La cryoconservation du tissu ovarien (CTO) permet aujourd'hui de préserver la fertilité des femmes et jeunes filles devant subir un traitement potentiellement gonadotoxique ou souffrant de pathologie à l'origine d'une insuffisance ovarienne prématurée. En vue d'optimiser les procédures de CTO, l'objectif de notre travail a porté d'une part sur la validation d'un protocole original de congélation lente du tissu ovarien applicable en thérapeutique et d'autre part, sur le développement d'un protocole de vitrification. L'analyse statique du tissu ovarien congelé selon notre protocole de congélation lente montre des résultats similaires en comparaison à ceux obtenus avec le tissu frais en termes de qualité des follicules et de l'endothélium vasculaire ovarien. Néanmoins, le stroma semble plus sensible aux effets délétères de la congélation. L'analyse fonctionnelle montre que le tissu ovarien congelé/décongelé présente une folliculogenèse active après 12 jours de culture in vitro. Les mesures des concentrations en oestradiol dans les milieux de culture et l'étude immunohistochimique du facteur de prolifération PCNA (proliferating cell nuclear antigen) témoignent en effet de l'activité fonctionnelle des follicules décongelés en culture. Dans un second temps, nous avons développé un protocole de vitrification du tissu ovarien. Pour évaluer son efficacité, nous avons réalisé une analyse statique du tissu ovarien vitrifié selon ce protocole versus notre protocole de congélation lente validé. Les résultats obtenus ne montrent aucune différence significative entre les deux méthodes, en termes de préservation de la morphologie et de l'intégrité nucléaire des follicules et du stroma ovarien. En conclusion, notre protocole de congélation lente préserve la qualité des différents compartiments constituant le tissu ovarien et la fonctionnalité de ce tissu. Ces données viennent compléter des travaux préliminaires de l'équipe et permettent de valider ce protocole envisageable maintenant en thérapeutique. La procédure de vitrification développée présente une efficacité similaire à la procédure de congélation lente en termes de préservation de la qualité des follicules et du stroma ovarien. Néanmoins, il serait nécessaire de poursuivre l'étude de l'efficacité de ce protocole par une analyse fonctionnelle. / No abstract available

La crête neurale (CN) dérive de la partie dorsale du tube neural des Vertébrés. Pendant le développement, ces cellules migrent et contribuent à la formation de différents tissus et organes. Le long de l'axe antéro-postérieur, la CN donne naissance aux neurones et cellules gliales du système nerveux périphérique, et aux mélanocytes. Par ailleurs, la CN céphalique est aussi à l’origine de tissus mésenchymateux qui constituent tous les os et cartilages de la face, la plus grande partie du crâne, le derme facial, et les adipocytes et cellules de muscles lisses dans la tête. Dans le tronc des Vertébrés amniotes, ces tissus dérivent du mésoderme. Chez les Vertébrés inférieurs, la CN troncale génère cependant des tissus mésenchymateux, comme les rayons des nageoires du poisson-zèbre. La question qui se pose est de savoir si la capacité de la CN à produire des cellules mésenchymateuses a été totalement perdue dans le tronc au cours de l’évolution, ou bien si elle peut encore se manifester chez les Amniotes dans des conditions spécifiques. Ce travail s’est intéressé à dévoiler le potentiel mésenchymateux de la CN troncale.Notre approche expérimentale a été d'examiner le potentiel de différenciation squelettogénique et adipogénique des cellules de la CN troncale de caille en culture in vitro, par hybridation in situ (HIS), immunocytochimie et RT-PCR. L’ostéogenèse a été initialement caractérisée par l'expression de Runx2, premier facteur de transcription des ostéoprogéniteurs, qui a été détectée par HIS à partir 5 jours de culture. Plus tard, nous avons observé la maturation des ostéoblastes, avec l’expression de la protéine collagen1, des ARNm de l'ostéopontine et de la phosphatase alcaline, jusqu’à l'étape de minéralisation de la matrice osseuse. Les cellules de CN troncale ont effectué également un processus de chondrogenèse, mis en évidence par l'expression des ARNm de Sox9, aggrecan et collagène10, et par la coloration au bleu Alcian. L'observation des zones minéralisées et des régions chondrogéniques suggère que les cellules de la CN troncale in vitro effectuent une ossification de types endochondral et intramembranaire. Dans les mêmes conditions de culture, les cellules se sont aussi différenciées en adipocytes, identifiés à partir de 10 jours de culture par le colorant Oil Red O. Les ARNm des facteurs de transcription CEBP et PPAR, essentiels pour l'adipogenèse, et de la protéine FABP4, ont été détectés par RT-PCR dès 3 jours de culture. Afin de caractériser les précurseurs des cellules osseuses et adipocytaires, nous avons examiné le potentiel de différenciation des cellules individuelles de la CN troncale. L'analyse des types cellulaires dans les cultures