The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under non-inflammatory conditions NOS generates Nitric Oxide. However, in an inflammatory environment, such as the oxidative tumor microenvironment, NOS’s cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2). NOS bound to BH2 is said to be uncoupled and produces superoxide O2-and peroxynitrite (ONOO-). Previous work in our and other’s labs have shown that increased production of ROS/RNS leads to the activation of pro-inflammatory/proliferative molecules such as NFκB, Stat3, β-Catenin and Akt. NOS can be re-coupled by supplementing cells and animals with BH4 or its precursor Sepiapterin (SP). Herein we show that recoupling NOS with SP in HCT116, Caco-2 and HT29 cells, decreased tumor cell proliferation, increased β-Catenin degradation and decreased Akt activity. We also see increased tumor cell death measured by in vitro clonogenic assay, as well as decreased metabolic uptake in Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) induced colorectal cancer in vivo measured by [18F]-fluorodeoxyglucose ([18F]-FDG) positron emitted topography (PET) imaging. We believe by recoupling NOS both in vivo and in vitro we are modulating Wnt signaling via Akt and GSK-3β. Lastly, we conducted studies to determine a mechanistic explanation of how tumor cells maintain a decreased BH4:BH2 ratio.

Selon la FAO, 2 milliards de personnes souffrent de la « faim cachée » qui correspond aux carences en micronutriments incluant la carence en vitamine A, principale cause de cécité dans le monde. Par ailleurs, une alimentation excédentaire est source de stress oxydant pour le corps, facteur de risque de nombreuses maladies non transmissibles comme le diabète et les maladies cardiovasculaires. Contenus dans les produits végétaux, certains caroténoïdes représentent une source indirecte de vitamine A et pourraient participer aux effets bénéfiques de la consommation de fruits et légumes via des propriétés antioxydantes. Dans cette thèse, le comportement in vitro des caroténoïdes et rétinoïdes pendant la digestion a été étudié, depuis leur libération de la matrice alimentaire jusqu'à leurs interactions avec les cellules intestinales. Mes recherches s'articulent autour de la micellisation : processus clé de l'absorption des caroténoïdes et rétinoïdes. Dans un premier temps, la stabilité et la bioaccessibilité en digestion in vitro de ces composés purs ont été comparées à celles de leurs homologues issus d'un jus de carotte, d'épinards crus et cuits et d'une farine fortifiée. Dans un deuxième temps, un nouveau modèle d'oxydation de nano-émulsions intestinales à base de sels biliaires (SB) a été développé. Les relations entre structure des micelles mixtes, réactivité à l'oxydation et protection par des antioxydants ont été étudiées grâce notamment au suivi de la dégradation des acides gras. Enfin, des expériences préliminaires ont été menées sur la réactivité enzymatique de cellules coliques (Caco-2) à divers oxydants et aux micelles mixtes. Pendant la digestion in vitro, le β-carotène et palmitate de rétinyl purs étaient particulièrement instables contrairement à la lutéine et à l'acétate de rétinyl. De plus, la matrice alimentaire protège ces composés et favorise leur bioaccessibilité surtout lorsqu'elle est broyée ou cuite. En effet, les prétraitements ont un impact conséquent sur la libération des caroténoïdes et doivent être considérés comme des moyens sérieux pour optimiser les apports. Les nano-émulsions intestinales mises au point se composaient de micelles mixtes sphériques ou cylindriques, ces dernières étant les moins résistantes à l'oxydation. En effet, la distribution des molécules de SB doit être différente selon la morphologie de la micelle, impactant ainsi leur réactivité. Dans nos conditions expérimentales, l'AAPH fut le seul oxydant efficace. L'α-tocophérol et la lutéine ont significativement ralenti la dégradation des acides gras contrairement au β-carotène. Leur place au sein de la micelle (cœur ou bordure) pourrait expliquer ces observations. Enfin, aucun des oxydants testés n'a significativement modifié l'activité catalase des cellules Caco-2. En revanche, la mise en contact avec les micelles à base de SB ont significativement diminué l'activité des 3 enzymes suivies. L'effet était positivement corrélé à la concentration en SB dont la conjugaison fut un élément déterminant. Finalement, le β-carotène, la lutéine, et l'acétate de rétinyl présents dans ces micelles ont en partie rétabli l'activité de la catalase contrairement au rétinol, palmitate de rétinyl et à l'α-tocophérol. / According to the FAO, two billions of people suffer from the « hidden hunger » which corresponds to micronutrient deficiencies and includes the vitamin A deficiency, principal cause of blindness throughout the world. Besides, an excessive food consumption entails an oxidative stress for the organism which increases the risk to develop non communicable diseases such as diabetes and cardiovascular diseases. Present in numerous fruits and vegetables, some carotenoids are an indirect source of vitamin A and could also contribute to the health benefits of the consumption of fruits and vegetables via antioxidant properties. In this thesis, in vitro behaviour of carotenoids and retinoids during in vitro digestion (IVD) has been studied, from their release from dietary matrices to their interactions with intestinal cells. My research is structured around micellisation: key process of carotenoid and retinoid absorption. First, stability and bioaccessibility during IVD of these pure compounds have been compared with those of their homologues from carrot juice, raw and cooked spinach and fortified flour. Second, a new model of oxidation of intestinal bile salt (BS)-based nanoemulsions has been developed. Relations between mixed micelles structure, reactivity to oxidation and protection by antioxidants have been studied using fatty acid degradation monitoring. Finally, preliminary