O consumo de álcool tem sido considerado um problema de saúde pública. Entretanto, também existem evidências de eventuais benefícios do consumo leve a moderado de algumas bebidas alcoólicas, no que se refere a doenças crônicas não transmissíveis. As doenças periodontais, sendo a sexta doença crônica mais prevalente, também têm sido associadas ao consumo de álcool com resultados controversos. Esta tese avaliou, através de três artigos a relação entre consumo de álcool e doenças periodontais. O primeiro artigo de divulgação, tem por objetivo alertar a profissão a respeito do estado atual do conhecimento em relação a associação entre álcool e doenças periodontais. O segundo artigo é um estudo experimental em modelo animal que avaliou o efeito da dependência química álcool sobre desfechos periodontais. Os resultados não mostraram diferenças estatisticamente significativas na perda óssea alveolar e secreção de TNF-α. O terceiro artigo, também em modelo animal, abordou o consumo do vinho tinto e valeu-se de controles total, álcool a 12%, suco de uva e resveratrol. A análise de ocorrência de periodontite espontânea demonstrou que o vinho tinto tem potencial protetor para a perda óssea alveolar e secreção de TNF- α . Os resultados encontrados nessa tese são instigantes e estão em linha com a literatura que demonstra uma relação do tipo curva J entre álcool e doenças periodontais, na qual até um determinado nível, o fator comporta-se como protetor. Assim, essa relação deve ser considerada na abordagem clínica, sem incentivo ao consumo, mas compreendendo até onde este é tolerável e/ou benéfico no que se refere a doenças periodontais. / Alcohol consumption has been considered a public health problem. Otherwise, there is evidence of eventual benefits of a light to moderate consume of some alcoholic beverage, in relation to non-transmissible chronic disease. The periodontal diseases, being the sixth most prevalent chronic disease, also have been associated to alcohol consumption with controversial results. This thesis evaluated, through three articles the relationship between alcohol consumption and periodontal diseases. The first article has the objective of alerting the professionals about the actual knowledge about the relationship between alcohol and periodontal diseases. The second article is an experimental study in animal model which evaluated the alcohol dependence over periodontal outcomes. The results did not show statistically significant differences in alveolar bone loss and TNF- α secretion. The third article, also in animal model, studied the red wine consumption and used total controls, 12% alcohol, grape juice and resveratrol. The analysis of the occurrence of spontaneous periodontitis demonstrated that red wine has a protector potential for alveolar bone loss and TNF- α secretion. The results found in this thesis are curious and they are in line with the literature that demonstrates a relationship like J-curve between alcohol and periodontal diseases, until some level, the factor behave as protector. So, this relation must be considered in clinical approaches, without encouraging the consumption, but understanding how much is tolerable and/or benefit according to periodontal diseases.

A obesidade é uma doença multifatorial fortemente associada com a síndrome metabólica e com as doenças hepáticas. A distribuição da gordura corporal é um fator relevante, e especificamente o tecido adiposo visceral parece ser o elo entre o aumento de peso, a síndrome metabólica e o acúmulo de gordura hepática. Os adipócitos estão envolvidos na regulação do balanço energético e podem ser modulados por hormônios, citocinas e nutrientes, entre eles o resveratrol. São dois os objetivos principais deste trabalho: (1) identificar os níveis basais de expressão do mRNA de SIRT1, adiponectina, FOXO1, PPARg e PPARb/d em diferentes tecidos adiposos (retroperitoneal, subcutâneo e visceral) de indivíduos obesos grau III com diferentes níveis de esteatose hepática; e (2) identificar a ação do resveratrol sobre a expressão do mRNA dos genes citados em adipócitos isolados do tecido adiposo visceral de indivíduos obesos grau III. Os tecidos adiposos foram obtidos durante cirurgia bariátrica. O RNA total foi extraído usando o reagente TRIzol e o mRNA foi quantificado por PCR em tempo real. Para a análise da expressão basal dos genes, dividimos os pacientes em dois grupos: aqueles com esteatose simples ou moderada e aqueles com esteatose severa associada ou não à fibrose e inflamação. Comparando os dois grupos estudados, os pacientes com esteatose severa apresentaram uma menor expressão do mRNA de SIRT1 (p=0,006), apenas no tecido adiposo visceral. Os níveis de adiponectina, FOXO1, PPARg1-3 e PPARb/d não diferiram