clonales a montré que 76% des clones contiennent des ostéoblastes Runx2-positifs. De plus, les cellules de CN troncale comprennent des progéniteurs multipotents dotés à la fois de potentiels neuraux et ostéogénique. Dans une autre condition de culture clonale, les adipocytes ont été trouvés dans la descendance de 35,3% des cellules, et environ la moitié de ces cellules possédaient aussi un potentiel glial et/ou mélanogénique. Ces résultats montrent que, in vitro, les cellules de la CN troncale sont capables de se différencier non seulement dans ses dérivés traditionnels trouvés in vivo (mélanocytes, neurones et cellules gliales), mais aussi dans des phénotypes mésenchymateux, y compris adipocytes et ostéoblastes. Comme dans les cellules de la CN céphalique, ces phénotypes mésenchymateux se différencient à partir de progéniteurs multipotents. Ceci suggère que, au cours de l’évolution, les cellules souches de la CN, dotées de potentiels à la fois mésenchymateux et neuraux, ont eu l'expression de leur potentiel mésenchymateux inhibée dans le tronc. Ainsi, chez les Vertébrés amniotes, même s’il ne se manifeste pas et reste dormant in vivo, un potentiel de différenciation mésenchymateuse est bien présent dans les cellules de la CN troncale et peut être révélé in vitro. / The neural crest (NC) derives from the dorsal borders of the vertebrate neural tube. During development, the NC cells migrate and contribute to the formation of different tissues and organs. Along the anteroposterior axis, the NC gives rise to neurons and glia of the peripheral nervous system and to melanocytes. Furthermore, the cephalic NC yields mesenchymal tissues, which form all facial cartilages and bones, the large part of skull, facial dermis, fat cells and smooth muscle cells in the head. In the trunk of amniotes Vertebrates, these tissues are derived from the mesoderm, not from the NC. In lower Vertebrates, however, the trunk NC generates some mesenchymal tissues, such as in the dorsal fins of zebrafish. The question therefore is raised whether the ability of the NC to produce mesenchymal cells was totally lost in the trunk of amniote Vertebrates during evolution, or if it can still be achieved under specific conditions. This work is interested in uncovering the mesenchymal potential of the avian trunk NC, with special interest in the differentiation into osteoblasts and adipocytes.Our experimental approach was to examine the skeletogenic and adipogenic differentiation potentials of quail trunk NC cells after in vitro culture. Cell differentiation was evidenced by the analysis of lineage-specific genes and markers using in situ hybridization (ISH), immunocytochemistry and RT-PCR. The established culture conditions allowed observation of both skeletogenesis and adipogenesis. Osteogenesis was initially characterized by expression of Runx2, the first transcription factor specific of the osteoprogenitors, which was detected by ISH from 5 days of culture. Later, we observed osteoblast maturation, with the expression of collagen1 protein, osteopontin mRNA and alkaline phosphatase mRNA, until the bone matrix mineralization stage. The trunk NC cells also underwent chondrogenesis, as demonstrated by Sox9, aggrecan and collagen10 mRNA expression, and Alcian blue staining. The observation of the mineralized areas and chondrogenesis suggested that the trunk NC cells in vitro are able to perform endochondral and membranous ossifications. In same culture conditions, the cells differentiated also into adipocytes, identified from 10 days of culture by Oil Red O staining. The mRNAs of the CEBP, PPAR and FABP4 adipogenic markers were detected by RT-PCR from 3 days of culture. For the characterization of bone and adipocyte progenitors, we evaluated the differentiation potential of individual trunk NC cells. The phenotypic analysis of these clonal cultures showed that 76% of the cells generated Runx2-positive osteoblasts. Moreover, most of the clone-forming trunk NC cells were multipotent progenitors endowed with both neural and osteogenic potentials. Furthermore, in another clonal culture condition, adipocytes were found in 35.3% of the clones, and approximately half of them also contained glial and/or melanogenic cells.These results show that the trunk NC cells in vitro are able to differentiate not only in their classical derivatives found in vivo (melanocytes, neurons and glial cells), but also in mesenchymal phenotypes, including adipocytes and osteoblasts. Importantly, as in cephalic NC cells, mesenchymal phenotypes differentiated from multipotent progenitor cells, suggesting that, during evolution, the NC stem cells intended for both mesenchymal and neural fates, had the expression of their mesenchymal potential inhibited in the trunk. Thus, although at the dormant state and not expressed in vivo, a significant mesenchymal potential is present in the trunk NC cells of amniotes Vertebrates and can be disclosed in vitro

