Orientadores: Mary Ann Foglio, Ana Lúcia Tasca Gois Ruiz, João Ernesto de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:38:49Z (GMT). No. of bitstreams: 1 Zago_PatriciaMariaWiziack1982-_D.pdf: 5120519 bytes, checksum: a581f776ecdecf5822427a828d170cc1 (MD5) Previous issue date: 2014 / Resumo: A Osteonecrose Maxilar Induzida por Bisfosfonatos (OMIB) está associada à terapia com bisfosfonatos como o ácido zoledrônico (ZA), que são medicamentos importantes para o manejo de patologias ósseas. Estudos in vitro demostraram que os bisfosfonatos podem alterar a viabilidade e proliferação de células epiteliais, o que justificaria situações de deficiente cicatrização em feridas de OMIB. Arrabidaea chica (H&B.) Verlot é uma Bignoniaceae com propriedades cicatrizantes, e que apresenta as antocianidinas como principais compostos identificados. Este estudo avaliou a atividade antimicrobiana, citotóxica e citoprotetora, in vitro, do extrato hidroalcoólico de A. chica (AC) na presença de ZA. Foi realizado estudo de estabilidade nas condições de 40 °C e 75% umidade, do extrato microencapsulado em mistura de goma arábica com maltodextrina (1:1) e do extrato livre correspondente. Queratinócitos (HaCaT), fibroblastos (PT64-04HF) e osteoblastos (MC3T3-E1) foram submetidos a 2 protocolos de tratamento: a exposição a ZA 10 µM por 24h e posteriormente AC por 48h e vice-versa; ou o co-tratamento de ambos. Os parâmetros estudados foram viabilidade, ocorrência de apoptose e ativação de caspases 3/7. A viabilidade de células de câncer de próstata (C4-2B) foi avaliada após tratamento com AC. Streptococcus mutans foram submetidos ao co-tratamento ZA e AC. Microcápsulas contendo AC foram produzidas por processamento em spray dryer, aliquotadas e armazenadas em câmara climática para o estudo de estabilidade acelerada, segundo protocolo da ANVISA (RE 01/2005). Os resultados foram submetidos à análise estatística (ANOVA e Teste T de Student, a=0,05). Após 48 h, ZA reduziu a viabilidade de queratinócitos, fibroblastos e osteoblastos em, respectivamente, 34%, 47% e 51%. O co-tratamento ZA e AC 5 µg mL-1 aumentou a viabilidade celular (82%, 83% e 55%), o mesmo ocorrendo para ZA e AC 10 µg mL-1 (69%, 70% e 64%). Após 48 h de tratamento com ZA, não houve apoptose celular ou ativação de caspases em fibroblastos, mas verificou-se a ativação de caspase 3/7 em osteoblastos. As concentrações de 5 e 10 µg mL-1 de AC não alteraram a viabilidade de células C4-2B, porém observou-se uma redução de mais de 40% em concentrações maiores que 10 µg mL-1. O crescimento de S. mutans foi aparentemente aumentado após o tratamento com ZA em concentrações maiores que 50 µM, sem que AC ocasionasse qualquer efeito. Finalmente, o material formador de parede (mistura de goma arábica com maltodextrina 1:1) empregado para microencapsular AC, foi incapaz de inibir a degradação das antocinidinas, cujas concentrações relativas foram reduzidas após 15 dias de incubação. Assim, baixas concentrações de AC (5 e 10 µg mL-1) demostraram promissora atividade citoprotetora contra os efeitos prejudiciais de ZA, em células epiteliais e osteoblastos, sem estimularem o crescimento das células cancerígenas (C4-2B) e de S. mutans. Outros materiais formadores de parede serão avaliados, buscando-se aumentar a estabilidade química de AC. Mais estudos são necessários para a comprovação dos efeitos benéficos de AC como um auxiliar no tratamento de OMIB / Abstract: The bisphosphonate related osteonecrosis of the jaw (BRONJ) is a condition associated to bisphosphonates, medicines used to treat bone disorders, as zoledronic acid. In vitro studies showed epithelial cells viability and growth can be altered by bisphosphonates, and may cause a deficient wound healing in BRONJ. Arrabidaea chica (H&B.) Verlot is a Bignoniaceae with wound healing properties, however, anthocyanidins, the main compounds of the plant extract, can be easily degraded under environmental conditions. This in vitro study aimed to evaluate the antimicrobial, citotoxic and citoprotective actions of A. chica hydroalcoholic extract (AC) in the presence of ZA therapy. Stability studies at 40 °C and 75% humidity conditions, from microencapsulated into arabic gum plus maltodextrin (1:1) and free crude extract samples were conducted. Keratinocytes (HaCaT), fibroblasts (PT64-04HF) and osteoblasts (MC3T3-E1) were submitted to 2 treatment protocols: zoledronic acid 10 µM (ZA) therapy during 24 h and then AC during 48 h and vice-versa; or co-treatment with both. Parameters as viability, apoptosis and caspase 3/7 activation were evaluated. Cancer cells (C4-2B) viability was determined after AC treatment. Streptococcus mutans were submitted to co-treatment with ZA and AC. Microcapsules containing AC were obtained after spray dryer processing, aliquoted and stored into climatic chamber for stability study according to ANVISA protocol (RE 01/2005). ANOVA and T Student test, ?a=0,05, were applied for statistical analysis. After 48 h, ZA treatment decreased viability of keratinocytes, fibroblasts and osteoblasts repectively by 34%, 47% and 51%. Co-treatments ZA and AC 5 µg mL-1 (82%, 83% and 55%), and ZA and AC 10 µg mL-1 (69%, 70% and 64%) increased cell viability. No