experiment were carried out on enzymatic reactivity of colon cells (Caco-2) to various oxidants and mixed micelles. During IVD, pure β-carotene and retinyl palmitate were particularly unstable conversely with lutein and retinyl acetate. Moreover, the dietary matrix protected these compounds and favoured their bioaccessibility especially when it was grounded or cooked. Indeed, pre-treatment have a significant impact on carotenoid release and should be considered as serious means to optimize intake. Developed intestinal nanoemulsions constituted of spherical or cylindrical micelles, the latter being less resistant to oxidation. Distribution of BS molecules is likely to differ according to the micelle morphology, thereby influencing their reactivity. In our experimental conditions, AAPH only was efficient as oxidant. Lutein and α-tocopherol significantly slowed fatty acid degradation, conversely with β-carotene. Their location within the micelle (core or border) could explain these observations. Besides, none of the tested oxidant significantly modified the catalase activity of Caco-2 cells. However, the contact with BS-based nanoemulsions significantly decreased the activity of the three tested enzymes. This effect was positively correlated with BS concentration whose conjugation revealed determining. Finally, β-carotene, lutein and retinyl acetate in micelles partly re-established catalase activity, conversely with retinol, retinyl palmitate and α-tocopherol. To conclude, IVD is an easy tool which allows independent comparisons of several parameters on micronutrient behaviour during digestion. Furthermore, the confrontation of different matrices may help to develop relevant strategies against vitamin A deficiency. Proposed nanoemulsions displayed various physical and chemical characteristics, thereby influencing their reactivity to oxidation. The antioxidant capacity of lutein and α-tocopherol could be observed in this new model. Lastly, the preliminary results on Caco-2 cells revealed some interesting interactions between micellised BS and antioxidant enzymes. Numerous questions arose that could not be addressed in this work but open perspectives for the future.

Les maladies cardiovasculaires (MCV) sont en augmentation au niveau mondial et sont désormais un problème de santé publique coûteux. La suralimentation et le manque d'activité physique sont des facteurs clé dans le développement pathologique. Ces dernières années, les études sur la pathogenèse des MCV ont mis en évidence de nombreux facteurs contribuant au développement de ces pathologies complexes, notamment le surpoids, l'obésité centrale, le stress oxydant, l'inflammation vasculaire et systémique, la résistance à l'insuline ou encore la dysfonction endothéliale. La prévention de ces désordres est donc la cible des stratégies pharmaceutiques et diététiques et les polyphénols ont d'ores et déjà démontré des effets bénéfiques et préventifs. Parmi les 8000 composés phénoliques décrits à ce jour, le resvératrol a émergé en tant que candidat robuste dans la prévention des pathologies liées à la nutrition. L'objectif de ce travail a été d'étudier les potentialités d'action d'un extrait de sarment de vigne (Vineatrol®) et de vins rouges riches en resvératrol et ses oligomères, sur un modèle animal d'athérosclérose nutritionnellement induite. La première partie de cette étude a consisté à mettre au point un régime alimentaire déclenchant au mieux l'athérosclérose précoce chez le hamster Syrien doré. A partir de ce modèle, un effet préventif du Vineatrol® a été mis en évidence : le Vineatrol® induit une diminution des dépôts lipidiques aortiques mais améliore également le statut oxydatif et inflammatoire des animaux. Dans une troisième partie, des vins rouges enrichis en Vineatrol® ont démontré des effets préventifs sur certains facteurs de risque de la pathologie athéromateuse et sur les désordres liés à la consommation d'un régime gras. Des mécanismes d'action possibles, expliquant les effets bénéfiques de ces vins, ont été envisagés et recherchés. Ces mécanismes pourraient impliquer une modulation de la voie du NF-κB et/ou de SIRT1. Le degré d'importance de ces différentes voies devra être confirmé. / Cardiovascular diseases (CVD) are increasing globally and are now an expensive public health problem. Overnutrition and lack of physical activity are key factors in the disease development. In recent years, studies on the pathogenesis of CVD showed many factors contributing to the development of these complex diseases including overweight, centralobesity, oxidative stress, vascular and systemic inflammation, insulin ressitance or endothelial dysfunction. Prevention of these disorders is the focus of pharmaceutical and dietarystrategies and polyphenols have already demonstrated beneficial and preventive effects. Among the 8000 phenolic compounds described to date, resveratrol has emerged as a strong candidate for the prevention of nutrition-related diseases.The objective of this work was to study the potential action of vine shoot extract (Vineatrol®) and red wines rich in resveratrol and its oligomers, in an animal model of nutritionallyinduced atherosclerosis.The first part of this study was to develop a diet triggering the best early atherosclerosis in theSyrian golden hamster. From this model, a preventive effect of Vineatrol® was highlighted: Vineatrol® induces adecrease in aortic lipid deposition but also improves the oxidative and inflammatory status of the animals. In the third part , Vineatrol®-enriched red wines showed preventive effects on risk factors foratherosclerotic disease and disorders related to the consumption of a high-fat diet. Possible mechanisms of action, explaining the beneficial effects of these wines have been consideredand sough. These mechanisms could involve modulation of the NF-κB and/or SIRT1pathways. The degree of importance of these different pathways will have to be confirmed.