It is recognised that the environment to which the fetus is exposed in utero, after implantation, can program longer term health outcomes and alter the possibility of disease onset later in life. It is becoming evident that the environment, to which the pre-implantation embryo is exposed, can also affect the ability of the embryo to form a viable pregnancy as well as altering fetal growth. Despite this understanding, little is known about the mechanism by which the environment can ‘program’ the pre-implantation embryo. Using model stress systems, either ammonium or DMO in the culture medium, this thesis addressed the hypothesis that suboptimal environmental conditions may alter mitochondrial homeostasis and function and/or epigenetic parameters and these are the possible mechanisms responsible for the altered fetal outcomes seen. While common measures of embryo quality such as on time blastocyst development were not affected by either stress, more in-depth investigations found several striking differences. Exposure to DMO significantly decreased blastocyst cell number and allocation to the inner cell mass and trophectoderm, as well as increased blastocyst apoptosis. After exposure to DMO, blastocysts were transferred to pseudopregnant recipients, and both the ability of the embryos to implant and develop into a fetus was impaired as well as fetal weights and crown rump length were significantly reduced indicative of altered growth. Similar results have also been demonstrated after pre-implantation embryos are exposed to ammonium in vitro. Exposure to ammonium during pre-implantation embryo development also altered placental gene expression and function, indicating a possible mechanism of the observed reduced fetal growth parameters. Interestingly, the pre-implantation embryo appears to be the most vulnerable to an environmental stress during the pre-compaction stage, in particular the zygote to 2-cell transition, as exposure to either stress during this stage alone shows similar perturbations to if the stress was present for the entire pre-implantation developmental period. At this early stage of embryo development, mitochondria are the sole energy generators and are therefore critical for embryo function. This study determined that either ammonium or DMO stress exposure, during the first cleavage division, significantly perturbed mitochondrial distribution, membrane potential and ATP/ADP levels. Removal of the stress did not allow these effects to be completely reversed, implicating mitochondrial perturbations as a possible mechanism behind altered embryo programming. During pre-implantation embryo development there are also significant epigenetic changes which are vital for re-programming the embryonic genome. Both in vitro stresses significantly altered DNA de-methylation at the 2-cell stage and reduced blastocyst gene expression levels of DNA methyltransferases (Dnmt3a and Dnmt3b), which are responsible for de novo methylation. Together these data highlight the importance of pre-implantation embryo development as a critical period of growth in which the presence of environmental stress can have an impact on metabolic homeostasis and critical epigenetic events that may be responsible for the downstream effects seen on fetal growth. These results are not only important for assisted reproductive therapy, where the presence of an in vitro laboratory stress can potentially alter embryo programming, but are also important for in vivo embryo development where the health and wellbeing of the mother can also potentially influence the in utero environment and thus the long-term health outcomes of her child. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522143 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2010

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Osteoporosis is a disease of the bone metabolism which is characterised with a decrease of bone substance. The cause of this disease is the imbalance between the creation of a new bone substance by osteoblasts and the resorption of a bone tissue by osteoclasts, in favour of the bone resorption. The risk group of the development of this disease are women after menopause, who naturally register a decline of the estrogen hormone. Estrogen operates as an inhibitor of proosteoclastic factors such as receptor activator of NFκB ligand (RANKL), interleukin (IL)-1, IL-6 or TNF-α. The imbalance of the bone metabolism can also be caused by a disbalance in the production of Prostaglandin E2 (PGE2) and 1α,25-dihydroxyvitamin D3. They are strong mediators which can both stimulate and inhibit an osteoclastogenesis in vitro in concordance with the conditions of the culture/co-culture. This thesis focuses on the examination of an influence of those mediators (PGE2 in the concentration of 10-6 M and 10-8 M; 1α,25-dihydroxyvitamin D3 in the concentration of 10-8 M and 10-9 M) on the osteoclastogenesis from the rat PBMC at the presence of osteoblasts, with or without the combination of proosteoclastic factors macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclastogenesis was stimulated if PGE2 and...