apoptosis or caspase activation were detected on fibroblasts, however, caspase 3/7 was active on osteoblasts after ZA 48 h treatment. Concentrations higher than 10 µg mL-1 of AC were toxic to cancer cells and showed a decrease by 40% in cell viability, while no effect was seen with AC 5 or 10 µg mL-1. Higher ZA concentrations (50 µM) seemed to induce S. mutans growth, while AC showed no effect on bacteria. Finally, the studied core matherial (arabic gum plus matodextrin 1:1) was ineffective on protecting AC extract against degradation, since anthocyanidins concentration was reduced after 15 days incubation. Therefore, lower AC concentrations (5 and 10 µg mL-1) were promising on treating BRONJ wounds, as they were effective in protect epithelial cells against harmfull effects of ZA, without affecting cancer cells or S. mutans growth. Different types of core matherials must be evaluated for improving AC chemical stability. More studies are required to prove AC beneficial effects on treating BRONJ / Doutorado / Farmacologia, Anestesiologia e Terapeutica / Doutora em Odontologia

Orientador: Hernandes Faustino de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T21:24:31Z (GMT). No. of bitstreams: 1 Damas-Souza_DaniloMarchete_D.pdf: 2083384 bytes, checksum: f2c3f40fb23b6b092127e3ba4a576d24 (MD5) Previous issue date: 2013 / Resumo: O crescimento e a fisiologia da próstata é dependente da expressão de receptores esteróides em alguns tipos celulares e de interações parácrinas estabelecidas após a resposta inicial a esses hormônios, em associação com hormônios somatotróficos como a insulina. O presente trabalho tem o objetivo de caracterizar componentes moleculares relacionados aos mecanismos de adaptação das células epiteliais prostáticas de rato na ausência andrógeno e/ou insulina e possíveis conexões com as vias gerais de regulação do metabolismo celular, monitorando diferentes pontos da via de sinalização integradas pelos TSC1-TSC2 e mTORC1 e mTORC2. Foram empregados 54 ratos Wistar adultos jovens com idade entre 75 e 105 dias. Os animais foram induzidos diabetes por injeções intraperitoneal de aloxana, em seguida, os ratos foram divididos em não diabético (ND), diabético (DIA) e diabético tratado com insulina (INS), somados a castração cirúrgica e sacrificados 48 e 72 horas após a castração. Utilizando a técnica de Western Blotting foram analisados os conteúdos e fosforização de mTOR, p70S6K, eIF4B, 4E-BP, eIF4E e p53. Os resultados obtidos no presente trabalho identificam uma nítida função do estímulo androgênico na regulação negativa de eIF4E (via 4E-BP1) e positiva de eIF4B. Ficou claro também que a insulina regula positivamente a fosforilação de 4E-BP1 e que a privação androgênica promove aumento de p53. Assim dois mecanismos parecem essenciais para a manutenção e seleção das células epiteliais prostáticas de ratos: (I), p53/4E-BP/eIF4E na ausência de andrógeno; (II), p70S6K/eIF4B na ausência de insulina. Apoio financeiro: CNPq, Capes / DS e FAPESP / Abstract: The growth and physiology of the prostate is dependent on steroid receptors in certain types of cells and the paracrine interactions established after the initial response to these hormones in combination with somatotropic hormones such as insulin. The current work aims to characterize the molecular components related to the adaptation mechanisms of the epithelial cells in the prostate of rats. In addition to the absence of androgen and/or insulin and possible connections with the general pathways regulating cell metabolism, monitoring different points of the signaling pathways that integrate TSC1- TSC2 and mTORC1 and mTORC2. 54 male Wistar rats aged between 75 and 105 days were used for the current work. Diabetes was induced by intraperitoneal injection of alloxan and the rats were divided into non-diabetic (ND), diabetic (DIA) and diabetic treated with insulin (INS) groups. Every group was divided into three sub groups and the rats were sacrificed non castrated, surgically castrated 48 and 72 hours. Using Western blotting the contents and phosphorylation of mTOR, p70S6K, eIF4B, 4E- BP, eIF4E and p53 were analyzed. The results obtained in this study identify a clear function of androgenic stimulation in the negative regulation of eIF4E (by 4E-BP1) and positive regulation eIF4B and p53. It also became clear that insulin positively regulates the phosphorylation of 4E-BP1. In conclusion two mechanisms seem to be essential for the maintenance and selection of prostatic epithelial cells in rats: (I) p53/4E-BP/eIF4E in the absence of androgen, (II ) p70S6K/eIF4B in the absence of insulin. Financial support: CNPq , CAPES / DS and FAPESP / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Les cellules souches embryonnaires humaines (CSEh), par leurs caractéristiques de pluripotence et de prolifération illimitée, représentent une alternative à l’utilisation de cellules issues de patients : leur différenciation en cellules épithéliales respiratoires pourrait permettre la production illimitée d’épithélium pour le criblage de molécules thérapeutiques.L’objectif de notre travail a été de mettre au point un protocole simple et financièrement acceptable afin de différencier les CSEh en cellules