Les flavonoïdes sont des composés naturels appartenant à la famille des polyphénols, qui sont étudiés depuis de nombreuses années pour leur caractère antioxydant ainsi que leurs propriétés thérapeutique. La mise en évidence, pour certaines d’entre eux, d’une activité vasorelaxante variant en fonction de leur structure, en fait un modèle d’étude de choix pour la prévention des maladies cardiovasculaires. Le 3’,4’-dihydroxyflavonol (DiOHF) est le dérivé le plus décrit pour son fort potentiel en terme de propriétés cardioprotectrices et antioxydantes et est considéré comme un composé d’intérêt dans le traitement anti-ischémique, bien que sa faible solubilité dans l’eau soit un facteur limitant pour son utilisation. Dans ce contexte, notre premier objectif a été de synthétiser une trentaine de dérivés du DiOHF, fonctionnalisés par un groupement hydrophile, tout en préservant ses propriétés antioxydantes et pour certaines des structures obtenues, ses propriétés vasorelaxantes. Pour cela, nous avons développé une méthode de synthèse rapide utilisant les irradiations micro-onde, qui nous a permis d’optimiser les rendements, de réduire les temps de réaction et qui ne conduit à aucune formation de sous-produits. Notre second objectif a été de déterminer l’activité antioxydante de ces dérivés flavonoïques par des tests mettant en œuvre des réactions de compétition avec des radicaux libres. Faisant suite à ce premier screening, des études biologiques chez le rat ont été réalisées sur les composés montrant les meilleures performances. A partir d’essais in vitro et in vivo nous avons démontré, l’innocuité de ces dérivés flavonols, et le fort impact des modifications de structure sur l’activité vasorelaxante et antioxydante, ainsi que sur la récupération fonctionnelle et métabolique consécutive à une ischémie/reperfusion myocardique. Un autre champ d’action de ces flavonols hydrosolubles pourrait être d’explorer leur utilisation dans le traitement du diabète de type 2. Pour cela nous avons greffé deux motifs antidiabétiques, correspondant à des molécules actives déjà utilisées dans le traitement de l’insulinorésistance, sur les dérivés flavonols les plus cardioprotecteurs et/ou vasorelaxants de notre série, ainsi que sur des dérivés de l’acide cinnamique. Ces composés ont été testés pour leur activité antioxydante, laissant supposer un potentiel significativement supérieur aux analogues antidiabétiques non fonctionnalisés, en termes de protection antioxydante et fonctionnelle. / Flavonoids are natural polyphenolic compounds, and epidemiological and pharmacological studies have shown that flavonoids intake is associated with many beneficial effects. An important example is the 3’,4’-dihydroxyflavonol (DiOHF) which showed interesting therapeutic properties in vitro so it could be considered as an important lead compound to the treatment of cardiovascular diseases. However is poor water solubility prevents it use as a drug. Starting from this model we have synthesized a series of new flavones functionalized by a hydrophilic group in order to increase water solubility. To obtain these derivatives we have set up a fast, simple and clean method without formation of by-products, using micro-waves irradiation to optimize yield and to decrease reaction time. Our second purpose was to investigate the antioxidant activity of the synthesized compounds by undergoing several assays. The most promising compounds were tested in biological studies, in vitro and in vivo. The results showed a significant enhancement of the post-ischemic mechanical function recovery in vivo and in vitro. Another purpose was to designed new flavonols linked to selected antidiabetic moieties already used in the treatment of insulinoresistance. It was shown that these new compounds retain the antioxidant properties of the parent flavonols, and can be of interest in the development of new diabete 2 treatments.

This thesis is concerned with the synthesis and evaluation of various tellurium-based chain-breaking antioxidants. The purpose is to find novel regenerable compounds with improved radical-trapping capacity. In the first part of this work, we explore the possibilities to incorporate tellurium into tocopherols and aromatic amines. Overall, tocopherols carrying alkyltelluro groups are better radical-trapping agents than the corresponding sulfur- and selenium analogues. Among them, 7-octyltelluro δ-tocopherol showed a ca. 17-fold higher reactivity than recorded for α-tocopherol and much better regenerability. Even longer inhibition times were recorded for the corresponding bis(tocopheryl) tellurides. In the aromatic amine series, diphenyl amines carrying alkyltelluro groups were shown to function as efficient radical-quenchers capable of inhibiting peroxidation for 460 min in the presence of N-acetylcysteine. Thiol-consumption experiments suggested that the long inhibition times are due to efficient quenching of in-situ formed alkoxyl radicals in a solvent cage. In the second part of the thesis, we study how the antioxidant properties are affected by variations in the electron density at tellurium and the number of alkyltelluro substituents in the molecule. Evaluation of a series of aryltelluro phenols carrying electron donating and electron withdrawing groups in the para-position of the aryl moiety suggested that a high electron density at the heteroatom prolonged the inhibition time. Among alkyltelluro phenols, alkyltelluro resorcinols and bis(alkyltelluro) phenols, phenols carrying alkyltelluro groups in both ortho positions were superior when it comes to radical-trapping activity and regenerability.