estatisticamente entre os dois grupos de pacientes nos três depósitos de gordura corporal. O valor de HOMA-IR foi diferente estatisticamente entre os grupos estudados, sendo maior no grupo de pacientes com diagnóstico de esteatose severa associada ou não a fibrose e necroinflamação (p=0,006). Neste mesmo grupo de pacientes, a expressão de SIRT1 e o valor de HOMA-IR correlacionaram-se positivamente no tecido adiposo visceral (r=0,607; p=0,048). Quanto à modulação por resveratrol, houve aumento significativo nos níveis do mRNA de SIRT1 (p=0,021), adiponectina (p=0,025) e FOXO1 (p=0,001) nos adipócitos isolados do tecido adiposo visceral de obesos grau III. A expressão do mRNA de PPARg1-3 foi modulada negativamente (p=0,003) nas células estudadas. Considerando a expressão do PPARb/d, não houve modulação por resveratrol na concentração, tempo e modelo celular estudados. Analisando a literatura, verificamos que valores diminuídos de SIRT1 já foram associados à progressão da esteatose, em modelos experimentais. Neste trabalho, observamos menor transcrição de SIRT1 no tecido adiposo visceral de obesos com esteatose severa, o que poderia prejudicar a biogênese mitocondrial e a oxidação de ácidos graxos, contribuindo com o aumento de ácidos graxos livres na circulação portal, favorecendo o acúmulo hepático de lipídeos nestes pacientes. Desta forma, sugerimos que o aumento da expressão de SIRT1 no tecido adiposo visceral pode ter efeito protetor contra a evolução da esteatose em obesos grau III. Também constatamos que resveratrol ativa a expressão do mRNA de SIRT1, adiponectina e FOXO1 e diminui a expressão de PPARg1-3. Os resultados indicam que resveratrol pode modular o metabolismo dos adipócitos de obesos grau III, possivelmente favorecendo o metabolismo das células estudadas. / Obesity is a complex disease, strongly associated with metabolic syndrome and hepatic disease. The distribution of body fat is important, and specifically visceral adipose tissue seems to be the link between weight gain, metabolic syndrome and hepatic fat accumulation. Adipocytes are involved in the regulation of energy balance and can be modulated by hormones, cytokines and nutrients as resveratrol. The objectives of this study were: (1) to determine the expression pattern of SIRT1, adiponectin, FOXO1, PPARg1-3 and PPARb/d mRNA in different adipose tissues (retroperitoneal, subcutaneous and visceral) of morbidly obese with different levels of hepatic steatosis; and (2) analyze whether resveratrol could modulate the mRNA expression of the cited genes in isolated visceral adipocytes of morbidly obese. The adipose tissue was obtained by bariatric surgery. Total RNAs were extracted using TRIzol reagent and genes reverse transcripts were determined by quantitative polymerase chain reaction. For analysis of basal genes expression, we divided patients in two groups: those with slight or moderate steatosis and other comprising individuals with severe steatosis associated or not with necroinflammation and fibrosis. When comparing the two groups of patients, we found that the amount of SIRT1 mRNA in the visceral adipose tissue of morbidly obese with severe steatosis was decreased (p=0.006). The levels of adiponectin, FOXO1, PPARg1-3 and PPARb/d did not differ statistically between the two groups of patients in the fat depots studied. We found that HOMA-IR value was significantly higher in severe steatosis patients group (p=0.006). Our results also show that the mRNA expression of SIRT1 and HOMA-IR was positively correlated in the visceral adipose tissue of patients with severe steatosis (r=0.654; p=0.048). Regarding the modulation by resveratrol, there was significant increase in SIRT1 (p=0.021), adiponectina (p=0.025) and FOXO1 (p=0.001) mRNA in isolated visceral adipocytes. The mRNA expression of PPARg1-3 was negatively modulated by resveratrol (p=0.003). Considering PPARb/d, there was not a significantly modulation by resveratrol, in the studied model. Reduced quantities of SIRT1 mRNA have been described in severe steatosis using experimental models. We found lower transcription of SIRT1 expression in the visceral adipose tissue of obese patients with severe steatosis, which may impair mitochondrial biogenesis and fatty acids oxidation, contributing to the increase of free fatty acids in portal circulation, impairing liver function. Thus, we suggest that the increased SIRT1 mRNA expression may have a protective role on steatosis in visceral adipose tissue of morbidly obese individuals. Also, we observed that resveratrol activated the expression of SIRT1, FOXO1 and adiponectin and decreases PPARg1-3, which indicate that resveratrol can control adipocytes metabolism, possibly favoring the metabolism of studied cells.

