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The overall objectives of this thesis were to develop a short-term culture system and to examine the effects of vitrification and short-term culture on the viability of fresh and vitrified bovine ovarian tissue and the follicles within. The first objective was to compare the health and development of preantral follicles in bovine ovarian tissue, as well as the neovascularization of these tissues, subjected to avian chorioallantoic membrane (CAM) culture with the traditional in vitro culture system. We hypothesized that the chorioallantoic membrane (CAM) of the chicken embryo is a 
more suitable culture system than traditional in vitro culture. Bovine ovaries were retrieved from a local abattoir and cortical pieces (1-2mm3) were randomly assigned to one of the following groups; control (fixed immediately), CAM or in in vitro culture. Ovarian tissue fragments from both groups were removed on D1, D3 and D5 of culture, fixed, sectioned (5μm) and stained with H&E. The numbers of healthy and degenerated follicles, primordial and activated preantral (primary and secondary), and the number of infiltrated bovine and avian blood vessels were determined using standard stereological procedures. All grafts placed on the traumatized CAM demonstrated increased neovascularization over time. The healthy primordial follicle density decreased over time concomitant with an increase in degenerated (primordial and activated preantral) follicles in both treatment groups. Healthy activated preantral follicle density did not differ between the two culture systems at a given time. In CAM group, blood vessel density increased over time (p = 0.015). The second objective of this thesis was to develop a suitable vitrification protocol for bovine ovarian tissue. The viability of bovine ovarian tissue vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryotop and cryovial) was assessed. We hypothesized that during vitrification the higher cooling rate on the cryotop (open vitrification method) will yield better post-thaw viability of bovine ovarian tissue as compared to the cryovial (closed vitrification method). We also hypothesized that trehalose is a superior non-permeating cryoprotectant to sucrose for vitrification of bovine ovarian tissue. The ovarian tissue was fragmented (1-2mm3) and divided into 6 different treatment groups. Tissues were vitrified in TCM199 supplemented with 15% EG, 15% DMSO, 20% calf serum and 0.5M sucrose or trehalose then placed in a cryovial or on a cryotop. After warming, the vitrified tissues were either immediately placed in 10% formalin (control) or on the chorioallantoic membrane of a 10-day old chicken embryo for 5 days. Follicles from control and vitrified tissue were observed under a light microscope for normal morphology and the total, normal and degenerated follicle densities were determined by standard stereological procedures. Sucrose and trehalose did not differ, nor was a difference observed between the cryovial and the cryotop for total, healthy or degenerated follicle density. Proportion of healthy follicles was higher in the control than all treatment tissues grafted to the CAM. All grafts placed on the traumatized CAM demonstrated presence of avian erythrocytes in the blood vessels after 5 days, but no difference was observed for blood vessel density among treatments. Lastly, the cooling rate of bovine ovarian tissue subjected to open and closed system devices for vitrification was evaluated. A thermocouple wire was used to determine the cooling velocity of 1-2mm3 fragments of bovine ovarian tissue placed on a cryotop (open system) or in a sealed cryovial (closed system). The cooling rate of tissues on the cryotop and in the cryovial was 7481±205.9° C/min and 664±26.0° C/min respectively. In conclusion, the CAM supported the bovine ovarian tissue, thus the CAM culture system may be considered an acceptable alternative to traditional in vitro culture system for bovine ovarian tissue. Furthermore, angiogenesis may be an additional indication of ovarian tissue health. The hypotheses of our second study were refuted. Results indicated that sucrose and trehalose, and the cryotop and cryovial were equally effective in vitrifying bovine ovarian tissue.

