Lectinas são proteínas cuja principal característica é a de se ligar específica e reversivelmente a carboidratos. BanLec é a lectina presente na polpa de bananas, que se liga especificamente a manose e glicose, e é capaz de induzir a proliferação de células T, podendo estimular a resposta imune. Existem indícios de que o teor de BanLec pode variar dependendo do estádio de amadurecimento e do tipo de cultivar, o que pode afetar a quantidade de BanLec existente na fruta quando consumida in natura e a possível resposta imune frente ao consumo de banana. Por este motivo, um dos objetivos desse trabalho foi determinar os teores e a atividade hemaglutinante de BanLec em extratos de farinha de banana verde, além de bananas das cultivares Pacovan, Figo, Terra, Mysore e Nanicão, nos estádios de maturação verde e maduro, e submetidas a tratamento com 1-MCP e baixa temperatura (para cv. Nanicão). Com vista a atender ao objetivo de avaliar seus efeitos imunomoduladores in vivo, a BanLec foi purificada da cultivar Nanicão e administrada por via oral a camundongos BALB/c. Os ensaios de atividade hemaglutinante dos extratos de banana apontaram para maior quantidade de BanLec no fruto maduro, quando comparado ao verde, e ausência dessa proteína na cultivar Figo. Os parâmetros imunológicos analisados após administração de BanLec aos camundongos demonstram que a resposta imune gerada após ingestão de BanLec é dose dependente, além disso, a administração de 50 µg de BanLec aos animais foi capaz de modular citocinas importantes na resposta imunológica, provavelmente causando um efeito que pode ser interpretado como mais protetor do que patogênico. Com base nos resultados obtidos, podemos concluir que existem diferenças nos teores de BanLec dependendo da cultivar e estádio de maturação analisado, sendo que essa proteína não está presente na polpa de todas as variedades de banana e finalmente, que ela tem grande potencial imunomodulador in vivo, uma vez que ativou citocinas de resposta anti-inflamatória. / Lectins are proteins which bind specifically and reversibly to carbohydrates. BanLec is the lectin present in banana pulp, and it binds to mannose and glucose, being capable of inducing T-cell proliferation, and to stimulate the immune response. There are some evidence that the amount of BanLec may vary depending on the maturation stage of the fruit and the cultivar (cv.), which may affect the amount of BanLec and the possible immune response after consumption of banana. Thus, this study aimed to evaluate the amount of BanLec and its hemagglutinating activity in crude extracts of bananas from cultivars Pacovan, Figo, Terra, Mysore and Nanicão, in both unripe and ripe maturation stage, and also fruits which were treated with 1-MCP and low temperature. In addition, in order to access their immunomodulatory effects in vivo, BanLec was purified by affinity chromatography and administered orally to BALB/c mice. The hemagglutinating activity assays indicate higher amount of BanLec in ripe fruit. Moreover, the possible was undetectable in the pulp of banana Figo. The immunological parameters of mice orally fed with BanLec showed that the immunological response is dependent on the amount of protein administrated, in agreement to previous in vitro studies. Besides, 50 µg of BanLec, were able to modulate some cytokines in immune response, causing an effect that seems to be more protective than pathogenic. We conclude that there are important differences in amount of BanLec depending on the cultivar and the maturation stage, and BanLec has a dose-dependent immunomodulatory effect in vivo.