épithéliales de voies aériennes et de produire un épithélium complet. Pour ce faire, nous avons suivi deux voies potentielles de différenciation des CSEh : une voie passant par la production d’endoderme définitif, feuillet embryonnaire à l’origine de l’épithélium respiratoire, et une voie passant par un progéniteur potentiel commun aux lignages respiratoire et épidermique. Différentes combinaisons de protéines matricielles, d’inducteur de différenciation, de temps d’induction et de milieux de culture ont été testées. Nos résultats montrent que la culture des CSEh sur cellules nourricières STO dans un milieu optimisé pour les cellules bronchiques, le BEGM, en présence de Bone Morphenetic Protein 4 et d’acide rétinoïque pendant 6 jours puis en BEGM seul pendant 30 jours conduit à l’obtention de plus de 76% de progéniteurs épithéliaux respiratoires exprimant des marqueurs spécifiques tels que CK13, P63, CXCR4, FOXA2, SOX17, NKX2.1, SOX2 et SOX9. Le passage par la production de cellules de l’endoderme définitif n’a pas permis d’améliorer l’efficacité de ce protocole. L’isolement de ces progéniteurs et la reconstitution d’un épithélium complet restent à mettre au point. / Human embryonic stem cells (hESCs), for to their characteristics of pluripotency and unlimited proliferation, represent an alternative to the use of primary cells from patients: their commitment and differentiation into airway epithelial cells could help to overcome the lack of patient’s cells and could allow the unlimited production of epithelium for the screening of therapeutic molecules.The objective of our work was to develop a simple and financially acceptable protocol to differentiate hESCs into airway epithelial cells and to produce a complete epithelium. To do this, we followed two potential routes of hESC differentiation: a route through the production of definitive endoderm, the germ layer at the origin of the respiratory epithelium, and a route through a common potential progenitor to the respiratory and epidermal lineages. Various combinations of matrix proteins, differentiation inducers, induction time and culture media were tested.Our results show that hESC culture on STO feeder cells in an optimized medium for human bronchial epithelial cells, the BEGM medium, in the presence of Bone Morphenetic Protein 4 and retinoic acid for 6 days then in BEGM medium alone for 30 supplementary days led to the differentiation of more than 76% of respiratory epithelial progenitors expressing specific markers such as CK13, P63, CXCR4, FOXA2, SOX17, NKX2.1, SOX2 and SOX9. The application of these culture conditions to definitive endoderm cells, previously obtained from hESC, failed to improve the effectiveness of this protocol. The isolation of these progenitors and the reconstruction of a complete airway epithelium remain to be developed.

Abstract Epithelial cell-cell adhesions have a critical role in morphogenesis, establishment and maintenance of tissue architecture, cell-cell communication, normal cell growth and differentiation. These adhesions are disrupted during malignant transformation and tumour cell invasion. Several kinases, phosphatases and small GTPases regulate cell-cell contacts. In the present work we investigated the dynamics of cell-cell adhesion structures after microinjection of fluorophore tagged vinculin, during transformation caused by an active Src tyrosine kinase and during Helicobacter pylori infection. The regulatory role of Rac GTPase as well as the behaviour of actin and cadherin were analysed in all these conditions. Microinjection of vinculin into bovine kidney epithelial MDBK cells induced release of actin, cadherin and plakoglobin to cytoplasm of the cells, caused disruption of protein complexes at adherens and tight junctions that finally led to formation of polykaryons. Activated Rac GTPase, in turn, enhanced accumulation of cadherin to membranes and thereby diminished the formation of polykaryons, whereas inactive Rac removed cadherin from membranes. Incorporation of vinculin to lateral membranes took place also in acidifying and depolarising conditions where cell fusions were prevented. Thus, the membrane potential seemed to control fusion ability. In src-MDCK cells, activation of Src kinase led to disintegration of adherens junctions. Clusters of junctional components and bundles of actin were seen at the basal surface already within 30 min after Src activation. p120ctn was the only component of adherens junction whose relocation correlated to its phosphorylation. Inhibition of Src by a specific inhibitor PP2 restored the cubic morphology of the cells and accumulated cadherin back to lateral walls. Still p120ctn remained in cytoplasm and thereby was not responsible for the epithelial phenotype. Activation of Rac GTPase by Tiam1 also increased the amount of cadherin at lateral membranes and maintained the morphology of src-MDCK cells practically normal after activation of Src kinase. In the same way, actin cytoskeleton was reorganised in gastric carcinoma cells in response to infection with H. pylori via activation of Rac signalling pathway. Hence, Rac and cadherin seem to be the major players in the maintenance of epithelial cell morphology.