La doxorubicine (DOX) est un médicament très efficace utilisé en chimiothérapie pour traiter de nombreuses tumeurs. Cependant, son utilisation clinique est limitée par des effets secondaires cardiotoxiques sévères causés par une augmentation de production de radicaux libres de l'oxygène (RLO) et de l'apoptose. De surcroit, une chimiothérapie pourrait être indiquée alors qu'une ischémie cardiaque est déjà installée. Dans cette thèse, nous avons donc examiné au niveau du cœur isolé de lapin et des cardiomyocytes en culture; d'une part, la cardiotoxicité de la DOX dans les conditions de l'ischémie/reperfusion (IR) myocardique et d'autre part, la cardioprotection assurée dans ces conditions par un antioxydant naturel puissant, le safran (SAF).D'abord nous avons procédé à une extraction méthanol-Eau des stigmates du safran cultivé au Liban. Une analyse par HPLC des principaux constituants a été réalisée afin de s'assurer de la qualité des extraits, suivie par des tests (xanthine/xanthine oxydase et électrolyse) comme sources de RLO pour rechercher le pouvoir antioxydant de ces extraits.La toxicité du SAF a été évaluée sur des cœurs isolés de lapin selon la technique de Langendorff et la dose thérapeutique optimale établie est de 10 μg/mL. Ensuite, la toxicité aigüe de la DOX a été testée sur des cœurs isolés sains traités ou non par la DOX et le SAF en comparaison avec des cœurs soumis à l'électrolyse ou à une période d'ischémie globale. La toxicité chronique de la DOX a été évaluée chez des lapins traités pendant 21 jours avec des extraits de SAF (par voie orale) et la DOX (par voie ip). Les cœurs ont été par suite isolés et soumis à une ischémie globale. D'après les études cardiodynamiques, anatomopathologiques et biochimiques; il apparaît que les extraits de safran protègent le cœur de la toxicité aigüe et chronique de la doxorubicine et assurent une protection optimale lors de la reperfusion. De plus, le SAF a inhibé l'apoptose induite par IR et DOX en activant la voie de survie AKT/mTOR, en augmentant le niveau de Troponine T et en inhibant l'expression de p-P38 tel que montré par les analyses de Western blot. Le safran contribuerai donc à améliorer nettement le retour des fonctions myocardiques vers la normale suite à l'IR et au traitement par la DOX.Les effets de la DOX et du SAF ont aussi été étudiés dans les cardiomyocytes H9c2 soumis à une IR simulée. Les effets des différents traitements ont été évalués par les tests MTT, LDH, Western blot, TMRM et caspases. Nos données prouvent que DOX et IR diminuent significativement la viabilité cellulaire ; inhibent la voie RISK ; réduisent les protéines contractiles ; activent les caspases 3 et provoquent la perte du potentiel de membrane mitochondrial. Ces effets ont été remarquablement inhibés par le traitement avec le SAF à la reperfusion. Les résultats suggèrent que le SAF a un effet protecteur contre la DOX et l'IR par un mécanisme impliquant la réduction de l'apoptose cardiaque, des RLO et du stress oxydatif. La cardioprotection du SAF est médiée par l'activation des voies de survie AKT/P70S6K et ERK ; augmentation de l'expression des protéines contractiles ; inhibition de la perméabilité des pores de transition mitochondrial et diminution de l'activité des caspases 3. En outre, nous avons montré que l'effet protecteur du SAF serait attribué à la crocine, son métabolite actif principal. Pris ensemble, nos résultats indiquent que la DOX exacerbe les lésions myocardiques de l'IR. Le traitement par le SAF à la reperfusion pourrait exercer des effets cardioprotecteurs contre la toxicité induite par la DOX via son activité antioxydante. Ainsi, nos travaux sur le safran se révèlent fort intéressants dans le développement d'une stratégie thérapeutique antioxydante potentielle afin de limiter la toxicité cardiaque de la DOX, ouvrant la voie à de futurs essais cliniques. / Doxorubicin (DOX) is a highly active chemotherapeutic drug used to treat many tumors. However, its clinical use is limited by severe cardiotoxic side effects. Previous studies have shown that DOX-Induced cardiotoxicity is mainly mediated through increased oxidative stress and apoptosis. In addition, DOX could be indicated in case of pre-Established myocardial ischemia. In this thesis we investigated, in isolated rabbit heart and cultured cardiomyocytes, the cardiotoxic effects of DOX in conditions of ischemia/reperfusion (IR). We also studied whether saffron (SAF), a natural compound that showed potent antioxidant activity, could exert cardioprotection against DOX and IR toxicity.First, the extraction of saffron stigmas grown in Lebanon was performed using methanol-Water mixture. HPLC analysis of the main components (safranal and crocines) was used to ensure the quality of the extracts; followed by tests (xanthine/xanthine oxidase and electrolysis) as sources of free radicals to determine the antioxidant activity of the sample.The toxicity of SAF was evaluated on isolated rabbit hearts according to Langendorff technique and the optimal therapeutic dose established is 10 μg/mL. Then, the acute toxicity of DOX was tested on isolated hearts treated with or without DOX and SAF compared with hearts subjected to electrolysis or a period of global ischemia (I 30 min/R 40 min). The chronic toxicity of DOX was evaluated in rabbits treated for 21 days with SAF extracts (orally) and DOX (ip). The hearts were afterward isolated and subjected to global ischemia (I 30 min/R 30 min). According to cardiodynamic, pathological and biochemical studies, it appears that saffron extracts protect the heart from the acute and chronic toxicity of doxorubicin, and ensure optimal protection during reperfusion. Furthermore, SAF inhibited IR and DOX-Induced apoptosis by activating the AKT/mTOR survival pathway, increasing Troponine T and inhibiting p-P38 expression as shown by Western blot analysis. Saffron could therefore significantly improve myocardial function to normal state following IR and DOX treatment.The effects of DOX and SAF were also investigated in H9c2 cardiomyocytes subjected to simulated IR injury. We evaluated the effects of the treatments by MTT assay, LDH release measurements and Western blot analysis. Apoptosis was assessed by Hoechst 33258 staining, TMRM probes and caspase activity. The results proved that DOX exacerbates cardiac cell injury when an underlying IR heart condition is present. Our data showed that DOX and IR significantly decreased cardiac cells viability; inhibited RISK cardioprotective pathway; reduced contractile proteins (α-Actinine, Troponine C and MLC); increased caspase 3 expression and induced loss of mitochondrial membrane potential. These effects were remarkably inhibited by treatment with SAF (10 μg/mL) at reperfusion. Our data suggested that SAF has a protective effect against DOX and IR induced cardiac apoptosis through a mechanism involving scavenging free radicals and reducing oxidative stress. SAF cardioprotection is mediated by activating survival pathways including AKT/P70S6K and ERK; increasing the expression of contractile proteins; inhibiting of mitochondrial permeability transition pore and decreasing caspase 3 activity. In addition, studies showed that protective activities of SAF belong to crocin, its major active metabolite.Taken together, our findings indicate that DOX exacerbates IR myocardial injury. The SAF treatment at reperfusion could exert cardioprotective effects against DOX induced toxicity by its antioxidant activity. Thereby, saffron offers a potential novel antioxidant therapeutic strategy to counteract doxorubicin cardiotoxicity, paving the way for future clinical trials.