Lectinas são proteínas cuja principal característica é a de se ligar específica e reversivelmente a carboidratos. BanLec é a lectina presente na polpa de bananas, que se liga especificamente a manose e glicose, e é capaz de induzir a proliferação de células T, podendo estimular a resposta imune. Existem indícios de que o teor de BanLec pode variar dependendo do estádio de amadurecimento e do tipo de cultivar, o que pode afetar a quantidade de BanLec existente na fruta quando consumida in natura e a possível resposta imune frente ao consumo de banana. Por este motivo, um dos objetivos desse trabalho foi determinar os teores e a atividade hemaglutinante de BanLec em extratos de farinha de banana verde, além de bananas das cultivares Pacovan, Figo, Terra, Mysore e Nanicão, nos estádios de maturação verde e maduro, e submetidas a tratamento com 1-MCP e baixa temperatura (para cv. Nanicão). Com vista a atender ao objetivo de avaliar seus efeitos imunomoduladores in vivo, a BanLec foi purificada da cultivar Nanicão e administrada por via oral a camundongos BALB/c. Os ensaios de atividade hemaglutinante dos extratos de banana apontaram para maior quantidade de BanLec no fruto maduro, quando comparado ao verde, e ausência dessa proteína na cultivar Figo. Os parâmetros imunológicos analisados após administração de BanLec aos camundongos demonstram que a resposta imune gerada após ingestão de BanLec é dose dependente, além disso, a administração de 50 µg de BanLec aos animais foi capaz de modular citocinas importantes na resposta imunológica, provavelmente causando um efeito que pode ser interpretado como mais protetor do que patogênico. Com base nos resultados obtidos, podemos concluir que existem diferenças nos teores de BanLec dependendo da cultivar e estádio de maturação analisado, sendo que essa proteína não está presente na polpa de todas as variedades de banana e finalmente, que ela tem grande potencial imunomodulador in vivo, uma vez que ativou citocinas de resposta anti-inflamatória. / Lectins are proteins which bind specifically and reversibly to carbohydrates. BanLec is the lectin present in banana pulp, and it binds to mannose and glucose, being capable of inducing T-cell proliferation, and to stimulate the immune response. There are some evidence that the amount of BanLec may vary depending on the maturation stage of the fruit and the cultivar (cv.), which may affect the amount of BanLec and the possible immune response after consumption of banana. Thus, this study aimed to evaluate the amount of BanLec and its hemagglutinating activity in crude extracts of bananas from cultivars Pacovan, Figo, Terra, Mysore and Nanicão, in both unripe and ripe maturation stage, and also fruits which were treated with 1-MCP and low temperature. In addition, in order to access their immunomodulatory effects in vivo, BanLec was purified by affinity chromatography and administered orally to BALB/c mice. The hemagglutinating activity assays indicate higher amount of BanLec in ripe fruit. Moreover, the possible was undetectable in the pulp of banana Figo. The immunological parameters of mice orally fed with BanLec showed that the immunological response is dependent on the amount of protein administrated, in agreement to previous in vitro studies. Besides, 50 µg of BanLec, were able to modulate some cytokines in immune response, causing an effect that seems to be more protective than pathogenic. We conclude that there are important differences in amount of BanLec depending on the cultivar and the maturation stage, and BanLec has a dose-dependent immunomodulatory effect in vivo.

Introdução: Com o crescente aumento de hipersensibilidades alimentares e doenças do trato gastrintestinal, o mercado de lácteos vem ampliando a variedade de produtos com diferentes matrizes alimentares. A expansão da criação bubalina no país incentivou o desenvolvimento de produtos derivados do leite bubalino, os quais contém propriedades nutricionais e físico-químicas diferentes da matriz bovina, ganhando maior participação no atual mercado consumidor. As bactérias probióticas são capazes de modular a resposta imune inata e adaptativa de acordo com a cepa, matriz e tecnologia empregada no desenvolvimento do produto. Assim como a modulação imune também pode ser alterada de maneira epigenética por interferência da matriz alimentar empregada. Método: Sendo os microRNAs de leite e leite bubalino homólogos aos do leite humano, foram preparados leites fermentados em matriz bovina e bubalina usando a cepa Bifidobacterium animalis subsp. lactis HN019 com o intuito de identificar a diferença entre o perfil físco-químico, viabilidade da cepa probiótica utilizada, e presença de microRNAs imunomoduladores homólogos com o intuito de verificar se as modificações sofridas nas matrizes bovina e bubalina durante o processo de fermentação por bactérias probióticas teria interferência na presença de miRNAs imunomoduladores e na resposta imune de mucosa. Conclusão: Os resultados obtidos mostraram maior teor de proteínas, sólidos totais e gordura na matriz bubalina em relação a matriz bovina. Enquanto na matriz bubalina fermentada apresentou maior teor de ácidos graxos comparado a matriz bovina fermentada, o perfil de acidificação e pós acidificação foi semelhante em ambas as matrizes. O processo de fermentação em matrizes lácteas distintas, proporcionou modificação do perfil celular imunológico na mucosa intestinal, porém levou a destruição dos miRNAs do processamento térmico, identificando que esta modificação da resposta imune independe dos microRNAs homólogos estudados. / Introduction: With the increasing increase of food hypersensitivity and diseases of the gastrointestinal tract, the dairy market has been increasing the variety of products with different food matrices. The expansion of buffalo breeding in the country encouraged the development of products derived from buffalo milk, which contain different nutritional and physicochemical properties of the bovine matrix, gaining greater participation in the current consumer market. Probiotic bacteria are able to modulate the innate and adaptive immune response according to the strain, matrix and technology employed in product development. Just as immune modulation can also be altered epigenetically by interference of the food matrix employed. Method: Since the microRNAs of milk and buffalo milk were homologous to those of human milk, fermented milks were prepared in bovine matrix and buffalo using the strain Bifidobacterium animalis subsp. Lactis HN019 in order to identify the difference between the physico-chemical profile, viability of the probiotic strain used, and the presence of homologous immunomodulatory microRNAs in order to verify if the modifications suffered in the bovine and buffalo matrices during the fermentation process by probiotic bacteria would interfere with the presence of immunomodulatory miRNAs and the mucosal immune response. Conclusion: The results showed higher protein content, total solids and fat in the buffalo matrix in relation to the bovine matrix. While in the fermented buffalo matrix presented higher content of fatty acids compared to fermented bovine matrix, the profile of acidification and post acidification was similar in both matrices. The fermentation process in distinct milk matrices provided a modification of the immunological cell profile in the intestinal mucosa but led to the destruction of the miRNAs of the thermal processing, identifying that this modification of the immune response is independent of the homologous microRNAs studied.

