Células tronco hematopoéticas (CTH) de sangue de cordão umbilical (SCU) possuem grande potencial em terapia celular. Mesmo sendo bem caracterizadas quanto às suas propriedades funcionais e fenotípicas, a regulação da auto-renovação de CTH e os genes envolvidos são pouco conhecidos. Investigou-se através da curva de crescimento, ensaio clonogênico e citometria de fluxo, a expansão e diferenciação de CTH cultivadas sem e com suplementação dos mitógenos estradiol e LiCl. A expressão da subunidade da telomerase teve aumento significativo em todas as condições, bem como a expressão de Nanog e Oct4 relacionados a pluripotência e auto-renovação. Observou-se também a expressão de Nanog, Oct4, Sox2 e FoxD3 em células CD133, células CD3 de sangue periférico e células de colônias hematopoéticas. Concluiu-se que o meio sem suplementação já é suficiente na expansão de CTH, mantendo suas características, relacionadas à proliferação, auto-renovação e pluripotência celular. / Hematopoietic stem cells (HSC) from umbilical cord blood (UCB) have a great potencial for hematopoietic reconstitution. Although these stem cells have been well characterized by their functional and fenotipics properties, self-renewal regulation and genes involved are still unknown. Analyses of cell growth, clonogenic assay and flow cytometry revealed the expansion and differentiation of HSC grown in medium with or without suplementation of the mitogens estradiol and LiCl. Expression of the subunit of telomerase increased in all treatments. As well as the expression of Nanog and Oct4, related to plutipotency and self-renewal. Nanog, Oct4, Sox2 and FoxD3 expression was also high in CD133 cells, in CD3 cells from peripherical blood and in clonogenic assay derived cells. Conclusion: medium without the suplementation is sufficient for expansion of HSC, keeping their characteristcs, realted to proliferation, self-renewal and pluripotency.

La ménopause est un facteur de risques associé au développement des maladies métaboliques. Du fait de ce déficit hormonal, les femmes ménopausées pourraient présenter des risques particuliers une fois exposées à des agents chimiques capables de perturber le système endocrinien. Pour en savoir plus, nous avons mis au point un modèle de souris ovariectomisées supplémentées ou non en œstradiol (E2), et exposées de manière chronique à un mélange de polluants (TCDD, PCB153, DEHP et BPA) ajoutés à une alimentation obésogène. Chaque polluant est utilisé à une dose équivalente à la Dose Journalière Tolérable, et l’exposition recouvre différents stades de développement, incluant ou non la période maternelle (gestation + lactation). Nos résultats indiquent qu’en condition d’obésité induite par le régime, l’ovariectomie accentue la prise de poids, l’intolérance au glucose et l’insulino-résistance chez les souris femelles adultes. Ce phénotype est associé à une diminution de la signalisation œstrogénique dans le foie et une dérégulation de l’adipogenèse et de l’inflammation dans le tissu adipeux. De manière intéressante, le mélange de polluants réduit l’impact délétère de l’ovariectomie sur le phénotype métabolique, possiblement par une amélioration de la signalisation œstrogénique dans le foie et le tissu adipeux. Nos résultats suggèrent fortement que ces effets dépendent d’une exposition maternelle. En conclusion, nous montrons que notre mélange de polluants possède une activité œstrogéno-mimétique. Ceci pourrait avoir une implication dans la compréhension du rôle des polluants environnementaux dans le développement des maladies métaboliques chez la femme en transition ménopausique / Menopause is a risk factor associated with the development of metabolic diseases. Because of estrogen deficiency, postmenopausal women may be at particular risk when exposed to chemical agents that can disrupt the endocrine system. To get more insight, we have developed a model of ovariectomized mice supplemented or not with estradiol (E2), and chronically exposed to a mixture of pollutants (TCDD, PCB153, DEHP and BPA) added to an obesogenic diet. Each pollutant was used at a dose close to the Tolerable Daily Intake dose, and the exposure had covered different stages of development, including or not the maternal period (gestation + lactation). Our results indicate that in diet-induced obesity, ovariectomy triggered weight gain, increase glucose intolerance and insulin resistance. This phenotype was associated with decreased estrogenic signaling in liver and deregulation of adipogenesis and inflammation in adipose tissues. Interestingly, the mixture of pollutants reduced the deleterious impact of ovariectomy on the metabolic phenotype, possibly through an alleviation of estrogenic signaling in both liver and adipose tissues. These data were not reproduced in mice not exposed to pollutants during the maternal life. In conclusion, the mixture of pollutants displayed some estrogeno-mimetic activity. This could have implications in understanding the role of environmental pollutants in the development of metabolic diseases in women in menopausal transition.