Les cellules épithéliales produisent des facteurs influençant la réponse inflammatoire intestinale. L'étude de la contribution de ces facteurs à l'homéostasie des tissus est devenue essentielle à la compréhension des maladies inflammatoires chroniques intestinales (MICIs). Vanin-1 est une enzyme épithéliale qui produit des facteurs solubles dans les tissus dont l'intestin en réponse à un stress. Nous avons identifié Vanin-1 comme facteur de susceptibilité et marqueur des MICIs chez l'homme. La création d'une souris mutante nous a permis d'identifier des mécanismes d'action potentiels de Vanin-1 dans les tissus. Vanin-1 pourrait un jour être exploité pour développer une stratégie thérapeutique contre les MICIs. / Intestinal epithelial cells have recently been identified as producing factors influencing grandly the curse of intestinal inflammation. It became of a great importance, therefore, to identify the contribution of epithelial molecules to inflammatory bowel diseases (IBD). Vanin-1 is an epithelial enzymes which release soluble factors in tissues such as gut. This study identifies Vanin-1 as a marker and a susceptibility factor of IBD. By producing a mutant mice, we identified potential mechanisms by which Vanin-1 could act in tissues. It is hoped that Vnn1 can be exploited to develop future therapeutic strategies against IBD.

Le cancer du sein est une pathologie hétérogène au plan clinique et au moins 5 sous-types moléculaires ont pu être définis sur la base de différences d’expression ARNm. Ces sous-types présentent des différences de profils d’anomalies génomiques et de méthylation des cytosines. Ces différences génétiques et épigénétiques s’expliqueraient par des types cellulaires d’origines distincts au sein de l’épithélium mammaire, toutefois, ceci n’a pas été confirmé clairement à ce jour. Alternativement, il a été proposé que l’activation de voies oncogéniques différentes pouvait avoir un impact significatif sur les modifications génétiques ou épigénétiques. Dans ce travail nous avons voulu vérifier cette hypothèse en l’appliquant à un modèle de cellules épithéliales mammaires normales primaires humaines, que nous avons isolé des à partir de glandes mammaires. Ces cellules ont été transformées en deux étapes par transduction avec (i) un shARN ciblant TP53, (ii) un oncogène. Nous avons sélectionné 3 oncogènes qui activent des voies de signalisations distinctes CCNE1, HRAS-v12 et WNT1. Nous avons établi un modèle de transformation tumorale en trois étapes, cellules normales, immortalisées et transformées, permettant de suivre les modifications moléculaires associées à chaque étape et de vérifier si l’activation de voies oncogéniques distinctes produisait des profils d’anomalies différents. Les différents modèles ont été analysés par CGH-array, RRBS, transcriptome et miRNA à des temps de culture définis.Nos résultats montrent que l’activation de la voie RAS aboutit à des profils d’anomalies génétiques et de méthylation des CpG radicalement différents de ceux obtenus après surexpression des gènes CCNE1 et WNT1. Ces différences apparaissent très rapidement après transduction des oncogènes alors que les profils des cellules CCNE1 et WNT1 divergent plus tardivement. Enfin, l’inactivation de p53 n’induit pas par elle-même une instabilité élevée, mais produit un contexte de plasticité favorable aux modifications génétiques et épigénétique.Par ailleurs, nous avons noté des différences phénotypiques entre les HMEC RAS (mésenchymateuses) et les HMEC CCNE1 et les HMEC WNT1 (épithéliales). Dans ce travail, je montre que les HMEC shp53 immortalisées présentent une plasticité phénotypique, une partie des cellules entrant en EMT spontanément, l’autre restant épithéliales. J’ai montré que la transduction RAS sélectionnait les cellules ayant effectué une EMT, alors que la transduction de CCNE1 ou WNT1 sélectionnait les cellules épithéliales. J’ai cherché à identifier les déterminants de ces changements phénotypiques et mes résultats suggèrent qu’ils résultent d’une balance entre une signalisation TGFB1/BMP1, qui favorise l’EMT, et BMP4/WNT7 qui favorise la MET. / Breast cancer is a heterogeneous pathology. Based on the differences of mRNA expression, at least five molecular subtypes have been defined. These subtypes show differences in profiles of genomic abnormalities and CpG methylation. These molecular subtypes are thought to originate from different cell lineages in the mammary gland. However, this has not yet been clearly demonstrated. Alternatively, it has been proposed that the activation of different oncogenic pathways could have a significant impact on genetic or epigenetic changes.We wanted to verify this hypothesis by applying it to a normal primary human mammary epithelial cells (HMEC) model, which we isolated from normal mammary explants. These cells were transformed in two step process by sequential transduction of (i) a shRNA targeting TP53, (ii) an oncogene. We selected 3 oncogenes that activate distinct signaling pathways CCNE1, HRAS-v12 and WNT1. Our tumor transformation model was established in three-step, normal, immortalized and transformed cells, allowing us to monitor the molecular changes associated with each step and to verify whether the activation of distinct oncogenic pathways produced different profiles of genetic and epigenetic modifications. The different models were analyzed at defined culture times by CGH-array, RRBS, transcriptome and miRNA. Our results show that genetic abnormalities and CpG methylation profiles are different between cells where the RAS pathway was activated and cells overexpressing WNT1 or CCNE1. These differences appear rapidly after oncogene transduction, whereas the profiles of the CCNE1 and WNT1 cells diverged later. Finally, inactivation of p53 by itself does not induce high instability, but produces a context of plasticity favorable to genetic and epigenetic changes.In addition, we noted phenotypic differences between HMEC RAS (mesenchymal) and HMEC CCNE1 and HMEC WNT1 (epithelial). In this work, I show that the immortalized HMEC shp53 exhibit a phenotypic plasticity, where some cells enter a spontaneous EMT and the others remain epithelial. I showed that RAS transduction selected cells that are undergoing an EMT, whereas transduction with CCNE1 or WNT1 selected the epithelial cells. I have sought to identify the determinants of these phenotypic changes and my results suggest that a balance exists between TGFβ1 / BMP1 signaling, which promotes EMT, and BMP4 / WNT7a which promotes TEM.