Os astrócitos são células conhecidas por sua capacidade dinâmica e versatilidade, participando não somente da manutenção da homeostase cerebral em condições fisiológicas, mas também respondendo em condições patológicas, como por exemplo, na encefalopatia hepática (EH). Esta patologia neurológica está associada principalmente à hiperamonemia, decorrente de uma falência hepática aguda ou crônica. A toxicidade da amônia no sistema nervoso central (SNC) é mediada por uma série de alterações celulares e metabólicas, principalmente nas células astrogliais. Nesse sentido, a busca por moléculas com potencial terapêutico no SNC é extremamente relevante. Trabalhos do nosso grupo mostraram que o resveratrol e o ácido lipoico, duas moléculas conhecidas pelos seus efeitos antioxidantes, modulam importantes funções gliais, relacionadas principalmente ao metabolismo glutamatérgico, à defesa antioxidante e à resposta inflamatória. Neste sentido, esta tese teve como objetivo avaliar os efeitos do resveratrol e do ácido lipoico em células astrogliais e neuronais expostas à amônia, assim como seus possíveis mecanismos protetores. Primeiramente, nós observamos que o resveratrol e o ácido lipoico foram capazes de prevenir as alterações induzidas pela amônia em células astrogliais C6, sobre parâmetros do metabolismo glutamatérgico, dentre os quais podemos destacar a captação de glutamato, a atividade da enzima glutamina sintetase (GS) e o conteúdo de glutationa (GSH). Além disso, o ácido lipoico também exerceu um efeito antiinflamatório nestas células, prevenindo a liberação de citocinas pró-inflamatórias (TNFα, IL- 1β, IL-6, IL-18) e da proteína S100B, através da diminuição da ativação do fator de transcrição NFκB. Nós também verificamos que a maioria dos efeitos protetores do resveratrol e do ácido lipoico foram dependentes da heme oxigenase 1 (HO1), uma enzima associada com a defesa celular em situações de estresse. Por fim, foram avaliados os efeitos do resveratrol e do ácido lipoico sobre células neuronais expostas à amônia. Novamente, ambos apresentaram um efeito benéfico, prevenindo tanto o aumento da produção de espécies reativas de oxigênio (ERO) quanto a diminuição do conteúdo de GSH induzidos pela amônia. Nas células neuronais, a proteína HO1 também participou dos efeitos protetores do resveratrol e do ácido lipoico. Em conjunto, esses resultados nos mostram que o resveratrol e o ácido lipoico são capazes de modular positivamente o funcionamento tanto de células astrogliais quanto de células neuronais em situações de hiperamonemia, compartilhando também alguns mecanismos de ação, como a indução da HO1. Este estudo in vitro sugere que o resveratrol e o ácido lipoico são potenciais agentes terapêuticos para doenças do SNC, as quais envolvam produção de espécies reativas e resposta inflamatória, como a EH. / Astrocytes are dynamic and versatile cells which participate in the maintenance of brain homeostasis in physiological conditions and also in response to pathological conditions, such as hepatic encephalopathy. This neurological disease is mainly associated with hyperamonnemia, resulting from acute or chronic liver failure. Ammonia toxicity in the central nervous system (CNS) is mediated by several cellular and metabolic alterations, primarily in astroglial cells. In this sense, the search for molecules with therapeutical potential for CNS becomes highly relevant. Our group has shown that resveratrol and lipoic acid, two molecules known for their antioxidant activities, are able to modulate important glial functions mainly related to glutamate metabolism, antioxidant defense and inflammatory response. In this sense, this study aimed to evaluate the effects of resveratrol and lipoic acid in astroglial and neuronal cells exposed to ammonia, as well as their possible protective mechanisms. Firstly, we observed that resveratrol and lipoic acid were able to prevent ammonia-induced alterations in C6 astroglial cells functioning, such as glutamate uptake, glutamine synthetase (GS) activity and intracellular GSH content. Moreover, lipoic acid also exerted an anti-inflammatory effect in C6 astroglial cells, preventing the ammonia-stimulated release of pro-inflammatory cytokines (TNF, IL-1β, IL-6, IL-18) and S100B protein, by decreasing the activation of the transcription factor NFκB. We also verified that the most protective effects of resveratrol and lipoic acid involved the heme oxygenase 1 (HO1), an enzyme associated with protection against stressful conditions. Finally, we evaluated the effects of resveratrol and lipoic acid on neuronal cells exposed to ammonia. Again, both showed beneficial roles, preventing the increase of reactive oxygen species (ROS) and decrease of GSH content induced by ammonia. In neuronal cells, HO1 also mediated the protective effects of resveratrol and lipoic acid. Taken together, these results show that resveratrol and lipoic acid are able to positively modulate the functioning of both astroglial and neuronal cells in hyperamonemmia conditions, sharing some mechanisms of action, such as HO1. This study in vitro suggests that resveratrol and lipoic acid may represent potential therapeutic agents for CNS, during oxidative and inflammatory damages, as the induced by ammonia.