Ce travail de thèse s'inscrit dans un projet régional, initié par le Parc du Lubéron et en collaboration avec le Laboratoire de Pharmacognosie et d'Ethnopharmacologie de l’Université de Marseille. Il avait pour objectif d'étudier la possibilité de domestiquer une plante sauvage, Inula montana L. (Asteraceae) connue dans la pharmacopée provençale pour ses effets anti traumatiques semblables à ceux d'Arnica montana L. et de la proposer comme nouvelle production agricole. Inula montana produit notamment des lactones sesquiterpènes, identifiées comme les métabolites secondaires responsables de ses aptitudes biologiques anti inflammatoires. Il s’est agit dans cette étude1) de déterminer les caractéristiques de croissance et de développement de la plante sauvage largement inconnue et d’identifier en conditions naturelles les facteurs les plus favorables à la production de métabolites secondaires, 2)d’étudier sa capacité à se multiplier végétativement in vitro et à former des cultures cellulaires aptes à synthétiser les molécules d’intérêt 3), de proposer un itinéraire technique applicable agronomiquement et 4) de tester les effets de divers facteurs environnementaux (fertilisation, apport de NaCl, modification du rythme circadien de l’éclairement,rayonnement UVB, ablation de feuilles, application de méthyl jasmonate ) sur la production qualitative et quantitative des lactones sesquiterpènes et des composés phénoliques. Les traits phénologiques de la plante sauvage sont impactés par l’altitude qui induit un retard dans la croissance végétative et la phase reproductrice ainsi que des modifications physiologiques et morphologiques. Les teneurs en métabolites secondaires (certaines lactones sesquiterpènes, les polyphénols totaux et flavonoïdes) varient en fonction de la saison et sont plus importantes dans le site qui présente les conditions climatiques les plus contraignantes du point de vue hydrique (sol drainant,température plus élevée et présence d’une période sèche en été). L’observation microscopique a indiqué la présence de deux types de trichomes : glandulaires (bisériés) et non glandulaires (des poils) qui sont potentiellement les structures porteuses des molécules d’intérêt. I. montana est apte à former des cals in vitro à partir d’explants racinaires, foliaires et caulinaires sur lesquels des pousses feuillées se forment. La domestication d’Inula a été réussie à partir de semences issues des plantes sauvages et en conditions agronomiques, les teneurs en lactones sesquiterpènes (costunolide, artémorine, eldarine et hydrocostunolide) et en composés phénoliques sont généralement plus élevées que chez les plantes sauvages. Les différentes contraintes appliquées pour tester les effets des facteurs environnementaux sur la production des métabolites ont montré : 1) qu’il ne peut pas être établi de corrélation entre la présence d’un stress oxydatif et une augmentation des teneurs en métabolites chez Inula 2) que l’accumulation des lactones et composés phénoliques semblent principalement favorisée lorsque la plante dispose d’un surplus de squelettes carbonés, non utilisés pour la croissance 3) enfin, les deux conditions les plus favorables à l’accumulation des métabolites chez Inula, sont : dans les feuilles, une alternance rapide de lumière et d’obscurité durant la photopériode et dans les capitules, l’application de méthyl jasmonate. Ce travail augure de bonnes perspectives en termes de valorisation d’Inula dans le secteur pharmaco-cosmétologique. Il reste à poursuivre la description du profil phytochimique de la plante et à localiser précisément les organes et/ou sous structures anatomiques concentrant les composés considérés. Ayant démontré que cette plante présente une