L’objectif de cette thèse était d’améliorer les connaissances fondamentales sur le tissu adipeux, organe qui est au cœur de la pratique des chirurgiens plasticiens. En effet, ce tissu peut être transplanté de façon autologue afin de combler une perte de substance (rôle volumateur des adipocytes) mais également servir à des fins de régénération tissulaire en lien avec les cellules de la fraction vasculaire stromale (FVS) et tout particulièrement des cellules stromales mésenchymateuses (CSM). Ces cellules s’obtiennent après lipoaspiration du tissu par une digestion enzymatique de la graisse obtenue. Il s’avère que les connaissances disponibles sur ces CSM sont essentiellement issues d’études in vitro après une phase de culture cellulaire plus ou moins longue et ainsi les propriétés in vivo sont mal connues. Ce travail a donc consisté à caractériser l’hétérogénéité du compartiment stromal natif du tissu adipeux obtenu après digestion enzymatique. Nous avons isolé deux populations stromales natives distinctes : les ASC (CD34+), en grande majorité, et des cellules péricytaires (CD146+). Ces 2 types cellulaires différaient dans leurs phénotypes, leurs potentiels de clonogénécité et leurs propriétés immunomodulatrices in vitro et in vivo. Nous avons ensuite comparé la digestion enzymatique du tissu aux techniques de digestion mécanique utilisable au sein de nos blocs opératoires. Nous avons démontré que ces nouvelles techniques permettaient bien de produite les cellules de la FVS dont des CSM, cellules particulièrement intéressante pour des gestes de régénération tissulaire. De plus, l’ensemble des techniques de laboratoire acquise au cours de ce travail nous ont permis d’investiguer le rôle des techniques de lipoaspiration utilisé en chirurgie plastique sur le tissu adipeux. Nous avons démontré par cytométrie de flux et par immunofluorescence in situ qu’une partie de la trame microvasculaire était conservée. L’ensemble de ces résultats viennent s’ajouter aux données cliniques démontrant que la lipoaspiration du tissu est un geste permettant d’être plus conservateur pour le tissu et pourrait expliquer des taux de complications moindre après chirurgie de contour de la silhouette. / The aim of this work was to improve the knowledge on adipose tissue, organ that is at the heart of the practice of plastic surgeons. Indeed, this tissue can be transplanted autologously in order to fill a defect (volumizing role of the adipocytes) but also to be used for tissue regeneration in connection with the cells of the stromal vascular fraction (SVF) and especially the mesenchymal stromal cells (MSCs). These cells are obtained after liposuction of the tissue by enzymatic digestion of the extracellular matrix. It turns out that the knowledge available on these CSM is essentially derived from in vitro studies after a cell culture phase and thus the in vivo properties are poorly known. This work consisted in characterizing the heterogeneity of the native stromal compartment of adipose tissue obtained after enzymatic digestion. We isolated two distinct native stromal populations: the ASC (CD34 +), for the most part, and the pericyte cells (CD146 +). These 2 cell types differed in their phenotypes, their clonogenecity potentials and their immunomodulatory properties in vitro and in vivo. We then compared the enzymatic digestion of the tissue with the techniques of mechanical digestion usable within our operating room. We have demonstrated that these new techniques made it possible to produce the cells of the FVS including MSC, cells particularly interesting for regenerative surgery. In addition, all the laboratory techniques acquired during this work allowed us to investigate the role of liposuction techniques used in plastic surgery on adipose tissue. We have demonstrated by flow cytometry and confocal microscopy, that part of the microvasculature framework is conserved after liposuction. All of these results are in addition to clinical data demonstrating that liposuction of the tissue is a gesture to be more conservative for the tissue and could explain lower rates of complications after contour surgery.

