. Nous avons montré, que l?inhibition de l'expression d'IkBa, par ARN interférence restaure en partie les propriétés invasives des cellules tumorales mammaires métastatiques surexprimant DDB2, en association avec une réexpression de MMP-9. Dans un 3ème temps, nous avons également montré dans les cellules tumorales mammaires métastatiques, que l?expression constitutivement élevée de la SOD Mn, en l'absence de DDB2, dépend de l'activité conjointe des facteurs de transcription NF-kB et Sp1, révélant ainsi un autre mécanisme moléculaire impliqué dans les propriétés invasives de ces cellules. L'ensemble de ce travail contribue ainsi à mieux comprendre comment les cellules tumorales mammaires progressent vers un statut invasif et renforce également l'idée que DDB2 présente un intérêt clinique potentiel, comme marqueur prédictif de la progression métastatique des tumeurs mammaires. Enfin, la relation entre la DDB2, NF-kB et la SOD Mn représente une voie intéressante pour le développement de nouvelles thérapies anticancéreuses / The Damaged DNA-Binding 2 protein (DDB2) is known to play a role in repair of UV-induced DNA damages. However, the laboratory has shown that this protein is overexpressed in nonmetastatic breast tumor cells and stimulates their proliferation by favouring their entry in G1/S transition phase of cell cycle. This novel biological activity of DDB2 depends on its ability to modulate transcription of target genes, such as that encoding the manganese superoxide dismutase (MnSOD) antioxidant enzyme. The fact that DDB2 is not expressed in metastatic breast tumor cells led us to focuse this work on the role of DDB2 in the invasive abilities of these cells. In a 1st time, we have shown that highly metastatic breast tumor cells (MDA-MB231 et SKBR3), when they overexpress DDB2 after introduction of its gene, have a strong decrease in their in vitro migratory and invasive abilities, and in their properties to develop in vivo lung metastasis, associated with a highly reduced expression of matrix metalloprotease 9 (MMP-9). In addition, DDB2 expression was analyzed in a cohort of 92 breast samples from patients. An inverse correlation is observed between DDB2 level and the high-grade (SBR>=3) breast tumors. In a 2nd time, we identified the molecular mechanism by which DDB2 controls negatively the invasive abilities of breast tumor cells. We have shown that DDB2 plays a positive role in the expression of gene encoding I kappa B alpha gene (IkB?), though its binding to a specific DNA sequence localized in the proximal promoter, and which promotes a strong decrease in the NF-kB activity. This transcription factor is well known to play a role in migratory and invasive abilities of metastatic breast tumor cells by regulating many target genes, such as that encoding MMP-9. We have shown that inhibition by RNA interference of I?B? expression restores in part the invasive properties of DDB2-overexpressing metastatic breast tumor cells, associated with an induction of MMP-9 gene expression. In a 3rd time, we have also shown in metastatic breast tumor cells, that the high basal MnSOD expression, when DDB2 is lacking, depends on the related activity of the NF-kB and Sp1 transcription factors, considering that this other molecular mechanism is involved in invasive properties of these cells. Taken together, this work contributes to a better understanding how breast tumor cells progress toward an invasive phenotype and underlines also the idea that DDB2 has a clinical relevance as a good potential marker for predicting breast tumor progression toward metastasis. Finally, the relationship between DDB2, NF-kB and MnSOD may be considered as an interesting pathway for development of new anticancer therapies

Submitted by Cristiane Chim (cristiane.chim@ucpel.edu.br) on 2016-09-13T16:35:46Z No. of bitstreams: 1 carolina - Copia.pdf: 1129007 bytes, checksum: deaef0e96e4837a47c36de1f6dc1e109 (MD5) / Made available in DSpace on 2016-09-13T16:35:46Z (GMT). No. of bitstreams: 1 carolina - Copia.pdf: 1129007 bytes, checksum: deaef0e96e4837a47c36de1f6dc1e109 (MD5) Previous issue date: 2010-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES# / #2075167498588264571# / #600 / Objectives: The aim of this study was evaluate uric acid level in a population–based sample of young adults (18 to 24 years old). People with bipolar and depression disorder were compared with healthy psychiatric subjects without a history of mood episodes. Methods: Case-control study nested in a population-based sample. The diagnosis was confirmed with the Structured Clinical Interview for DSM-IV. The uric acid was assessed by commercial kits Results: The sample consisted of 216 subjects, 49 with diagnosis for bipolar disorder, 76 with depression and 91 population control. The uric acid levels were similar (control = 4.08 ± 1.27 mg/dL, depression = 3.78 ± 1.00 mg/dL e bipolar disorder =3.86 ± 1.33 mg/dL, p= 0,251). For maniac/hippomaniac and depressive episodes, were observed a tendency to increase in acid uric levels in maniac/hippomaniac episodes (4.43 ± 1.74 mg/dL) and a decrease in depressive episodes (3.70 ± 0.99 mg/dL) when compared to control subjects (4.08 ± 1.27 mg/dL)(p= 0.106). Conclusion: Uric acid levels is not correlated with diagnosis group, but the uric acid level have a tendency to presented increased in manic and/ or mixed episodes and decreased in depression episode, these result observed that levels of uric acid in mood disorders are altered according to the manifestation of symptoms. / Objetivo: O objetivo deste estudo é avaliar o nível de acido úrico em pacientes com transtornos de humor oriundo de uma amostra de base populacional com jovens adultos (18 a 24 anos), para este fim jovens com transtorno de humor bipolar e depressão foram comparados com jovens sem histórico de transtorno de humor. Métodos: Trata-se de um estudo de caso-controle aninhado a um estudo de base populacional, o diagnostico foi realizado através da Entrevista Clínica Estruturada para o DSM-IV (SCID) e os níveis de acido úrico séricos foram dosados utilizando-se kits comerciais. Resultados: A amostra foi constituída de 216 jovens, 49 com diagnostico de transtorno bipolar, 76 com depressão e 91 controles sem historia psiquiátrica prévia, os níveis de acido úrico entre os três grupos foram similares (controle = 4.08 ± 1.27 mg/dL, depressão = 3.78 ± 1.00 mg/dL e transtorno bipolar =3.86 ± 1.33 mg/dL, p= 0,251). Quando avaliados os níveis de ácido úrico entre os episódios mania/hipomania e depressivo, observa-se uma tendência no aumento do ácido úrico no episódio maníaco/misto (4,43 ± 1,74 mg/dL) e uma diminuição no episódio depressivo (3,70 ± 0,99 mg/dL) quando comparados aos jovens do grupo controle (4,08 ± 1,27 mg/dL)(p= 0,106). Conclusão: Os níveis de ácido úrico não foram significativos entre os grupos diagnósticos, porém os níveis de acido úrico apresentaram uma tendência para o aumento nos episódios maníacos e/ou mistos e uma diminuição no episódio depressivo, com estes resultados podemos observar que os níveis de acido úrico nos transtornos de humor podem estar alterados de acordo com a manifestação dos sintomas