bonne réponse à la domestication, il est également proposé de poursuivre l’étude des leviers environnementaux susceptibles d’influencer positivement et significativement le profil chimique d’Inula. / This thesis work is part of a regional project, initiated by the Luberon Park and in collaboration with the Laboratory of Pharmacognosy and Ethnopharmacology of the University of Marseille. It aimed to study the possibility of domesticating a wild plant, Inula montana L. (Asteraceae) known in the Provençal pharmacopoeia for its anti-traumatic effects similar to those of Arnica montana L. and to propose it as a new agricultural production. Inula montana produces lactones sesquiterpenes, identified as the secondary metabolites responsible for its biological anti-inflammatory properties. In this study, 1) to determine the growth and development characteristics of the largely unknown wild plant and to identify under natural conditions the most favorable factors for the production of secondary metabolites, 2) to study its characteristics. ability to multiply vegetatively in vitro and to form cell cultures able to synthesize the molecules of interest 3), to propose an agronomically applicable technical itinerary and 4) to test the effects of various environmental factors (fertilization, NaCl supply, modification circadian rhythm of illumination, UVB radiation, ablation of leaves, application of methyl jasmonate) on the qualitative and quantitative production of sesquiterpene lactones and phenolic compounds. The phenological characteristics of the wild plant are impacted by the altitude which induces a delay in the vegetative growth and the reproductive phase as well as physiological and morphological modifications. Levels of secondary metabolites (certain sesquiterpene lactones, total polyphenols and flavonoids) vary according to the season and are more important in the site which has the most water-constraining climatic conditions (draining soil, higher temperature and presence of a dry period in summer). Microscopic observation indicated the presence of two types of trichomes: glandular (biseriate) and non-glandular (hair) which are potentially the carrying structures of the molecules of interest. I. montana is able to form calli in vitro from root, foliar and shoot explants on which leafy shoots are formed. The domestication of Inula has been successful from seed from wild plants and under agronomic conditions, sesquiterpene lactone (costunolide, artemorine, eldarin and hydrocostunolide) and phenolic compounds are generally higher than in wild plants. The different constraints applied to test the effects of environmental factors on the production of metabolites have shown: 1) that there can be no correlation between the presence of oxidative stress and an increase in metabolite levels in Inula 2) that the accumulation of lactones and phenolic compounds seems mainly favored when the plant has a surplus of carbon skeletons, not used for growth; 3) finally, the two most favorable conditions for the accumulation of metabolites in Inula, are: in the leaves, a rapid alternation of light and darkness during the photoperiod and in the flower heads, the application of methyl jasmonate. This work augurs good prospects in terms of valuation of Inula in the pharmaco-cosmetological sector. It remains to continue the description of the phytochemical profile of the plant and to precisely locate the organs and / or anatomical substructures concentrating the compounds in question. Having demonstrated that this plant has a good response to domestication, it is also proposed to continue the study of environmental levers likely to positively and significantly influence the chemical profile of Inula.