Les préparations de Viscum album (VA), connu sous le nom vernaculaire de gui européen, sont fréquemment utilisées en support des traitements anticancéreux, principalement pour améliorer la qualité de vie des malades et réduire la croissance des tumeurs. Elles sont connues pour exercer des effets anti-tumoraux. Il existe de plus en plus de données scientifiques faisant état de liens étroits entre cancer et inflammation. Étant donné que la prostaglandine E2 (PGE2) induite par la cyclo-oxygénase 2 (COX-2) joue un rôle clef dans l’inflammation, j’ai exploré la régulation du système COX-2-PGE2 par VA et ses mécanismes sous-jacents. J’ai montré que VA exerce ses effets anti-inflammatoires en inhibant sélectivement l’expression de COX-2 et en diminuant la production de PGE2 qui en découle, par le biais d’une déstabilisation de l’ARNm de COX-2. En plus de leurs propriétés cytotoxiques, il a été montré que les préparations de VA ont également des effets immunostimulants. Les différentes préparations de VA sont hautement hétérogènes du fait de leurs compositions biochimiques qui varient selon la récolte, l’espèce de l’arbre hôte et les méthodes de préparation qui peuvent influer sur leur efficacité clinique. De ce fait, j’ai réalisé une étude comparative sur cinq préparations de VA dans le but d’analyser leurs capacités de maturation et d’activation des cellules dendritiques (DC) qui peuvent à leur tour présenter une réponse immunitaire anti-tumorale. Les résultats ont montré que parmi les cinq préparations,VA Qu Spez induit de manière significative l’activation des DC et la sécrétion de cytokines pro-inflammatoires telle que l’IL-6, l’IL-8 et le TNF-α qui induisent la production d’IFN-γ,orientant de ce fait la réponse immunitaire vers une réponse Th1. L’orchestration de la11fonction des cellules myélomonocytiques est un élément central à l’interface entre inflammation et cancer. Il constitue un paradigme expliquant la plasticité et la fonction des macrophages. Mon étude met en évidence l’influence de VA Qu Spez sur la polarisation des macrophages qui passent d’un état alternatif (M2) à un état dit classique (ou M1). Les macrophages M2 sont connus pour polariser les réponses immunitaires Th2, pour participer à l’élimination des parasites, pour diminuer l’inflammation, pour promouvoir le remodelage tissulaire et la progression des tumeurs et pour avoir des fonctions immunorégulatrices. Les macrophages M1 sont impliqués dans la réponse Th1, favorisent la résistance aux pathogènes intracellulaires et aux tumeurs et promeuvent des réactions de désagrégation tissulaires. L’ensemble de ces résultats permet de comprendre les propriétés anti-inflammatoires et immunostimulantes des préparations de VA. Des recherches complémentaires permettront d’améliorer les stratégies d’utilisation thérapeutique de VA et son utilisation dans les soins de support aux traitements anticancéreux. / Viscum album (VA) preparations, commonly known as European mistletoe, are frequentlyused as complementary therapy in cancer, mainly to improve quality of life of the patients andto reduce the tumor growth. They are known to exert anti-tumoral effects. There is increasing evidence of the convoluted connection of cancer and inflammation. As cyclooxygenase-2(COX-2)-induced prostaglandin E2 (PGE2) plays a key role in the inflammation, I explored the regulation of COX-2-PGE2 axis by VA and underlying mechanisms. I found that VA exerts anti-inflammatory effects by selectively inhibiting COX-2 expression and ensuing PGE2 production. Inhibition of COX-2 expression implicates COX-2 mRNA destabilisation. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. Each VA preparations are highly heterogeneous because oftheir chemical composition which varies depending on the time of harvest, species of host treeand manufacturing methods, together which might influence clinical efficacy of VA.Therefore I performed a comparative study involving five different preparations of VA concerning maturation and activation of dendritic cells (DCs) which in turn may manifestanti-tumoral immune response. Results showed that among all five preparations, VA Qu Spez significantly induces DC activation, secretion of pro-inflammatory cytokines such as IL-6, Il-8 and TNF-α, enhancing IFN-γ production hence promoting Th1 immune response. The orchestration of myelomonocytic cell function is a key element that links inflammation and cancer and provides a paradigm for macrophage plasticity and function. My study reveals the effect of VA Qu Spez in switching the M2 macrophages which are known to participate inpolarizing Th2 responses, help with parasite clearance, dampen inflammation, promote tissue remodelling and tumor progression and have immunoregulatory functions, towards classicallyactivated M1 macrophages which are part of a polarized Th1 response and mediate resistance13to intracellular pathogens and tumors and elicit tissue-disruptive reactions. These results together should assist in understanding the anti-inflammatory and immunostimulatory properties of VA preparations and further research is warranted to improve the therapeutic strategies of use of VA and their role as complimentary therapy in cancer.