It is known that some psychiatric disorders may deteriorate in relation to the menstrual cycle. However, in some conditions, such as premenstrual dysphoric disorder (PMDD), symptomatology is triggered mainly by the variations in ovarian steroid hormones. Although symptoms induced by fluctuations in ovarian steroids often are affective, little is known about how emotion processing in women is influenced by variations, or actual levels, of ovarian steroid hormones. The general aim of this thesis was to evaluate menstrual cycle effects on reactivity in emotion generating and controlling areas in the corticolimbic system to emotional stimulation and anticipation, in healthy controls and women with PMDD. A second aim was to evaluate corticolimbic reactivity during long-term administration of exogenous ovarian steroids. In study I, III and IV effects of the menstrual cycle on emotional reactivity in women with PMDD was studied. In study I, women with PMDD in displayed higher amygdala reactivity than healthy controls to emotional faces, not in the luteal phase as was hypothesised, but in the follicular phase. No difference between menstrual cycle phases was obtained in women with PMDD, while healthy controls had an increased reactivity in the luteal phase. The results of study I was further elaborated in study III, where women with PMDD were observed to have an increased anticipatory reactivity to negative emotional stimuli. However, no differences in amygdala reactivity to emotional stimuli were obtained across the menstrual cycle. Finally, in study IV the hypothesis that amygdala reactivity increase in the luteal phase in women with PMDD is linked to social stimuli rather than generally arousing stimuli was suggested, tested and supported. In study II, re-exposure to COC induced mood symptoms de novo in women with a previous history of COC-induced adverse mood. Women treated with COC reported increased levels of mood symptoms both as compared to before treatment, and as compared to the placebo group. There was a relatively strong correlation between depressive scores before and during treatment. The effects of repeated COC administration on subjective measures and brain function were however dissociated with increased aversive experiences accompanied by reduced reactivity in the insular cortex.

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Les cellules dérivées de la moelle osseuse, principalement les cellules endothéliales progénitrices, sont réduites chez les patients souffrant de maladies cardiovasculaires. Leur mobilisation et leur incorporation aux sites de lésion vasculaire sont des évènements prépondérants dans l’accélération des processus de réendothélialisation. Dans un modèle murin, le 17β-estradiol favorise les processus de guérison vasculaire par la mobilisation et le recrutement des cellules endothéliales progénitrices dérivées de la moelle osseuse. Il existe présentement plusieurs stratégies afin d’augmenter la mobilisation des cellules progénitrices ainsi que leur incorporation à la paroi vasculaire. Cependant, peu d’études privilégient la livraison locale d’un nombre élevé de cellules progénitrices fonctionnelles par un véhicule biodégradable et leur maintien au site de lésion afin de favoriser la réendothélialisation ciblée. Un polymère d’intérêt pour cette application s’avère être le chitosan. Ce biopolymère non toxique et biodégradable est couramment utilisé dans l’ingénierie tissulaire et, depuis peu, est utilisé dans la guérison vasculaire. Le chitosan complexé à la phosphorylcholine voit sa solubilité s’accroître dans les solutions aqueuses ainsi que sa biocompatibilité cellulaire en condition physiologique. Le projet de ce mémoire visait donc : 1) à étudier in vitro, la capacité d’un polymère de chitosan complexé à la phosphorylcholine à influencer l’adhésion, la survie, la différenciation et la fonctionnalité cellulaire dans un modèle murin de culture mixte de cellules dérivées de la moelle osseuse et 2) de déterminer l’impact de la présence du 17β-estradiol sur ces mêmes comportements cellulaires. Nos travaux démontrent que la matrice de chitosan-phosphorylcholine s’avère compatible avec notre modèle de culture cellulaire. En