Les Dioscorea cultivées, dont la reproduction sexuée est aléatoire, sont multipliées essentiellement par voie végétative, ce qui entraîne la dissémination dagents pathogènes dans les plantations, provoquant une baisse de rendement et de qualité des récoltes. Dans ces conditions, les besoins en semences sont rarement comblés et les possibilités dextension de la culture restent limitées. En effet, devant une demande quantitative toujours croissante et qualitative de plus en plus restrictive, les techniques classiques encore employées aussi bien pour la multiplication que pour lamélioration de la production des végétaux sont relativement lentes. En revanche, les opportunités offertes par les cultures de tissus peuvent remédier efficacement aux insuffisances et offrir des améliorations irréalisables par les autres méthodes. La multiplication en alternance par bourgeonnement axillaire à partir de nuds pendant 28 semaines et par mise en germination des microtubercules découpés pendant 16 semaines peut remédier à cette situation. Plusieurs facteurs peuvent avoir un impact sur lefficacité de cette approche: la présence ou labsence de régulateurs de croissance, la teneur en saccharose ou en éléments minéraux du milieu de culture. Dans le cadre de cette étude, les tests réalisés ont montré une formation plus précoce du tubercule en présence de polyamines et dacide jasmonique. Si les teneurs en polyamines endogènes et leur métabolisme sont significativement affectés par les polyamines exogènes, les modifications des teneurs en polyamines endogènes, quant à elles, ne peuvent être directement corrélées avec la formation du tubercule. Un retard dans la formation des tubercules lors dune réduction de la teneur en sucre du milieu de culture a aussi été constaté. Ce retard dans nest pas lié à une réduction de losmolarité du milieu de culture, comme nous avons pu le montrer en remplaçant partiellement le saccharose par du sorbitol. La putrescine et ses précurseurs larginine et lornithine favorisent aussi le développement des tubercules, ceux- ci sont plus longs et plus lourds lorsque ces composés sont ajoutés au milieu de culture à faible concentration. Une augmentation de la teneur endogène en putrescine et en auxine a été observée dans ces conditions. Laddition dacide jasmonique a un effet similaire. Une réduction du développement des tubercules est, par contre, observée en présence dune teneur en saccharose réduite. La réduction de la teneur en sucre dans le milieu de tubérisation a aussi un effet négatif sur la germination ultérieure des microtubercules. Pour pouvoir utiliser les microtubercules comme semences, il faut être assuré dun taux de germination élevé et dun stockage possible. Les microtubercules récoltés après 9 mois de culture et transférés sur un nouveau milieu sans régulateur de croissance germent très rapidement. Aucune dormance nest observée. Les microtubercules peuvent aussi être stockés pendant au moins 18 semaines. Les meilleures conditions pour une germination élevée sont une conservation à lobscurité, sous ± 50% dhumidité relative et à 25°C. Une période de dormance secondaire sinstalle une fois le stockage en cours qui varie entre 20 et 28 semaines respectivement pour les microtubercules les plus rapides et les plus lents. Seuls les tubercules de taille supérieure à 350 mm devront être utilisés pour la germination in vitro ou ex vitro.

Açoita-cavalo , Luehea divaricata Martius et Zuccarini, pertaining to the Tiliaceae family, is a forest species that suffered great entropic action in the last decades, fact that contributed a lot to the reduction of the natural populations, becoming necessary the conservation of germ plasma in vitro. It presents slow and irregular, changeable germination between 20% and 75%, and viability of the seeds very unevenness. These characteristics contribute for one reduced frequency of the species in natural forests. As form of vegetative propagation, the micropropagation becomes an alternative for the regeneration of plants that present difficulty of natural reproduction, beyond presenting itself as a conservation way for the species. The work had as objective to establish a protocol of disinfestations for aseptic germination of açoita-cavalo seeds, to determine the more efficient type of explants and way of culture for the establishment in vitro, to verify the influence of the orientation of the explants in the bottle on the development in vitro and to observe the influence of different concentrations of cytokine BAP in the multiplication of nodes segments of açoita-cavalo . The works had been carried through in the Laboratory of Tissue Culture of the Núcleo de Biotecnologia e Melhoramento, Departamento de Fitotecnia, Centro de Ciências Rurais, Universidade Federal de Santa Maria, in Santa Maria, RS. The seeds had been collected and conserved by Fundação Estadual de Pesquisa Agropecuária - Fepagro/Florestas in Santa Maria, RS, in 2004 and 2005, and seedlings gotten in had been used as source of explants for the studies vitro. During the disinfestations experiments it was observed that the immersion of the seeds in hot water is basic for the control (partial) of microorganisms associated to the seeds, as well as promoting a bigger tax of germination. The mercury chloride controlled the fungal contamination partially and total the bacterial contamination, however it demonstrated toxicity to the development of seedling with the yellowish leaf appearance. For the establishment in vitro of açoita-cavalo can be used as many