A Doença Renal Crônica (DRC) é um importante problema de saúde pública ao redor do mundo e a proteinúria é um marcador de progressão da doença bem estabelecido. A própolis, uma resina natural produzida por abelhas a partir de resíduos e seivas de diferentes partes de plantas, possui propriedades anti-inflamatória, imunomoduladora, anti-câncer, anti-oxidante, bem como demonstrou ter efeito antiproteinúrico em modelo experimental de DRC. O objetivo deste estudo foi avaliar o efeito do extrato de Própolis verde brasileiro na proteinúria e ritmo de filtração glomerular estimada (RFG) em pacientes com DRC. Este foi um estudo clínico, randomizado, duplo-cego, controlado por placebo que reuniu pacientes com DRC de etiologias diabética e não-diabética, com idade entre 18 e 90 anos, com RFG estimado entre 25-70 ml/min/1.73 m2 e proteinúria (excreção urinária de proteína > 300mg/dia) ou microalbuminúria ou macroalbuminúria (taxa urinária de albumina-creatinina > 30mg/g Cr ou > 300mg/g Cr, respectivamente). Nós pesquisamos 148 pacientes e selecionamos randomicamente 32 para receberam, por 12 meses, extrato de própolis verde brasileiro na dose de 500mg/dia (n=18) ou placebo (n=14). No final do tratamento, a proteinúria foi significantemente menor no grupo Própolis do que no grupo Placebo--695 mg/24 h (IC de 95%, 483 a 999) x 1403 mg/24 h (IC de 95%, 1031 a 1909); P=0.004--independente de variações no RFG e pressão arterial, os quais não foram diferentes entre os grupos. O subgrupo de pacientes com DRC de etiologia diabética que recebeu Própolis apresentou redução significativa da albuminúria ao final do estudo (de 981 mg/g uCr [IC de 95%, 223 a 1739] para 476 mg/g uCr [IC de 95%, -282 a 1235]; p= 0,031), ao passo que o subgrupo de diabéticos que recebeu Placebo apresentou elevação da albuminúria (de 1261 mg/g uCr [IC de 95%, 569 a 1953] para 1451 mg/g uCr [IC de 95%, 758 a 2143]; p = 0,999). O Monocyte Chemoattractant Protein-1 (MCP-1) urinário também foi menor no grupo Própolis do que no grupo Placebo ao final do tratamento -- 58 pg/mg Cr [IC de 95%, 36 a 95] x 98 pg/mg Cr [IC de 95%, 62 a 155]; P=0.038. O extrato de própolis verde brasileiro foi seguro e bem tolerado, bem como reduziu significativamente a proteinúria em pacientes com DRC de etiologia diabética e não-diabética / Chronic kidney disease (CKD) is a public health problem worldwide, and proteinuria is a well-established marker of disease progression. Propolis, a natural resin produced by bees from residues and sap of different parts of plants, has anti-inflammatory, immunomodulatory, anti-cancer, anti-oxidant properties, as well as having been shown to have an antiproteinuric effect in experimental CKD. The aim of this study was to evaluate the effect that Brazilian green propolis extract has on proteinuria and on the estimated glomerular filtration rate (eGFR). This was a randomized, double-blind, placebo-controlled study including patients with CKD caused by diabetes or non-diabetic etiology, 18-90 years of age, with an eGFR of 25-70 ml/min per 1.73 m2 and proteinuria (urinary protein excretion > 300 mg/day) or micro- or macro-albuminuria (urinary albumin-to-creatinine ratio > 30 mg/g or > 300 mg/g, respectively). We screened 148 patients and selected 32, randomly assigning them to receive 12 months of Brazilian green propolis extract at a dose of 500 mg/day (n=18) or 12 months of a placebo (n=14). At the end of treatment, proteinuria was significantly lower in the propolis group than in the placebo group--695 mg/24 h (95% CI, 483 to 999) vs. 1403 mg/24 h (95% CI, 1031 to 1909); P=0.004--independent of variations in eGFR and blood pressure, which did not differ between the groups during follow-up. The subgroup of patients with CKD of diabetic ethiology who received Propolis presented a significant reduction of albuminuria at the end of the study (from 981 mg / g uCr [95% CI, 223 to 1739] to 476 mg / g uCr [95% CI, (P = 0.031), whereas the subgroup of diabetics receiving Placebo presented elevation of albuminuria (from 1261 mg / g uCr [95% CI, 569 to 1953] to 1451 mg / g uCr [CI 95%, 758 to 2143], p = 0.999). Urinary Monocyte Chemoattractant Protein-1 (MCP-1) was also significantly lower in the propolis group than in the placebo group -- 58 pg/mg creatinine (95% CI, 36 to 95) vs. 98 pg/mg creatinine (95% CI, 62 to 155); P=0.038. Brazilian green propolis extract was found to be safe and well tolerated, as well as to reduce proteinuria significantly in patients with diabetic and non-diabetic CKD