effet, ce polymère est capable de promouvoir l’organisation et le développement des cellules dérivées de la moelle osseuse de façon comparable à la matrice normalement utilisée dans la croissance in vitro des cellules endothéliales progénitrices, la fibronectine. De plus, ce polymère n’a nullement compromis l’activité migratoire des cellules, laissant supposer qu’il pourrait éventuellement être un véhicule approprié pour effectuer une livraison cellulaire à un site de lésion. Il s’avère que le 17β-estradiol, lorsqu’ajouté au milieu de culture ou complexé au polymère de chitosan phosphorylcholine, est capable de moduler le comportement cellulaire, et ce, de façon différente. Le 17β-estradiol complexé au polymère de chitosan-phosphorylcholine démontre, par rapport à sa forme soluble, une plus grande aptitude à accroître le nombre de cellules hématopoïétiques ainsi que des cellules endothéliales progénitrices dérivées de la moelle osseuse in vitro. De plus, le 17β-estradiol complexé au polymère de chitosan-phosphorylcholine permet une amplification marquée des cellules endothéliales progénitrices et leur offre un support adéquat afin de favoriser la guérison vasculaire. L’ensemble de nos travaux suggère que le polymère de chitosan complexé à la phosphorylcholine en présence ou non de 17β-estradiol est une matrice compatible avec les cellules progénitrices dérivées de la moelle osseuse in vitro. Le 17β-estradiol complexé au polymère est toutefois plus efficace que sa forme soluble à promouvoir l’amplification du nombre de cellules progénitrices. Ce polymère représente un outil thérapeutique attrayant et une matrice de livraison d’agent bioactif prometteuse pour le recrutement cellulaire dans l’accélération de la guérison vasculaire. / Bone marrow derived cells, including endothelial progenitor cells, are reduced in numbers in patient with cardiovascular disease or risk factors. Mobilization and incorporation of these cells at the vascular lesion site are important events in the reendothelialization process. 17β-estradiol was shown in a mouse model of injury, to favour this healing process through mechanisms which involve the mobilization and incorporation of endothelial progenitor cells derived from the bone marrow. At the moment, there are many strategies to increase endothelial progenitor cells mobilization as well as recruitment into the vascular wall. However, few studies favour local delivery of a large number of functional progenitor cells on a biodegradable scaffold and to maintain them at the lesion site in order to promote reendothelialization. An interesting biopolymer for this application is chitosan. This non toxic and biodegradable biopolymer is commonly used in tissue engineering and was recently used in vascular healing. Phosphorylcholine modified chitosan can increase the water solubility and cell biocompatibility of the biopolymer in physiological condition. This master project was thus designed to :1) evaluate, in vitro, the capacity of phosphorylcholine modified chitosan to influence cell adhesion, survival, differentiation and functionality in a mouse model of bone marrow mixed culture and 2) determine the impact of 17β-estradiol on these cell behaviours. Our results suggest an adequate biocompatibility of phosphorylcholine modified chitosan with our cell culture system. Indeed, this polymer was able to promote cell organization and development of bone marrow derived cells in the same way that fibronectin, the most commonly matrix used in the progenitor cells in vitro culture. Moreover, cell migratory activity was not compromised by the chitosan polymer. It appears that 17β-estradiol, when added to cell culture media or attached on phosphorylcholine modified chitosan is able to modulate differently cell behaviour. Our data suggest that 17β-estradiol coupled to the chitosan polymer was superior to increase the number of haematopoietic and endothelial progenitor cells derived from bone marrow in vitro compared to the soluble form. 17β-estradiol coupled to the polymer of phosphorylcholine modified chitosan allowed an increased amplification of progenitor cell number and provided adequate scaffold to favour vascular healing. We propose that phosphorylcholine modified chitosan in presence or not of 17β-estradiol is a compatible matrix with bone marrow derived progenitor cells in vitro. 