apexes shoot as nodes segments, independent of the way of culture WPM or MS. For the rooting, the half WPM was more efficient, in the two types of node explants, apexes shoot and segments. Aiming at to maximize the açoita-cavalo culture, the half WPM must be used for the establishment, for having reduced cost and explants of the type nodal segment, that are produced in bigger number for seedling. In does not have influence of the orientation of the explants in relation to the bottle in the development of cultures vitro of açoita-cavalo . In the tested concentrations, cytokine BAP inhibited the formation of shoot and leaves. The tested concentrations of BAP had been high and can have been toxic to the development of the node segments of açoita-cavalo . New studies must more be carried through for an ascertainment deepened of the culture in vitro of açoita-cavalo . / Açoita-cavalo, Luehea divaricata Martius et Zuccarini, pertencente à família Tiliaceae, é uma espécie florestal que sofreu grande ação antrópica nas últimas décadas, fato este que contribuiu muito para a redução das populações naturais, tornando necessária a conservação de germoplasma in vitro. Apresenta germinação lenta e irregular, variável entre 20% e 75%, e viabilidade das sementes muito desuniforme. Estas características contribuem para uma reduzida freqüência da espécie em florestas naturais. Como forma de propagação vegetativa, a micropropagação torna-se uma alternativa para a regeneração de plantas que apresentam dificuldade de reprodução natural, além de se apresentar como uma maneira de conservação das espécies. O trabalho teve como objetivos estabelecer um protocolo de desinfestação para germinação asséptica de sementes de açoitacavalo, determinar o tipo de explante e o meio de cultivo mais eficientes para o estabelecimento in vitro, verificar a influência da orientação do explante no frasco sobre o desenvolvimento in vitro e observar a influência de diferentes concentrações da citocinina BAP na multiplicação de segmentos nodais de açoita-cavalo. Os trabalhos foram realizados no Laboratório de Cultura de Tecidos do Núcleo de Biotecnologia e Melhoramento do Departamento de Fitotecnia, Centro de Ciências Rurais da UFSM, em Santa Maria, RS. As sementes foram coletadas e armazenadas pela Fundação Estadual de Pesquisa Agropecuária Fepagro/Florestas em Santa Maria, RS, em 2004 e 2005, e as plântulas obtidas foram utilizadas como fonte de explantes para os estudos in vitro. Durante os experimentos de desinfestação observou-se que a imersão das sementes em água quente é fundamental para o controle (parcial) de microrganismos associados às sementes, bem como promover uma maior taxa de germinação. O cloreto de mercúrio controlou parcialmente a contaminação fúngica e totalmente a contaminação bacteriana, porém demonstrou toxidade ao desenvolvimento das plântulas com o aparecimento de folhas amareladas. Para o estabelecimento in vitro de açoita-cavalo pode-se empregar tanto ápices caulinares como segmentos nodais, independente do meio de cultivo WPM ou MS. Para o enraizamento, o meio WPM foi mais eficiente, nos dois tipos de explantes, ápices caulinares e segmentos nodais. Visando maximizar o cultivo de açoita-cavalo, deve-se utilizar para o estabelecimento o meio WPM, por ter custo reduzido e explantes do tipo segmento nodal, que são produzidos em maior número por plântula. Não há influência da orientação do explante em relação ao frasco no desenvolvimento de culturas in vitro de açoita-cavalo. Nas concentrações testadas, a citocinina BAP inibiu a formação de brotações e folhas. As concentrações de BAP testadas foram altas e podem ter sido tóxicas ao desenvolvimento dos segmentos nodais de açoita-cavalo. Novos estudos devem ser realizados para uma averiguação mais aprofundada do cultivo in vitro de açoita-cavalo.

Background: Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. Results: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of feta bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS. Conclusions: In sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling.:Background; Results; Discussion; Conclusions

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L’ingénierie tissulaire vise à l’élaboration de prothèses biologiques par la régénération ou la culture, in vitro ou in vivo, de tissus ou d’organes. Dans la stratégie de culture in vitro, le développement de nouveaux outils, tels que des bioréacteurs, permettant la culture de cellules ou de tissus sous sollicitations mécaniques spécifiques au tissu est primordial. De plus, l’avancée de cette discipline dans la régénération des tissus nécessite de développer, dès à présent, des méthodes d’évaluation mécanique satisfaisantes permettant de comparer ces néo-tissus aux tissus sains selon des critères de sollicitations physiologiques. En effet, pour parvenir à une bonne évaluation de ces matériaux, il est nécessaire de pouvoir les tester sur des chargements représentatifs des sollicitations physiologiques auxquelles ils sont soumis. Nous avons ainsi, dans un premier temps, conçu et développé un bioréacteur de ligaments permettant la culture de cellules stimulées mécaniquement par des sollicitations cycliques de traction-torsion. Ce bioréacteur a été dimensionné afin de pouvoir obtenir des bio-prothèses de taille comparable aux ligaments et tendons à remplacer (4 à 5 cm de long). Nous avons, dans un deuxième temps, développé un modèle du comportement mécanique global de ces tissus à partir du formalisme thermodynamique développé