This study describes the development of a novel method of three dimensional perfused cell culture for in vitro toxicity testing. Multiple parallel perfused microbioreactors (TissueFlex^TM) were adopted to provide a well-controlled cell culture environment. Alginate and collagen type I, commonly used as hydrogel scaffolds to support cell culture, were tested as the scaffolding materials for this application. Alginate supports cell proliferation, but does not support cell attachment. Collagen gel (type I), good for cell attachment but with poor mechanical strength, could be used at the high concentration of 5mg/ml to prevent the degradation of the gel. Improvement of collagen biomechanical property by a purpose-designed compressor to physically induce cross-linking showed promising results and merits further study. The suitability of alamarBlue® assay, a common non-toxic non-destructive viability assay method, was confirmed for this study and the protocol was optimised. To demonstrate the effectiveness of three dimensional perfused cell culture, human mesenchymal stem cells (MSC) seeded in collagen type I were employed to test the cell inhibition of two antibiotics, trimethoprim and pyrimethamine. The results displayed the perfusion system has greater advantage and sensitivity than the static system, as does these of 3D scaffolds, compared with 2D. Such differences are related to the continuous supply of fresh culture medium to keep cells at a stable pH, temperature, oxygen, and a more physiological like environment. The cytotoxicity of two stereoisomer compounds, obtained confidentially from Pfizer. Ltd., was assessed using the developed method and compared to conventional 2D static and perfused culture by using rat adipose mesenchymal stem cells. The results successfully distinguished toxic and non-toxic compounds and also demonstrated that the 3D perfused system improved the prediction of drug toxicity over 2D culture. 3D perfused bioreactors were applied to hepatotoxicity study using freshly isolated rat hepatocytes. Only algimatrix^TM supported hepatocyte spheroid formation among those tested including collagen type I, alginate beads, poly lactic acid fibres, and Algimatrix^TM. A new variation of TissueFlex^TM bioreactor with micro-patterned surface, designed specifically for hepatocyte self-assembly culture without use of any scaffold, was tested. The results demonstrated that, compared with the standard sandwich culture, the self-assembly culture in the micro-patterned bioreactors showed high cell viability, biomarkers expression, as well as more physiological immunocytochemistry. Moreover, the differential gene expression indicated that self-assembly culture could provide more relevant information regarding metabolising processes than the 2D sandwich culture, which would potentially improve hepatotoxicity prediction. In conclusion, 3D perfused cell culture for in vitro toxicity testing improved the predictivity, reliability and physiological relevance of drug toxicity compared to traditional 2D culture.

Les plantes halophiles sont des plantes résistantes au stress salin, qui subissent une grande variété de stress dans leur environnement naturel. Ces conditions les ont menées à synthétiser des molécules de défense, qui peuvent présenter des activités biologiques intéressantes de par leur structure et diversité. Dans le cadre d’une collaboration avec la PME Salipouss, trois espèces ont été choisies sur la base de tests antioxydants préliminaires, avec pour objectif d’optimiser (i) la multiplication de plants in vitro pour des cultures industrielles en serre et (ii) d’améliorer le niveau d’activité de leurs extraits. La diversité des composés potentiellement actifs présents dans ces extraits est ensuite analysée par fractionnement bioguidé, afin d’isoler des molécules valorisables. Ce fractionnement est appuyé par des analyses de composés par RMN, permettant d’obtenir des informations sur la structure des composés bio-actifs. Les résultats obtenus montrent le fort potentiel de valorisation de ces trois espèces dans l’industrie, et plus particulièrement dans la nutrition et la cosmétique. / Halophytes are salt tolerant or salt-resistant plants which undergo high stress in their natural habitat. As a consequence of environmental stresses, they produce a number of active defense molecules which display interesting biological activities because of their diverse actions or structures. For the present study, three halophytic species were selected from preliminary antioxidant screening. In collaboration with Salipouss SME, objectives of the work were (i) to optimize in vitro halophyte multiplication in order to produce biomass under greenhouse and (ii) to elicit particular metabolic pathways in order to improve extract activities. To attempt to isolate molecules with potentially valuable activities, the variety of compounds from these extracts is reduced by successive fractionations. In addition, NMR analyzes allow to obtain indications on the nature and on the structure of the active compounds. First results highlight the strong activities of the selected halophytes, making them promising candidates for industrial uses, especially in nutrition and cosmetics.