The problem of cadmium (Cd) toxicity aggravates every year. However, the amount of plant species presenting some degree of tolerance to this metal is low. Therefore, it is of utmost importance to understand the Cd tolerance mechanisms. Two tomato cultivars exhibiting distinct levels of tolerance to Cd were investigated by exposing them to 35 μm de CdCl2 for eight days. The samples (roots, stems, and leaves) were collected at 0, 2, 4, 6 and 8 days. The root length and surface area were obtained using a WinRhizo PRO 2009c image analyzer. The ICP OES method was used for the quantification of metals in the different tissues. The translocation index (TrI), metal uptake and Cd distribution nutrient absorbed (NA), and nutrient efficiency (NUE) were calculated. The plant material used for stress indicators and proteomics analysis were collected in liquid nitrogen. Lipid peroxidation and H2O2 contents were obtained by spectrometry. For the proteomics data the gel-free method was used. The stress indicators showed the variation in both cultivars, evidencing the stress caused by the Cd. Chlorosis and necrotic spots were observed in plants exposed to Cd less significant way in the first days of exposure and more evident in the less tolerant cultivar (TTO) in the last days. The growth reduction can be observed in both cultivars, more evident in TTO. Regarding the Cd distribution, the two cultivars showed similar behavior, with the highest percentage of Cd in the roots. The Cd absorption could interfere with both cultivars in the accumulation absorption and efficiency in the use of most nutrients assessed. In general, the Cd interference was more evident at TTO than at YOSH. Highest percentages of TrI were also observed in TTO cultivar. Identification and evaluation of the differentially accumulated protein (DAP) in the two cultivars roots it is possible to observe that in the presence of Cd the cultivar TTO generates a more significant alteration in the amplitude of the accumulation and the number of proteins. These results suggest that the cultivar, which presents a higher degree of tolerance, has some form to make the Cd unavailable or a mechanism that makes the Cd transportation to the leaves more difficult. / O problema da toxicidade do cádmio (Cd) agrava-se a cada ano. No entanto, a quantidade de espécies de plantas que apresentam algum grau de tolerância a este metal é baixa. Portanto, é de suma importância entender os mecanismos de tolerância do Cd. Com esse intuito duas cultivares de tomate (TTO e YOSH) exibindo níveis distintos de tolerância ao Cd foram investigadas, expondo-as a 35 μm de CdCl2 por oito dias. As amostras (raízes, caules e folhas) foram coletadas aos 0, 2, 4, 6 e 8 dias. O comprimento da raiz e área de superfície foram obtidos usando um analisador de imagem WinRhizo PRO 2009c. O método ICP OES foi utilizado para a quantificação de metais nos diferentes tecidos. O índice de translocação (Trl), a absorção de metal e a distribuição de Cd dos nutrientes absorvidos (NA) e eficiência de nutrientes (NUE) foram calculados. O material vegetal usado para indicadores de estresse e análise proteômica foi coletado em nitrogênio líquido. A peroxidação lipídica e o teor de H2O2 foram obtidos por espectrometria. Para os dados proteômicos, foi utilizado o método livre de gel. Os indicadores de estresse mostraram a variação nas duas cultivares, evidenciando o estresse causado pelo Cd. A clorose e manchas necróticas foram observadas em plantas expostas ao Cd de maneira menos significativa nos primeiros dias de exposição e mais evidentes no cultivar menos tolerante (TTO) nos últimos dias. A redução do crescimento pode ser observada em ambas as cultivares, mais evidente no TTO. Quanto à distribuição de Cd, as duas cultivares apresentaram comportamento semelhante, com o maior percentual de Cd nas raízes. A absorção de Cd pode interferir nas duas cultivares na absorção, acúmulo e eficiência no uso da maioria dos nutrientes avaliados. Em geral, a interferência do Cd foi mais evidente no TTO do que no YOSH. As maiores porcentagens de TrI também foram observadas na cultivar TTO. Identificação e avaliação da proteína diferencialmente acumulada (DAP) nas raízes das duas cultivares é possível observar que na presença de Cd a cultivar TTO gera uma alteração mais significativa na amplitude do acúmulo e no número de proteínas. Estes resultados sugerem que a cultivar, que apresenta um maior grau de tolerância, tem alguma forma de tornar o Cd indisponível ou um mecanismo que dificulte o transporte de Cd para as folhas.