A espécie redox ativa resveratrol (3,5,4’-trihidroxi-trans-estilbeno), uma fitoalexina encontrada nas uvas e no vinho tinto, apresenta importantes efeitos biológicos como atividade antioxidante, anti-inflamatória, antitumoral e cardioprotetora. O sistema nervoso central (SNC) também é alvo do resveratrol que por sua vez pode atravessar a barreira hematoencefálica e exercer efeito neuroprotetor, modulando importantes funções neurogliais. Fisiologicamente os astrócitos controlam importantes funções cerebrais, principalmente, relacionadas ao metabolismo glutamatérgico, à plasticidade sináptica e neuroproteção; e em situações neuropatológicas, relacionadas ao estresse oxidativo. Assim, neste estudo nós investigamos o efeito do resveratrol sobre importantes parâmetros gliais em células C6 frente a dois modelos de insulto oxidativo: (I) 1 mM de H2O2/0,5 h e (II) 0,1 mM de H2O2/6 h. Avaliamos, nessas condições, o perfil das principais defesas antioxidantes enzimáticas celulares, a genotoxicidade celular e possíveis vias de sinalização que possam contribuir para o melhor entendimento do mecanismo de ação do resveratrol no SNC. Nós demonstramos que o resveratrol modula a captação de glutamato; a atividade da enzima glutamina sintetase; os níveis intra e extracelulares de glutationa; a secreção da proteína S100B; as enzimas SOD, CAT e GPx; a peroxidação lipídica; a frequência de micronúcleos; as enzimas heme oxigenase 1 e iNOS e o fator de transcrição NFκB, de maneira dependente das condições oxidativas do meio e do ambiente redox celular. Dessa forma, nossos resultados mostram que o resveratrol apresenta comportamentos anti e pró-oxidante e que sua ação neuroprotetora pode estar relacionada a modulação da atividade glial e da heme oxigenase 1. Enfim, o resveratrol pode representar um potencial agente terapêutico em patologias que envolvam déficits no sistema glutamatérgico associado ao estresse oxidativo. / The redox active compound, resveratrol (3,5,4 '-trihydroxy-trans-stilbene), a phytoalexin, found in grapes and red wine, has a wide range of biological effects, such as, antioxidant, antiinflammatory, anticancer and cardioprotective. The central nervous system (CNS) is also the target of resveratrol which is able to trespass the blood-brain barrier and exerts neuroprotective effects by modulating important glial parameters. Physiologically, astrocytes control important brain functions, mainly related to glutamatergic metabolism, synaptic plasticity and neuroprotection; and in neurophatology events related to oxidative stress. In this study, we investigated the effect of resveratrol on important parameters in C6 astoglial cells against oxidative insult in two models: (I) 1 mM H2O2/0.5 h and (II) 0.1 mM H2O2/6 h. We evaluated under these conditions, the activity of the major cellular enzymatic antioxidant defenses, the possible genotoxicity and cell signaling pathways that may contribute to better understanding the mechanism of resveratrol in the CNS. Resveratrol modulates glutamate uptake; glutamine synthetase activity; intracellular and extracellular levels of glutathione; S100B secretion; the enzymes SOD, CAT and GPx; lipid peroxidation; the frequency of micronuclei; the enzymes heme oxygenase 1 and iNOS and the transcription factor NFκB depending upon the oxidant conditions of the milieu and the cellular redox environment. Thus, our results show that resveratrol presents anti and pro-oxidant effects and their neuroprotective action may be related to modulation of glial function and heme oxygenase 1. Finally, resveratrol can represent a potential therapeutic agent against pathologies associated to glutamatergic system and oxidative stress.