nÃo hà / Macroalgas marinhas constituem um recurso vital para a economia de diversos paÃses e tem sido exploradas ao redor do globo devido a sua capacidade de produÃÃo de fico colÃides como o Ãgar, carragenina e alginatos, bem como pelo seu espectro de utilizaÃÃo, seja na indÃstria de alimentos, de fertilizantes e na medicina. Estudos sobre a induÃÃo, cultivo e reorganizaÃÃo de calos, tem representado um importante papel nas tÃcnicas de cultura de tecidos e suas aplicaÃÃes. Em diferentes tecidos cultivados in vitro, a utilizaÃÃo de reguladores de crescimento à de importÃncia primordial para o estabelecimento da competÃncia e determinaÃÃo, condiÃÃes necessÃrias para a formaÃÃo de calos e regeneraÃÃes. No presente estudo foram testados os efeitos de uma auxina e de uma citocinina, em diferentes concentraÃÃes de Ãgar em meio ASP 12-NTA. O efeito do Ãcido indolacÃtico (AIA) e da 6-benzilaminopurina (BAP) foram testados em explantes de macroalgas de espÃcie Gracilaria birdiae, em separado, com duas concentraÃÃes de 0,5 mg/L e 0,8 mg/L e em conjunto, nas concentraÃÃes de 1:5 mg/L e 5:1 mg/L. Com relaÃÃo a concentraÃÃo de Ãgar, foram testadas duas concentraÃÃes, com 0,5% e 0,8% para todos os tratamentos. Para o processo de esterilizaÃÃo foram aplicados um fungicida (nistatina) e um antibiÃtico (ciprofloxacina), alÃm de iodopovidona e hipoclorito de sÃdio. Todo o experimento foi conduzido em cÃmara de germinaÃÃo, com temperatura de 25  2oC e fotoperÃodo de 16hs de luz e 08hs escuro, salinidade de 30  2â e pH em torno de 7,6, durante aproximadamente 60 dias. Ao final de 50 dias de cultivo, foi observada a formaÃÃo de calos e de processos de regeneraÃÃo indireta dos mesmos. Os resultados foram submetidos a tratamentos estatÃsticos de anÃlise de contigÃncia atravÃs do Quiquadrado (χ2 ), onde foram observadas diferenÃas significativas entre a incidÃncia de calos e regeneraÃÃes e os nÃveis de Ãgar estudados. Ou seja, nos tratamentos onde foram utilizados nÃveis de Ãgar de 0,8%, apresentaram o maior nÃmero de regeneraÃÃes / Seaweed consists in a vital resource to the economy of many countries and it has been explored all over the world thanks to its capacity of producing colloids such as agar, carrageen and sodium alginate as well as the useful aspect either in food industry, fertilizers or medicine area. Studies on induction, cultivation and callus reorganization have been shown an important issue on tissue culture technique and its usage. In different tissue experience in vitro the usage of regulating growth is so fundamental to establish the competence and necessary conditions to callus formation and regeneration. In this present study has been tested the auxin and citocinin effects in different dosages of agar in ASP 12-NTA. The indolacetic acid effect and 6- benzilalaminopurine have been tested as well in seaweed in the species Gracilaria birdiae with two separated doses, 0,5mg/L and 0,8mg/L and together in concentration of 1:5mg/L and 5;1mg/L. Regarding to agar concentration, it has been tested two concentrations with 0,5% and 0,8% in all treatments. As to sterilization process has been done with fungicide (nistatina) and antibiotic (ciprofloxacin) besides iodopovidona and the sodium hypochlorite. The complete experiment had been lead in a germination chamber with 25Â2ÂC and photoperiod of more or less 16 hs of light and 08hs dark and 30,2â saltiness, pH around 7,6 within 60 days approximately. In more or less 50 days of cultivation had been noticed callus formation and regeneration process therein. The results had been underwent by statistic analyze treatments of contingent through (x2) where many meaningful different observations had been checked in callus and regeneration and the agar levels studied, that is to say the agar level treatments in 0,8% presented a major number of regeneration