A doença de Alzheimer (DA) é caracterizada pela perda progressiva de memória episódica relacionada à agregação do peptídeo ?-amiloide (A?) e à fosforilação anormal da proteína tau, levando à perda da função colinérgica. É bem conhecido que o comprometimento desta função devido à neurotoxicidade do peptídeo A? contribui significativamente para o declínio cognitivo associado à DA. Os inibidores da enzima acetilcolinesterase (AChE) compõem a principal classe de drogas usadas no tratamento da DA. No entanto, há uma grande necessidade de síntese de novas moléculas, uma vez que os fármacos já em uso pelos pacientes apresentam alta toxicidade hepática, além de vários efeitos colaterais devido à ação nos tecidos periféricos. A hipótese do presente projeto se baseia na atividade de indução de neurodiferenciação e neuritogênese de novos compostos inibidores de AChE, os quais são híbridos sintéticos de donepezila-tacrina, além de atuarem como neuroprotetores em culturas de células neuronais, sob condições experimentais de um estímulo neurotóxico e de estresse oxidativo induzidos pelo peptídeo A?(1-42) e peróxido de hidrogênio (H2O2). Assim, o objetivo deste trabalho foi avaliar a capacidade de neurodiferenciação e neuritogênese, assim como o potencial neuroprotetor de duas moléculas híbridas de donepezila-tacrina na linhagem SH-SY5Y, modelo de diferenciação neuronal. Foram realizados ensaios de citotoxicidade e hepatotoxicidade em células tratadas e coletadas em diferentes tempos (24, 48, 72 e 120 h) nas linhagens celulares SH-SY5Y e HepG2, respectivamente. Ensaios de diferenciação neuronal (análise morfológica), expressão proteica, cinética ciclo celular, proliferação celular, alterações mitocondriais e de estresse oxidativo foram realizados em linhagem neuronal SH-SY5Y. Ainda, foram realizados ensaios de viabilidade e morte celular para avaliar a capacidade de neuroproteção dos novos compostos híbridos na linhagem SH-SY5Y. Estes não se mostraram citotóxicos e não alteraram a viabilidade celular em todas as concentrações avaliadas: TA8Amino 0,0035 a 0,112µM e TAHB3 0,088 a 2,84µM; nos ensaios de hepatotoxicidade, o composto TAHB3 reduziu a viabilidade celular na maior concentração (2,84 µM) e no tempo de 24 h. Ambos os compostos híbridos foram capazes de induzir diferenciação neuronal e neuritogênese, cerca de 55% das células para TA8Amino e 43% para TAHB3. Apenas o composto TA8Amino induziu um aumento de aproximadamente 75% na expressão do marcador ?-III-Tubulina comprovando a formação de neurônios maduros. Nenhum dos tratamentos realizados causaram alterações significativas na distribuição das células nas fases do ciclo celular e no ensaio de proliferação celular. O composto TA8Amino foi capaz de induzir a produção de ROS intracelular (48,27%) e mitocondrial (67,60%) nas células diferenciadas. Ambos os compostos híbridos aumentaram a expressão de SOD1, indicando atuação na atividade antioxidante das células. Entretanto, os compostos não alteraram o potencial de membrana mitocondrial e massa mitocondrial. Esses dados demonstram que o estresse oxidativo induzido pelo TA8Amino não gerou danos por disfunção mitocondrial nas células diferenciadas. Na avaliação de alterações da expressão proteica de PTEN(Ser380/Thr382/383), AKT(Ser473) e COX2 ambos os compostos híbridos promoveram a indução de PTEN e AKT. TA8Amino e TAHB3 induziram um aumento na expressão de COX2 demonstrando umapossível atuação na via de sinalização PI3K/AKT/COX2, compatível com a diferenciação neuronal. Nos ensaios de viabilidade celular, os compostos TA8amino e TAHB3 apresentaram efeito de neuroproteção frente ao dano neurotóxico induzido pelo peptídeo A?(1-42), promovendo uma proteção de 91,93% para TA8Amino e 54,68% para TAHB3, embora o mesmo efeito não tenha sido observado nas células tratadas com o peróxido de hidrogênio. Entretanto, a indução de morte por apoptose pelas moléculas híbridas nas células tratadas com o peptídeo A?(1-42) foi apenas levemente alterada pelos compostos, havendo uma redução de 5,10 e 4,85%, respectivamente, para TA8Amino e TAHB3. Em geral, os resultados do presente trabalho demonstraram que os compostos híbridos donepezilatacrina (TA8amino e TAHB3), inibidores da AChE, apresentam vantagens como potenciais fármacos, visto não serem citotóxicos nas concentrações de inibição da enzima AChE, além de serem capazes de induzir diferenciação neuronal, neuritogênese e neuroproteção, diferentemente dos fármacos donepezila e tacrina, testados isoladamente. / Alzheimer\'s disease (AD) is characterized by a progressive loss of episodic memory related to aggregation of ?-amyloid peptide (A?) and to abnormal phosphorylation of tau protein, leading to loss of cholinergic function. It is well known that the impairment of this function due to A? peptide neurotoxicity contributes significantly to the cognitive decline associated with AD. Acetylcholinesterase (AChE) inhibitors are the main class of drugs used to treat AD. However, there is a great need for synthesis of new molecules, since current drug treatment presents high hepatic toxicity, besides several side effects due to the action in the peripheral tissues. The hypothesis of this project is based on the activity of AChE inhibitor compounds (synthetic hybrids of donepezil-tacrine) in terms of inducing neurodifferentiation and neuritogenesis, as well as neuroprotective effects in neuronal cells under experimental conditions of a neurotoxic stimulus and oxidative stress induced by the peptide A? (1-42) and hydrogen peroxide (H2O2). Thus, the objective of this work was to evaluate the neurodifferentiation and neuritogenesis abilities, as well as the neuroprotective potential of two hybrid molecules of donepezil-tacrine in the SH-SY5Y model. Cytotoxicity and hepatotoxicity assays