Orientador: Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T20:53:59Z (GMT). No. of bitstreams: 1 Andre_DianaMajolli_M.pdf: 1732006 bytes, checksum: b29eeed204f693e810f408917cd2340a (MD5) Previous issue date: 2014 / Resumo: Obesidade e asma são doenças de grande importância na saúde pública. Nos últimos 30 anos, a incidência de obesidade cresceu de forma dramática em todo o mundo enquanto que a prevalência de asma triplicou. Um número crescente de estudos clínicos e experimentais sugere a existência de uma associação entre a obesidade e asma. A obesidade é um fator de risco para asma, alterando esta doença para um fenótipo de difícil controle. Acredita-se que o estresse oxidativo associado à obesidade desempenha importante papel no desenvolvimento da asma. Estudo clínico mostrou uma correlação inversa entre a ingestão de polifenóis e incidência de asma. O resveratrol é um fitocomposto polifenólico capaz de reduzir a hiperreatividade das vias aéreas e o infiltrado eosinofílico, supostamente por ação antioxidante por meio da ativação da sirtuína 1 (SIRT 1) via fator de transcrição forkhead O (FOXO). O resveratrol é também capaz de ativar AMPK (proteína quinase ativada por AMP) e de inibir a fosfodiesterase do tipo 4 (PDE4), elevando os níveis de AMPc. Em vista disso, no presente estudo propomos estudar o efeito do tratamento do resveratrol na exarcebação da asma em camundongos obesos por indução de dieta hiperlipídica. Foram utilizamos camundongos C57BL/6 alimentados com dieta padrão (grupo controle) ou dieta hiperlipídica por 12 semanas para induzir obesidade (grupo obeso). Na 10ª. semana, os animais foram sensibilizados e desafiados com ovalbumina (OVA). A coleta do LBA e pulmão para análise histológicas foi realizada 48 h após o desafio com OVA. Camundongos obesos e controles foram tratados com resveratrol (100 mg/kg, gavage, 2 últimas semanas de dieta). Nossos resultados mostraram que o ganho ponderal, peso da gordura epididimal, níveis de glicose em jejum e taxa de desaparecimento de glicose (Kitt) foram significativamente maiores nos animais obesos em relação àqueles alimentados com dieta comercial padrão. O tratamento com resveratrol nos animais obesos promoveu reduções significativas do peso da gordura epididimal, dos níveis de glicose e do Kitt, não interferindo no peso corpóreo desses animais. O infiltrado pulmonar de células inflamatórias e eosinófilos dos camundongos obesos em resposta ao desafio com OVA foi 70% maior (P<0,05) do que nos animais controles. Contrariamente, no LBA dos animais obesos notamos número marcantemente menor de células inflamatórias totais e eosinófilos em relação aos controles. O tratamento com resveratrol preveniu de maneira significativa a elevação do infiltrado celular no parênquima pulmonar bem como a redução do número de células no LBA. Níveis elevados de TNF-? no LBA e de iNOS no tecido pulmonar foram encontrados no grupo obeso, sendo prevenidos pelo resveratrol. No tecido pulmonar de camundongos obesos, encontramos níveis menores de SOD e níveis maiores de espécies reativas de oxigênio (EROs), sendo ambos prevenidos pelo resveratrol. Os níveis de GSH total não foram modificados em nenhum grupo estudado. O resveratrol reduziu a expressão p47 phox e elevou a expressão da SOD-1 no pulmão de animais obesos. Em relação à AMPK, notamos que o resveratrol aumenta significativamente a expressão dessa enzima no pulmão dos obesos. Além disso, notamos redução da expressão da SIRT 1 a qual foi prevenida pelo resveratrol. Quanto à PDE-4, o resveratrol diminui a expressão da mesma tanto nos pulmões dos animais controle quanto obesos. No conjunto, sugerimos que o resveratrol ativa a AMPK em camundongos obesos, prevenindo a resistência à insulina e a exacerbação da resposta inflamatória pulmonar e produção de EROs. Ao corrigir a resistência à insulina nos camundongos obesos há aceleração do processo de resolução da inflamação pulmonar alérgica, possivelmente pela diminuição do estresse oxidativo em resposta à ativação de SIRT 1 / Abstract: Obesity and asthma are diseases of great importance in public health. Over the past 30 years, the incidence of obesity has grown dramatically around the world while the prevalence of asthma tripled. An increasing number of clinical and experimental studies suggest the existence of an association between obesity and asthma. Obesity is a risk factor for asthma, altering this disease to a phenotype difficult to control. It is believed that oxidative stress associated with obesity plays an important role in the development of asthma. Clinical study showed an inverse correlation between the intake of polyphenols and incidence of asthma. Resveratrol is a polyphenolic compound able to reduce airway hyperresponsiveness and eosinophilic infiltrate, supposedly by antioxidant action through activation of sirtuin 1 (SIRT 1) via forkhead transcription factor (FOXO). Resveratrol is also able to activate AMPK (AMP-activated protein kinase) and inhibit phosphodiesterase type 4 (PDE4) by increasing cAMP levels. In view of this, in this study we propose to study the effect of resveratrol in asthma exacerbation observed in sensitized obese-induced high fat diet mice. The study was conducted with C57BL / 6 mice fed with standard diet (control group) or high fat diet for 12 weeks to induce obesity (obese group). In 10th week, the animals