Slug est un membre de la famille des protéines Snail impliquées au cours du développement dans le contrôle de la forme et la différenciation cellulaire. Nous avons localisé Slug au cours du développement des glandes mammaires de souris dans les cellules qui participent aux mécanismes de croissance. Seule une sous-population de cellules épithéliales mammaires située dans le compartiment basal de la glande exprime Slug. Cette expression est maintenue pendant les différentes étapes du développement de la glande mammaire, de la puberté jusqu'au début de la gestation. Nous avons observé une perte d'expression dans lobules se différenciant en alvéoles sécrétoires, ensuite une re-expression au stade d' involution. La population exprimant Slug est positive pour la cytokératine 5, décrit comme un marqueur des cellules basales et myoépithéliales, également considéré comme un marqueur de cellules souches ou progénitrices, et elle est incluse dans la population CD24 positive et surexprimant le CD49, connue pour contenir les cellules souches de l'épithélium mammaire. Nous avons constaté que canaux epithéliaux des glandes mammaires de souris Slug knock-out envahissent moins le coussinet adipeux mammaire. En outre, Ils montrent un phénotype de branchements latéraux, ce qui suggère une différenciation précoce. Ce phénotype ressemble le phénotype des glandes mammaires de souris Knock-out pour la P-cadhérine. Nous avons également constaté une diminution de l'expression de la P-cadhérine in vivo dans les glandes mammaires des souris SlugLaZ, et in vitro, dans les cellules épithéliales mammaires transfectées avec un siARN ciblant Slug. Ces cellules montrent un retard de migration cellulaire. Ces observations valident à notre hypothèse que le facteur de transcription Slug contrôle la différenciation des cellules épithéliales au cours de la croissance physiologique de la glande mammaire murine. / Slug is a member of the Snail protein family involved during development in the control of cell shape and differentiation. We located Slug during mammary gland development in mouse in cells participating to the growth mechanisms. Only a distinct sub-population of mammary epithelial cells located in the basal compartment was found to express Slug. This expression is maintained during the various stages of mammary gland development, from puberty until the beginning of gestation. We observed a loss of expression in lobules differentiating into secreting alveoli, followed by re-expression at involution stage. The Slug expressing population was positive for Cytokeratin 5, described as a basal and myoepithelial cell marker, also considered as a stem/progenitor marker, and was included into the (CD24+ CD49++) population, known to contain the mammary epithelial progenitor cells. We found that mammary gland from Slug-deprived mice were slower to invade the mammary fat pad. In addition, they displayed increased lateral branching, suggesting precocious differentiation. This phenotype resembles the phenotype of mammary glands of P-cadherin Knock-out mice. We also found that P-cadherin is down regulated in vivo in SlugLaZ mice mammary gland, and in vitro, in mammary epithelial cells transfected with an siRNA targeting Slug. These cells show a delay in migration. These observations lead to our hypothesis that Slug controls an early epithelial cell differentiation stage during mammary gland physiological growth.

La leptine est une hormone peptidique ayant une action sur de nombreux tissus. Une dérégulation de la sécrétion de cette hormone est observée au cours de l’obésité. L’obésité est fréquemment associée à des troubles de santé dont les principaux sont le diabète de type II, l’hypertension artérielle et les maladies cardiovasculaires. Elle est également un facteur de risque du cancer du sein, particulièrement en post-ménopause favorisant la récidive et augmentant la mortalité. Ces perturbations, associées à un état de stress oxydant défini par un excès de production des espèces réactives de l’oxygène (ERO) par rapport aux systèmes de défense antioxydants, pourraient avoir un impact majeur dans le risque de carcinogenèse chez le sujet obèse. Il est clairement établi aujourd’hui que le statut oxydatif des cellules est directement corrélé aux capacités de prolifération mais aussi de survie des cellules dans leur environnement. A ce jour, très peu de données existent concernant le rôle de la leptine dans la modulation du statut oxydatif des cellules épithéliales mammaires saines et tumorales. L’objectif de cette thèse était d’étudier d'abord les mécanismes d’action et les effets de la leptine sur le statut oxydatif et inflammatoire des cellules épithéliales mammaires saines et néoplasiques ; puis dans un deuxième temps, une étude expérimentale a été conduite pour caractériser in vivo l’impact de l’obésité associée ou non à l’activité physique sur la croissance tumorale et le statut oxydatif et inflammatoire des tumeurs. Le projet avait également pour but de mettre en œuvre une nouvelle technique d’analyse basée sur la détection de fluorescence native induite par excitation laser à 224 nm afin d’évaluer la production de composés bio-actifs de la famille des éicosanoïdes, dont les isoprostanes, impliqués dans le processus inflammatoire. Nous avons exploré in vitro l’impact de la leptine sur le statut oxydatif des cellules épithéliales mammaires. Cette étude nous a permis d’établir que la réponse au signal leptinique varie en fonction du statut néoplasique de la lignée considérée, en