were performed on treated cells that were collected at different times (24, 48, 72 and 120 h) in SH-SY5Y and HepG2 cell lines, respectively. Neuronal differentiation assays (morphological analysis), protein expression, cell cycle kinetics, cell proliferation, mitochondrial changes and oxidative stress were performed on SH-SY5Y cell line. Further, viability and cell death assays were performed to evaluate the neuroprotection ability of the novel hybrid compounds in SH-SY5Y cells. The compounds did not present cytotoxic effects and did not alter cell viability at the following concentrations: TA8Amino 0.0035 to 0.112?M and TAHB3 0.088 to 2.84?M; in the hepatotoxicity assays, TAHB3 reduced cell viability only at the highest concentration (2.84 ?M) and at the time of 24 h. Both hybrid compounds were able to induce neuronal differentiation and neuritogenesis, around 55% after TA8Amino and 43% after TAHB3 treatment. Only TA8Amino induced approximately 75% increase in ?-III-Tubulin expression, compatible with the formation of mature neurons. None of the treatments performed caused significant changes in cell cycle kinetics. The synthetic hybrid TA8Amino induced the production of intracellular (48.27%) and mitochondrial ROS (67.60%). Both hybrid compounds increased SOD1 expression, which could be related to the increase of oxidative damage. However, both TA8amino and TAHB3 did not promote changes in mitochondrial membrane and mitochondrial mass potential. This data demonstrate that oxidative stress induced by TA8Amino did not generate damage due to mitochondrial dysfunction in differentiated cells. In the evaluation of changes in PTEN(Ser380/ Thr382/383), AKT(Ser473) and COX2 protein expression, both hybrid compounds were able to induce PTEN and AKT expression. TA8Amino and TAHB3 promoted an increase in COX2expression indicating a possible role in the PI3K/AKT/COX2 pathway, which is related to neuronal differentiation. In the cell viability assays, TA8amino and TAHB3 showed a neuroprotective effect against the neurotoxic damage induced by the A? peptide (1-42), promoting a protection of 91.93% for TA8Amino and 54.68% for TAHB3, although the same effect could not be observed in cells treated with hydrogen peroxide. However, the induction of apoptosis by the hybrid molecules in cells treated with the A?(1-42) peptide was only slightly altered by the compounds, with a reduction of 5.10 and 4.85%, respectively, for TA8Amino and TAHB3, respectively. In general, the results of the present work demonstrate that donepezil-tacrine hybrid compounds (TA8amino e TAHB3), AChE inhibitors, have advantages as potential drugs, since they are not cytotoxic at concentration levels that inhibit AChE enzyme, besides being able to induce neuronal differentiation, neuritogenesis and neuroprotection, unlike the drugs donepezil and tacrine, tested alone.

A hidrocefalia é uma doença que afeta não só a dinâmica do líquido cefalorraquidiano, mas também outras estruturas do sistema nervoso central. Apesar do tratamento com derivações liquóricas ser eficiente na redução da ventriculomegalia, muitos danos neurológicos não são revertidos com a cirurgia. Vários estudos demonstram que o estresse oxidativo está envolvido na gênese das lesões na hidrocefalia. Objetiva-se avaliar os efeitos da quercetina nas lesões secundárias à hidrocefalia experimental em ratos. Foram utilizados ratos machos com sete dias de vida, que recebem a injeção de caulim a 15% na cisterna magna para indução da hidrocefalia. Foram divididos em grupo controle (C), hidrocefálico não tratado (HN), hidrocefálico derivado (HD), hidrocefálico tratado com água destilada (HA), hidrocefálico tratado com água destilada e derivação (HDA), hidrocefálico tratado com quercetina peritoenal (HQp), hidrocefálico tratado com quercetina peritoneal e derivação (HDQp), hidrocefálico tratado com quercetina por gavagem (HQg), hidrocefalcio tratado com quercetina por gavagem e derivação (HDQg). A quercetina apresentou significativamente uma melhora nos marcadores imunoistoquimicos, principalmente caspase-3 e GFAP. Não houve alteração significativa na avaliação clinica/comportamental. A utilização da quercetina isolada não altera o volume e tamanho ventricular e a realização de derivação ventriculo-subcutânea em ratos recém-nascidos com hidrocefalia apresenta uma alta morbi-mortalidade / Hydrocephalus is a disease that affects not only the dynamics of the cerebrospinal fluid, but also other structures of the central nervous system. Although CSF derivation is effective in reducing ventriculomegaly, many neurological damage is not reversed with surgery. Several studies have shown that oxidative stress is involved in the genesis of hydrocephalus lesions. The objective of this study was to evaluate the neuroprotective response of quercetin in hydrocephalus. Male rats with seven days of life were used, which received the 15% kaolin injection in the cisterna magna for induction of hydrocephalus. They were divided into control group (C), untreated hydrocephalic (HN), derived hydrocephalic (HD), hydrocephalic treated with distilled water (HA), hydrocephalic treated with distilled water and shunt (HDA), hydrocephalic treated with quercetin peritoenal (HQp), hydrocephalic treated with quercetin peritoneal and derivation (HDQp), hydrocephalic treated with quercetin by gavage (HQg), hydrocephalus treated with quercetin by gavage and bypass (HDQg). Quercetin significantly improved the immunohistochemical markers, mainly caspase-3 and GFAP. There was no significant change in clinical / behavioral assessment. The use of isolated quercetin does not alter the volume and ventricular size and the realization of ventriculo-subcutaneous shunt in newborn rats with hydrocephalus presents a high morbi-mortality