were sensitized and challenged with ovalbumin (OVA). The collection of BALF and lung tissue for histological analysis was performed 48 h after OVA challenge. Obese and control mice were treated with resveratrol (100 mg / kg, oral gavage during the last 2 weeks of diet). Our results showed that the weight gain, weight of epididymal fat, fasting glucose levels and insulin tolerance test (Kitt) were significantly higher in obese animals compared to those fed a standard commercial diet. Treatment with resveratrol in obese animals reduced significantly the weight of epididymal fat, glucose levels and Kitt, no changes in body weight of these animals was observed. The lung infiltration of inflammatory cells and eosinophils of obese mice in response to challenge with OVA was 70% greater (P <0.05) than in control animals. In contrast, in BALF of obese animals noticed markedly lower number of total inflammatory cells and eosinophils compared to controls. The treatment with resveratrol significantly prevented the elevation of cellular infiltrate in pulmonary parenchyma as well as reducing the number of cells in BALF. Elevated levels of TNF-? in the BALF and iNOS in lung tissue observed in the obese group is prevented by resveratrol. In lung tissue of obese mice, we found lower levels of SOD and increased levels of reactive oxygen species (ROS), both prevented by resveratrol. The total GSH levels were not modified in any group studied. Resveratrol reduced the p47 phox expression and increased expression of SOD-1 in lung of obese animals. Regarding AMPK, we note that resveratrol significantly increases the expression of this enzyme in the lungs of obese mice. Furthermore, we noticed a reduction in expression of SIRT 1 which was prevented by resveratrol. As for the PDE 4, resveratrol decrease the expression in lungs of control and obese animals. Overall, we suggest that resveratrol activates AMPK in obese mice, preventing insulin resistance and exacerbation of pulmonary inflammatory response and ROS production. When correcting insulin resistance in obese mice accelerates the resolution of allergic airway inflammation, possibly by reducing oxidative stress in response to activation of SIRT 1 / Mestrado / Farmacologia / Mestra em Farmacologia

Ce travail porte sur la mise au point et l’étude d’un nouveau système de culture in vitro permettant une production efficace de dérivés de t-resvératrol. Pour cela, des lignées de racines transformées de Vitis vinifera L. ont été établies, stabilisées et criblées. Le faible taux de croissance a été amélioré par criblage de différents milieux de cultures et différentes concentrations en saccharose, montrant une préférence pour le milieu ½ SH avec 2% (p/v) de saccharose. Les cinétiques de croissance et de production de stilbènes ont ensuite été évaluées dans ces conditions. Nous avons mis en évidence une production basale de stilbènes par les racines, bien que celles-ci soient aussi fortement inductibles par des traitements d’élicitation par du méthyl jasmonate et des cyclodextrines. Dans ces conditions, les racines transformées de vigne ont montré une forte capacité de production et d’excrétion de différents stilbènes. Un profilage phytochimique des racines et de leur milieu de culture a été réalisé par CPC-RMN et LC-MS pour illustrer cette diversité. En parallèle des études sur un modèle simplifié, les cultures de cellules en suspension ont été réalisées pour rechercher des transporteurs candidats pour l’excrétion active du t-resvératrol vers son lieu d’action. Une approche de protéomique globale de la membrane plasmique par iTRAQ a permis de cibler des candidats de type ABC transporteurs, qui ont ensuite été caractérisés par des approches d’étude de l’expression de leurs transcrits. Ensemble, ces résultats soutiennent l’intérêt de cet outil pour l’étude du métabolisme ainsi que pour la bioproduction de stilbènes. / This work aims at the setting up and the study of a new in vitro culture for a cost-effective production of highly pure resveratrol derivatives. To answer that need, hairy root lines of Vitis vinifera L. were established, stabilized and screened. Their low growth rate was improved by testing various culture media and different sucrose concentrations. The best growth rate was obtained with ½ SH medium with 2% (w/v) sucrose. The growth and stilbene production kinetics were assessed in these conditions. A constitutive production of stilbenes was observed in roots, though they showed a strong response to eliciting treatments such as methyl jasmonate and cyclodextrines. In these conditions, the hairy roots yielded high stilbene production in terms of concentrations as well as diversity. The diversity of the stilbenes obtained has been described by biochemical profiling of both root and their culture medium extracts using CPC-NMR and LC-MS. Together with the study of hairy roots, we used cell suspensions cultures as a simplified model to study the excretion of t-resveratrol. Candidate transporters have been screened for using a global plasma membrane proteomic approach based of iTRAQ. ABC G transporters were pointed out as promising candidates and were further characterized by studying their gene expression. Together, these results support the interest of grapevine hairy root cultures for the study of stilbenes metabolism and their bioproduction.