fonction du temps de contact et non de la dose testée. Ensuite, nous avons étudié l’impact de l’obésité associée ou non à l’activité physique sur la croissance tumorale et sur le statut oxydatif et inflammatoire des tumeurs à l’aide d’un modèle de souris âgées C57BL/6 nourries avec un régime hyper-lipidique (HL) pendant 14 semaines, et hébergées soit dans un environnement enrichi (EE) pour favoriser l’activité physique et les interactions sociales, soit dans un environnement standard pendant 8 semaines, après quoi des cellules syngéniques de tumeur mammaire EO771 ont été implantées dans les quatrièmes coussinets adipeux mammaires. In vitro, la leptine a stimulé la production de ROS de façon indépendante de la dose et cette augmentation était dépendante de la production d'O2 cytosolique. Les résultats montrent une augmentation significative du poids dans les groupes recevant le régime HL à prise alimentaire journalière identique. La composition corporelle à 8 semaines montre une prise de masse grasse significative sous régime HL, majorée par l’ovariectomie et partiellement limitée par l’activité physique. Après implantation des tumeurs, le régime HL favorise la croissance tumorale et la perte de l’activité locomotrice. Par contre, l’EE prévient la perte d’activité physique des animaux. L’ensemble de ces travaux montre que la leptine contribue à l’apparition d’un stress oxydant en lien avec le statut tumoral des cellules épithéliales mammaires. Ceci peut expliquer en partie l’augmentation du risque de cancer mammaire associée à l’obésité en post-ménopause. Ces résultats permettront d'objectiver le bénéfice d'une intervention nutritionnelle ciblée afin de moduler la réponse des cellules aux stimulations des adipokines. A terme, cette étude doit contribuer à mieux comprendre l’intégration des signaux issus de l’environnement cellulaire. / Obesity is now considered, as a risk factor for developing breast cancer in postmenopausal women and for mortality in response to this pathology. Obesity, which is frequently associated with hyperleptinemia, induces cellular signalling pathways, some of which involving reactive oxygen species (ROS) as intracellular messengers. High levels of ROS contribute to oxidative stress, cellular damages and pathogenesis. Therefore, ROS production associated to obesity could be a major risk factor for mammary carcinogenesis. Furthermore, increased oxidative stress and inflammation characterised by infiltration of immune cells into adipocytes are described. This is associated with a lipid peroxidation and the production of bio-active compounds including isoprostanes.The aim of this study was to determine the impact of leptin in modulating the oxidative and inflammatory status of epithelial mammary cells and in tumor mammary tissue. Moreover, the purpose of this work was to develop a new analysis technique based on native fluorescence detection induced by laser excitation at 224 nm to evaluate the production of bio-active compounds from the family of eicosanoids, involved in the inflammatory process, including isoprostanes.Initially we identified in vitro the leptin effects on ROS production in 3 human epithelial mammary cell models which present different neoplastic status (healthy primary (HMEC) cells, MCF-7 and MDA-MB-231) in presence of two leptin concentrations (10 ng/ml close to physiological values, 100 ng/ml as obesity level). To better understand the potential involvement of adipocyte tumor microenvironment in mammary carcinogenesis, we secondly explored in vivo the impact of high fat diet (HFD) and of enriched environment (EE) on mammary tumor development. Female C57BL/6 mice were fed with a HFD versus a normo-caloric diet (NC) for 14 weeks. After 8 weeks mammary tumor syngeneic cells EO771 were implanted into the fourth mammary fat pads. Before injection, mice were housed in EE or in standard environment (ES) for 8 weeks. In vitro, leptin stimulated ROS production in dose-independent manner and this increase was dependent of cytosolic O2•- production. This ROS production contributed to a different antioxidative response depending of the neoplastic cell status. Leptin induced the antioxidative enzymes expression and activities such as heme-oxygenase or glutathione peroxidase only in HMEC cells. In neoplastic cells, these enzyme activities did not change whatever the leptin concentration used.Thus, high fat diet promoted mammary tumor development associated with a decrease in body fat and an increase in volume and weight of tumors that was not limited by physical activity. This diet induced a decrease of adiponectin and an increase of leptin plasma level compared to NC diet however, leptinemia was not influenced by EE.The native fluorescence isoprostanes determination method, turned out not to be quite sensitive. Therefore, the native fluorescence of these compounds is too low to allow their detection in biological media used. In contrast, the native fluorescence appears to be a potential cellular exploration tool.Through this work, we have shown that leptin contributes to the onset of oxidative stress linked to the status of mammary epithelial tumor cells. This may partly explained the increase of risk of breast cancer recurrence observed in situations of obesity. The results obtained in vivo eventually will support the benefit of a nutrition intervention to modulate cell response to adipokines stimulation. Ultimately, this study contributes to better understand the integration of signals from the cell environment.