Since decades, hematopoietic stem cell transplantation (HSCT) has become a well established treatment modality for hematological malignancies and non-malignant disorders. Autologous and allogeneic hematopoietic stem cells (HSCs) mobilized into the peripheral blood (PB) have been used as a preferred source of transplantable stem cells1-3. And umbilical cord blood (UCB) has been introduced as a more attractive HSC source for HSCT, because fetal stem cells in UCB are speculated to be more primitive in comparison to adult stem cells. However the limited amount of HSCs is limiting their application for stem cell therapy in clinic. Therefore, people started to utilize extra-embryonic tissue to harvest more fetal stem cells, while people also tried to optimize the clinical protocol to mobilize more adult stem cells out of adult bone marrow. The innovative strategies and feasible procedures were discussed in this thesis. The axis of the chemokine receptor CXCR4 and its ligand SDF-1 is important for trafficking and homing of HSCs. It has already been demonstrated that the bicyclam AMD3100, a CXCR4 antagonist, in combination with G-CSF is able to induce a significant mobilization of CD34+ cells4. And human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development5. The homing of HSCs to the placenta is probably also mediated by the expression of SDF-1 as demonstrated for the bone marrow niche. In this study (part 1 of the chapter “Results and discussions”), we utilized AMD3100 to mobilize HSCs from placenta. And we can demonstrate that the CXCR4 antagonist AMD3100 mobilise placenta derived CD34+ cells ex utero already after 30 min of incubation and may further enhance the efficacy of harvesting placenta-derived HSC. The alpha4 integrin CD49d is involved in migration and homing of hematopoietic stem cells (HSC). Therapeutic application of natalizumab, an anti-CD49d antibody, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. In our study (part 2 of the chapter “Results and discussions”), we compared circulating HSCs from MS patients after natalizumab treatment and HSCs mobilized by G-CSF in healthy volunteers, with regard to their migratory potential, clonogenicity and gene expression. CD34+ cells in the blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared to that of CD34+ cells mobilized by granulocyte-colony stimulating factor (G-CSF) (median 43.9% vs. 15.1%). This was associated with a more than doubled migration capacity towards a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more m-RNA for p21 and less MMP9 compared to G-CSF mobilised HSC. Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches. In order to further improve the clinical outcome of HSC transplantation, many groups are focusing on ex vivo maintain or expand HSC. Unfortunately, the maintenance of HSC in vitro is difficult to achieve because of their differentiation. This is presumably caused by a lack of appropriate cues that are provided in vivo by the microenvironment. Indeed, HSCs located in the bone marrow are interacting with a specific microenvironment referred to as the stem cell niche, which regulates their fate in terms of quiescence, self-renewal and differentiation. An orchestra of signals mediated by soluble factors and/or cell-to-cell contact keeps the balance and homeostasis of self-renewal, proliferation and differentiation in vivo. To investigate the communication between HSCs and the niche, coculture assays with mesenchymal stromal cells (MSCs) were performed in vitro. Here, we can demonstrate that cell-to-cell contact has a significant impact on hematopoietic stem cells expansion, migratory potential and stemness. In this study (part 3 of the chapter “Results and discussions”), we investigated in more detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex-vivo expansion. And we defined three distinct localizations of HSCs relative to MSC layer: (i) those in supernatant (non-adherent cells); (ii) cells adhering on the surface of mesenchymal stromal cells (phase-bright cells) and (iii) cells beneath the mesenchymal stromal cells (phase-dim cells). Our data suggest that the mesenchymal stromal cell surface is the dominant location where hematopoietic stem cells proliferate, whereas the compartment beneath the mesenchymal stromal cell layer seems to be mimicking the stem cell niche for more immature cells. Our data provide novel insight into the construction and function of three-dimensional HSC–MSC microenvironments. In summary, we provided a new method to isolate fetal stem cells from extra-embryonic tissue (i.e. placenta) in the first part, then we discussed an innovative strategy with CD49d blockade to improve clinical modality for adult stem cell mobilization in the second part, and finally we investigated HSC maintenance and expansion in vitro and provided feasible way to mimic HSC niche in vitro in the last part. This thesis contributes to HSC-based stem cell therapy in two aspects, i.e. 1) fetal and adult stem cell isolation holding great therapeutic potential for blood diseases; 2) ex vivo stem cell manipulation providing a valuable platform to model HSC niche regulation.