The work presented herein focus upon crystal engineering of nutraceutical cocrystals. Cocrystals are considered unique solid dosage form which has many advantages over other traditionally known solid forms. Furthermore, cocrystals have proven to improve stability, solubility and bioavailability of Active Pharmaceutical Ingredient (API) as shown in the case of carbamazepine and other APIs in previous studies. Crystal engineering is commonly used to design new solid forms based on the bases of supramolecular chemistry. In this study, crystal engineering based on intensive Cambridge Structural Database (CSD) analysis used to predict and design new cocrystals of targeted nutraceuticals. Two nutraceuticals were selected for this study; resveratrol and citric acid. The rationale behind selecting resveratrol was to improve its solubility and, accordingly, bioavailability. On the other hand, citric acid is known as a highly soluble and safe nutraceutical, and thus it can be used as a coformer. Five new cocrystals were prepared and characterized using a variety of techniques that include single crystal X-ray diffraction (XRD), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), FT-IR, and thermo-gravimetric analysis (TGA). Most of the reported cocrystals were obtained using different techniques; solvent slow evaporation, mechanichemical approach, slurry, and from melt. Moreover, dissolution test has been performed on resveratrol and two of its cocrystals, using UV-vis spectrophotometer, where the data demonstrate that through cocrystallization with different cocrystal formers, solubility of resveratrol could be greatly modified, and further controlled. The polymorphism phenomenon is encountered, and accordingly addressed, herein where four novel polymorphs were obtained during cocrystallization attempts. Polymorphism has a significant importance in industry, in general, and in pharmaceutical industry, in particular, due to the vast differences in physical properties of polymorphs. Furthermore, the study of polymorphism provides valuable information essential to understand how different crystal forms are attained.

Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations in PTEN frequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygous PTEN deficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizes PTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation in PTEN-deficient lipoma cells. Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis. PTEN expression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced in PTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primary PTEN wild-type preadipocytes were less affected compared to PTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes.

Obesity has been associated with accelerated cognitive decline during ageing and an increased risk for dementia (Fergenbaum et al., 2009; Fitzpatrick et al., 2009; Gustafson et al., 2009; Whitmer et al., 2008). However, recent discussions are questioning this detrimental relationship (Prickett et al., 2015). A protective effect of midlife obesity was found in a large epidemiologic study (Qizilbash et al., 2015), whereas a meta-analysis showed that midlife obesity increases the risk of dementia (Albanese et al., 2017). Methodological difference and reverse causation could be the reasons for such conflicting results (Gustafson, 2015; Kivimäki et al., 2018). For example, some studies used either body mass index (BMI) or waist-to-hip ratio (WHR); their results suggested abdominal or central obesity, which is measured by WHR or waist circumference, is more detrimental for cognitive decline than global obesity measured by BMI (Gustafson et al., 2009; Whitmer et al., 2008). Therefore, in order to assess the effect of body fat on cognitive function in a large group of community-dwelling healthy adults, we used both BMI and WHR as indicators for adiposity (Study 1). To better understand cognitive changes, it is important to investigate obesity-related brain changes. Regarding to brain structure, high body fat has been associated with decreased total brain volume and regional grey and white matter volume (Willette and Kapogiannis, 2015). However, the findings regarding white matter volume are inconsistent; obesity was found to be associated with decreased as well as increased white matter volumes (Bobb et al., 2014; Driscoll et al., 2012; Karlsson et al., 2013; Walther et al., 2010). Because of the inconsistency, diffusion-weighted imaging (DWI), which assesses the microstructural architecture, has been considered to have higher agreements among studies on obesity and decreased directional property of white matter (Willette and Kapogiannis, 2015). However, a recent study reported a positive association between obesity and white matter microstructure (Birdsill et al., 2017). The conflicting results may be explained by the relative small–medium sample size (n = 15– 268) among the studies and limited statistical power for data-driven whole brain voxel- wise analyses. Following the thought of increasing statistical power with bigger samples might be beneficial and more reliable, we investigated the link between body fat and white matter microstructure using a population-based sample of 1255 participants in Study 1. The obesity-related changes in the brain is considered to be a result of neuroinflammation. The neuroinflammation is likely caused by high fat intake- and excess adipose tissue-induced peripheral inflammation, which in turn leads to insulin resistance and hyperglycaemia; and these all together could also affect the hippocampus and result in memory inhibition (O’Brien et al., 2017; Pugazhenthi et al., 2016). A number of studies reported