Natural killer (NK) cells represent a crucial component of the innate immune system and are primarily regulated by the interactions of their activation and inhibitory receptors with ligands available on target cells. The genetically linked Ly49 and NKR-P1 family of receptors constitute two of the major regulatory receptor systems used by NK cells and have been shown to bind different ligands. Whereas the Ly49 receptors survey MHC-I ligands on target cells, the NKR-Pl receptor family members bind to various members of the C-type lectin-related (Clr) family. Interestingly, NKR-P1 and Clr haplotypes possess a stable genomic polymorphism across multiple mouse strains, suggesting that this inhibitory receptor:ligand relationship has an important role in the maintenance of host cellular cognate specificities. The NKR-P1 and Clr receptor-ligand pairs identified in mice include the NKR-P1B:Clr-b and the NKR-P1G:Clr-f interacting pairs. Previous RT-PCR and in situ RNA hybridization data generated by our laboratory determined that kidney tubular epithelium as well as the small and large intestinal epithelial cells specifically and highly expresses the Clr-f transcripts. Contrarily, the Clr-b transcripts were only detected on hematopoietic cells of various lymphoid organs and kidneys. Moreover, foregoing studies revealed that the loss of Clr-b following viral or chemical induced stress mediates NK cell killing of the target cell, suggesting a tissue-specific immune-surveillance mechanism in parallel with the global MHC-I-dependent missing-self model. However, the role of the NKR-P1B:Clr-b recognition-system have never been examined in the intestine. Additionally, the role of Clr-f in the kidney and intestines, where they are highly expressed, has not been investigated. For these reasons, I aimed in my thesis to provide a better understanding of the functional aspect of the NKR-P1B:Clr-b and NKR-P1G:Clr-f recognition systems in mediating gut mucosal and renal homeostasis, respectively. First, in order to determine the role of NKR-P1B and Clrb receptor:ligand pair as a “missing-self” immunosurveillance system in the gut, I started by identifying the expression pattern of both the receptor and ligand on various intestinal cells. My results demonstrate that NK cells do not represent the major NKR-P1B-expressing cells in the gut lamina propria. Instead, ILC3 subsets constituted the predominant cell population expressing the receptor, whereas γδT cells composed a small fraction of NKR-P1B+ lymphocytes. In addition, the NKR-P1B expression on myeloid cells was exclusive to colon macrophages and DC subsets. Interestingly, the highest percentage of NKR-P1B+ immune cells was found in the gut, which suggests the dominant role of NKR-P1B in regulating immune functions at the level of intestinal mucosa. As expected, the expression of the NKR-P1B ligand, Clr-b, appeared on all innate immune cell types in the gut. Next, using oral infection models of Salmonela typhimurium and Citrobacter rodentium, I showed that NKR-P1B-deficient NK cells, ILC3 and γδ T cells are hyporesponsive compared to their WT counterparts. In particular, gut NKR-P1B-deficient NK cells and γδT cells secreted low levels of IFNγ cytokine while infected with S.typhimurium. Importantly, the decreased IFNγ secretion by NK and γδT cells was associated with an increased dissemination of the bacterium into the knockout spleens at day 5 post-infection. Likewise, I detected a significant decrease in IL-22 cytokine production by NKR-P1B-deficient ILC3 compared to their WT counterparts at both steady state and following C.rodentium infection. Next, I address the potential role of Clr-f in the kidney. Renal tubular epithelial cells have been shown to express high levels of Clr-f transcripts. Epithelial cells constitute the major cellular component of kidney tubules and are well known to mediate metabolic waste excretion, reabsorption of essential molecules as well as other physiological functions, such as ions exchange and water retention. To determine the role of Clr-f in renal epithelial cells, I generated a Clr-f-deficient mouse with the help of two of my previous lab colleagues. Importantly, chemical analysis on urine and serum samples from knockout and WT littermates indicated that Clr-f-deficient kidneys display a decreased filtration capacity. In particular, higher creatinine levels were detected in the Clr-f deficient serum. In addition, Clr-f-deficient mice appeared to have a lower fractional excretion of sodium (FENa) in their urine filtrates in comparison to WT excreted urine. Blood pressure measurements on the same mice at 12 and 24 weeks of age revealed a hypotensive phenotype in the Clr-f-deficient mice. Furthermore, pathological assessment of Clr-f-deficient kidneys exhibited moderate and aggravated lesions of the tubular epithelium along with marked glomerular mesangiolysis. Lastly, flow cytometry analysis on isolated lymphocytes from Clr-f-deficient and WT mice demonstrated comparable immune infiltrates between the two mouse genotypes. Altogether, our data shows that the absence of Clr-f results in the development of glomerular and tubular lesions in an immune-independent manner leading to an abnormal kidney function. Additionally, the disruption of NKR-P1B:Clr-b recognition system results in abnormal innate immune cell number and function in the mouse intestine. These novel findings sheds light on the important role of Clr-f in maintaining healthy kidney morphology and function, as well as the crucial role for NKR-P1B:Clr-b interactions in mediating intestinal homeostasis at steady and infected states.