17β-estradiol enhances the amplification of progenitor cell in vitro when associated to the polymer compared to its soluble form. This biopolymer may be an attractive matrix and a promising vehicle in a drug delivery therapeutic system for progenitor cells recruitment and to promote vascular healing.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains x, 160 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Hibiscus tiliaceus L. (Malvaceae) é usado popularmente em desordens do pós-parto. O extrato metanólico de flores de H. tiliaceus apresentou atividade antidepressiva-like nos testes de nado forçado e de suspensão da cauda, ambos são amplamente usados como modelos animais preditivos da atividade antidepressiva. Além disso, o extrato demonstrou uma tendência de aumento do tempo gasto nos braços abertos no labirinto em cruz elevado. Considerando que o extrato de H. tiliaceus, que contém fitoesteróis como o estigmasterol, o stigmastadienol e o stigmastadienone, pode ser útil no tratamento ou prevenção da depressão pós-parto relacionadas à retirada crônica de altos níveis de hormônios associados à gestação. Nosso objetivo foi avaliar o efeito de extratos de H. tiliaceus em modelo animal de depressão pós-parto e ansiedade. Utilizamos ratas Wistar fêmeas submetidas ao modelo de depressão pós-parto induzido por hormônios (estradiol e progesterona), administrados por via subcutânea. Após período gestacional induzido, os animais receberam água, veiculo ou extrato de H. tiliaceus nas doses de 100 e 400mg/kg, via oral (gavagem). No período pós-parto, as ratas foram submetidas a testes comportamentais de nado forçado, o labirinto em cruz elevado e de preferência claro-escuro como modelos preditivo de depressão e ansiedade. Foi obsevado um aumento no tempo de imobilidade associado a uma diminuição na tentativa de fuga, no teste do nado forçado, em animais submetidos ao modelo de depressão pós-parto comparado ao grupo controle, sugerindo que este modelo é capaz reproduzir sintomas de depressão pós-parto. Não houve diferença significativa nos testes de ansiedade entre os animais submetidos ao modelo de depressão pós-parto comparado aos controles. A administração de extrato metanólico de H. tiliaceus não foi capaz de alterar o comportamento relacionado à depressão e ansiedade em ratas Wistar. / Postpartum affective disorders are rarely modeled. The depressive-like behavior of hormone withdrawal following hormone-simulated "pregnancy" was described in Long-Evans and Sprague Dawley rats. Our aim was to evaluate the validity of hormone withdrawal following hormonesimulated "pregnancy" method in Wistar rats as a model of depression and/or anxiety. Recently, it was demonstrated an antidepressant-like profile of methanol extract of Hibiscus tiliaceus L., a plant used in postpartum disorders, in adult male Swiss albino mice, then, we also investigated the antidepressant and anxiolytic-like activities of the methanol extract of H. tiliaceus flowers using this animal model of postpartum disorder. Ovariectomized rats received daily injections of the vehicle or hormones (estradiol and progesterone) to simulate the 23-day gestational period in the rat. Days 24-27 were considered the ''post-partum'' period, where the methanolic extract of H. tiliaceus or vehicle were administered by gavage. Rats were submitted to forced swimming, elevated plus-maze test and lightdark box tests. Rats submitted to ''post-partum depression model'' increased the immobility time in forced swimming. The methanolic extract of H. tiliaceus administration did not alter the immobility time in the forced swim test. In the light-dark box test, rats submitted to post-partum depression model showed decreases in number of rearing in dark compartment. In conclusion, our data indicate that Wistar rats may be an adequate model postpartum affective disorders, showing ''depressive-like'' symptoms in the forced swim test without any anxiogenic effect. Besides, we could suggest that decrease on number of rearing in the dark compartment may indicate the motivational state.