a negative association between obesity and hippocampal volume (Debette et al., 2011; Raji et al., 2010; Taki et al., 2008). However, others found a positive association (Widya et al., 2011) or no association (Bobb et al., 2014; Debette et al., 2011; Driscoll et al., 2012). This could be due to the fact that hippocampus comprises several subfields (cornu ammonis fields [CA1–4], the dentate gyrus [DG], and the subiculum), and these subfields have distinct functional properties (Deng et al., 2010; Strien et al., 2009; Yassa and Stark, 2011). It has also been reported that the hippocampal subfields are affected by ageing differently (Malykhin et al., 2017). Because the hippocampus is highly susceptible to degenerative processes (Pfefferbaum et al., 2013; Raz et al., 2010) and possesses neurogenesis ability in adults (Eriksson et al., 1998), it is often a key target in intervention studies. Several studies indicate that lifestyle interventions can be effective to combat obesity and to restore cognitive functions (Siervo et al., 2011). A double-blind randomised controlled trial of 1260 older individuals (aged 60–77 years) from the FINGER study showed that the intervention group obtained higher scores in a neuropsychological test battery compared to the control group after a 2-year multidomain intervention (diet, exercise, cognitive training, vascular risk monitoring) (Ngandu et al., 2015). Dietary nutrients that have anti-inflammatory or anti-oxidative effects, such as omega-3 fatty acids and polyphenols, have been associated with improved cognitive function and brain structural changes (Gómez-Pinilla, 2008; Huhn et al., 2015). Resveratrol is one type of polyphenols and occurs in variety of plants such as red grapes and blueberries (Baur et al., 2006). It has been reported to be associated with glucose metabolism and insulin sensitivity (Liu et al., 2014) as well as improved cognitive functions (Huhn et al., 2015; Marx et al., 2018). However, some studies reported no improvements in cognition after intervention (Köbe et al., 2017; Wightman et al., 2015). Besides the possibility that resveratrol does not affect cognition, another reason for these results could be that resveratrol is only effective in healthy ageing population (Evans et al., 2017; Witte et al., 2014) but not in young (Wightman et al., 2015) or patient population (Köbe et al., 2017). Therefore, replication studies with healthy older adults could help to evaluate whether there is an effect of resveratrol on cognitive function. Following this idea, we have conducted a double-blind randomised controlled trial (Study 2) with comprehensive neuropsychological test battery to assess the effect of resveratrol using 60 elderly subjects. In Study 1, we applied whole brain voxel-wise analysis to explore the correlations between overall obesity (measured by BMI) or abdominal obesity (WHR) and white matter microstructure. We found a negative correlation between BMI as well as WHR and fractional anisotropy (FA), a measure of microstructural architecture, in multiple white matter tracts independent of confounding factors. We further explored the indirect link of obesity and cognitive dysfunction using mediation analysis. In the mediation analysis, an indirect path through obesity-associated clusters was considered. We found that although obesity had no direct effect on executive functions and processing speed, it affected cognitive performance through lower FA in callosal and associative fibre tracts. We found the correlation between obesity and memory performance was not mediated by FA in the selected white matter tracts. In Study 2, we conducted a randomised trial for the effect of resveratrol on memory performance and hippocampal structure. We found that intake of resveratrol did not show any beneficial effect on either glucose metabolism or cognitive performance. Neither volume nor mean diffusivity (MD), another measure of microstructural architecture, showed changes after the intervention compared to the placebo group. However, subtle ageing- or lifestyle-related changes in the MD of the hippocampus were detected. This demonstrated that MD outperforms volumetric measures for detecting subtle changes of the hippocampus. The reason we could not observe any changes after resveratrol intake might be that this compound does not have an effect or that other lifestyle changes undermined the effect of resveratrol as neuroplasticity can be influenced by many factors. This thesis highlights that body fat is associated with lower FA in the white matter of the brain. This may indicate some widespread damage to the white matter; and mediation analysis indicates abdominal obesity is linked to poorer executive functions and processing speed through lower FA. Further this thesis shows that adding a dietary supplementation of resveratrol for six months does not improve memory or hippocampal structure in the present cohort of healthy adults with a large BMI range. Future studies should investigate longitudinal changes of body fat and brain structure in order to establish the causal relationship among obesity, white matter microstructure, and cognitive function. And more comprehensive lifestyle interventions combining diet, exercise, and cognitive training should be considered instead of one single approach to prevent and hopefully preserve obesity induced changes in cognition and in the brain.

Background: Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established. Scope of Review: EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells. Major Conclusions: Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways. General significance: These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.