The photobiomodulation therapy (PBMT) has many demonstrated applications in the health area including anti-inflammatory and wound healing effects. The main objective of this work is to verify if the PBMT causes measurable changes in the mechanical properties of cells, specifically in red blood cells, epithelial cells and fibroblasts. In addition, to contribute to the knowledge of the action mechanisms of the PBMT, this study intends to support applications of the PBMT during invasive procedures, such as the direct photo-treatment of the blood in surgical procedures with cardiopulmonary bypass, regarding security of the cellular integrity. For this analysis, three experimental techniques were used: optical magnetic twisting cytometry (OMTC), defocusing microscopy and confocal laser-scanning microscopy. Human bronchial epithelial cells were evaluated with OMTC. The epithelial cell culture was either photo-treated or not, with red laser (lambda=660 nm), and fixed power and time (power density of 153 mW/cm2, time 300 s). It was not possible to observe significant differences between photo-treated and control epithelial cells, for the hysteresivity (ratio between the cell loss and elastic shear moduli). The defocusing microscopy, similar to a phase contrast microscopy, was used to study human red blood cells from fresh blood. The red blood cells were either photo-treated or not, with red laser (lambda=660 nm), and different powers and times (power densities from 0 to 510 mW/cm2, times from 0 to 180 s). Some morphological and mechanical characteristics of individual red blood cells were evaluated, such as volume, radial profile of cell thickness, lateral and vertical membrane fluctuations, for the photo-treated and control red blood cells. It was not possible to detect differences between the two groups, for any of the parameters analyzed. For both techniques, the absence of detectable differences might be due to several factors, such as the non-action of the PBMT, with the parameters used, in the epithelial cells and red blood cells or to the small sensitivity of each technique. Confocal laser-scanning microscopy was used to evaluate the actin filaments of mouse fibroblasts. The fibroblast cell culture was either photo-treated or not, with red (lambda=625 nm) or infrared (lambda=808 nm) light and fixed power and time (power density from 113 to 158 mW/cm2, time 300 s). The nucleus and cell areas increased slightly when comparing photo-treated and control cells. On the other hand, the total actin, total actin density and the number of filaments decreased. These changes were detected for a short time after treatment, however, after 24 h they are not anymore detectable. The total branch length does not seem to suffer any modifications. In summary, with the data acquired with the three techniques, it was found that the PBMT, in the red range, with the parameters used, could not cause noticeable changes in red blood cells and epithelial cells, in vitro. On the other hand, the PBMT in the red and near-infrared range, with the power and times used, cause changes in actin filaments of fibroblasts, in vitro, in particular the decrease of the total actin density. / A terapia por fotobiomodulação tem muitas aplicações na área de Saúde devido a sua ação anti-inflamatória e de reparação tecidual. O objetivo geral desse trabalho é verificar se a terapia por fotobiomodulação provoca mudanças nas propriedades mecânicas de células, em particular em hemácias, células epiteliais e fibroblastos. Além de contribuir com o conhecimento dos mecanismos de ação da terapia por fotobiomodulação, este estudo pretende subsidiar aplicações da terapia por fotobiomodulação durante procedimentos mais invasivos, como a iluminação direta do sangue em procedimentos cirúrgicos com circulação extracorpórea, sob o ponto de vista da segurança quanto à integridade celular. Para essa análise foram utilizadas três técnicas experimentais: citometria óptica magnética de oscilação (OMTC), microscopia de desfocalização e microscopia confocal. Com a técnica de OMTC foram avaliadas células epiteliais brônquicas humanas em cultura, foto-tratadas com laser vermelho (lambda=660 nm), com potência e tempo fixos (densidade de potência de 153 mW/cm2, tempo 300 s). Não foi possível constatar diferenças significativas entre as células epiteliais foto-tratadas e as células controle, para a histerisividade (razão entre os módulos viscoso e elástico das células). Com a técnica de microscopia de desfocalização, semelhante a uma microscopia de contraste de fase, foram estudadas hemácias humanas de sangue recém coletado. As hemácias foram tratadas com laser vermelho (lambda=660 nm), com potências e tempos variados (densidade de potência de 0 a 510 mW/cm2, tempo de 0 a 180 s). Foram avaliadas algumas características morfológicas e mecânicas das hemácias individualmente, como o volume, perfil radial de espessura, flutuações lateral e vertical da membrana, tanto para hemácias foto-tratadas quanto para hemácias controle. Não foi possível detectar diferenças entre as hemácias foto-tratadas e controle para nenhum dos parâmetros avaliados. Para ambas as técnicas, a falta de mudanças observáveis poderia ser devida a diversos fatores, como a não ação da terapia por fotobiomodulação nas células epiteliais e nas hemácias, com os parâmetros aqui empregados, ou à falta de sensibilidade de cada uma das técnicas usadas. A microscopia confocal foi utilizada para avaliar os filamentos de actina de fibroblastos de camundongo em cultura, os quais foram foto-tratados com luz vermelha (lambda=625 nm) ou infravermelha (lambda=808 nm) e potência e tempo fixos (densidade de potência de 113 a 158 mW/cm2, tempo 300 s). Foi possível constatar ligeiro aumento nas áreas nuclear e celular das células foto-tratadas em relação aos fibroblastos controle. Também foi possível verificar a diminuição da quantidade total de actina, densidade de actina e do número de filamentos de actina nos fibroblastos foto-tratados. Essas mudanças são detectadas para tempos curtos após o tratamento, sendo que depois de 24 h elas desaparecem. O tamanho total dos filamentos parece não sofrer alterações. A partir dos dados coletados com as três técnicas, foi possível constatar que a terapia por fotobiomodulação, com os parâmetros utilizados, não consegue provocar mudanças perceptíveis em hemácias e em células epiteliais, in vitro. Porém, causa mudanças nos filamentos de actina de fibroblastos, in vitro, em particular a diminuição da densidade de actina total.