au sein de notre laboratoire et des observations faites sur des tendons d’Achille de lapin. Ce modèle a pour but d’approfondir la compréhension des structures intervenant de façon prépondérante dans la qualité mécanique de ces tissus ainsi que l’évaluation et l’optimisation des matrices de support et des néo-tissus devant s’y substituer / Tissue Engineering aims to fabricate bio-prostheses by regenerating or culture, in vivo or in vitro, tissues or organs. In the in vitro strategy, developing new tools such as bioréactors which allow the culture of cells or tissues under their specific mechanical solicitations is a huge point. Moreover, the last advances of this discipline in regeneration of tissues require new mechanical model allowing their evaluation and comparison to native tissue under physiological loading. Indeed, in order to obtain a good evaluation of their mechanical quality, it is important to be able to applied mechanical solicitations linked to physiological ones. As a first step, a bioreactor of ligament allowing the culture of cells under mechanical solicitations of cyclic traction-torsion was designed and developed. This bioreactor was sized to potentially obtain a bio-prosthesis comparable to native tissue in term of size (4 to 5 cm long). In a second time, a mechanical model was elaborated based on a thermodynamic formalism developed in our laboratory and the observation made on rabbit Achilles tendons. The goals of this model are to improve our knowledge on the mayor structures involved into the mechanical quality of theses tissues and to evaluate and optimise the scaffolds and neo-tissues of substitution

La conservation au niveau population est extrêmement nécessaire pour limiter la perte de biodiversité au sein d'une espèce ou d'un complexe d'espèces. Ainsi, l'évaluation de la variabilité inter-populationnelle dans le complexe est reconnue comme première étape importante pour bien définir les plans de conservation des espèces menacées. Arenaria grandiflora form un complexe d'espèces herbacées pérennes à courte durée de vie (4 ans en moyenne) menacé dans certains sites de ses zones de distribution en Europe. A ce jour, sa taxonomie n'est pas bien résolue, ce qui entraîne des problèmes potentiels pour mettre en oeuvre une conservation efficace de ce taxon. Une variation inter-populationnelle du complexe d'espèces A. grandiflora est présentée dans cette étude aux niveaux génétiques, cytogénétiques et morphométriques. Quatre méthodes ont été utilisées : des marqueurs microsatellites nucléaires, une approche cytogénétique, la cytométrie en flux, et enfin la morphométrie sur les feuilles. De plus, les études phénotypiques de variation de taux de germination entre stocks de graines ont été développées. Une différenciation significative entre les profils de variations moléculaires, cytogénétiques et phénotypiques a été détectée dans le complexe d'espèces. Deux cytotypes (diploïdes 2n=2x=22 et tétraploïdes 2n = 4x = 44) ont été mis en évidence en utilisant à la fois des méthodes classiques et des méthodes plus récentes (marqueurs microsatellites, nombres chromosomiques et cytométrie de flux). Le complexe d'espèces d'A; grandiflora présente une forte variation de la valeur de l'ADN 2C, la taille du génome varie de 2.11 ± 0.74 pg à 2.70 ± 0.11 pg pour les populations diploïdes et de 4.30 ± 1.51 pg à 5.27 ± 0.14 pg pour les populations de tétraploïdes. En outre, les grains de tétraploïdes germent significativement mieux que les graines des diploïdes. Les feuilles diffèrent considérablement entre les diploïdes (aciculaires et linéaires) et les tétraploïdes (lancéolées). Cette étude peut être considérée comme préliminaire pour une révision taxonomique de ce complexe d'espèces. D'autre part, grâce à l'ensemble des résultats obtenus, il est également possible de revisiter le concept d'unités évolutives significatives (ESUs) dans le complexe d'espèces A. grandiflora et donc de définir les groupes de populations devant faire l'objet de mesures distinctes. Ainsi, il est possible d'évaluer la pertinence de plans déjà entrepris et de proposer de nouveaux plans de restauration efficaces pour l'avenir de ce complexe d'espèces. / Population-level conservation is being extremely required to restrain the biodiversity loss within a species. So, the assessment of the variability within the species complex is being renowned as an important first step to well implement the future conservation settings for threatened species. The species complex of Arenaria grandiflora is a short-lived perennial herbaceous and a threatened taxon in certain of sites of its distribution areas in Europe, with unresolved gentics and taxonomy, which lead to potential problems in the conservation and utilization of the resource. A differenciation among populations of the species complex of A. grandiflora is presented in this study based on the genetic, cytogenetic and phenotypic patterns. Intraspecific ploidy level varaition is an important aspect of numerous species, so, the present study explores this phenomenon within the A. grandiflora species complex in some type of populations (27 natural populations). To infer the intraspecific genetic and cytogenetic patterns of variability among the studied natural populations of the investigated species complex (A. grandiflora), three methods were used : nuclear microsatellite markers, cytogenetic and flow cytometry approaches. Moreover, the phenotypic patterns of variation among both the stock of seeds and the herbarium materials of A. grandiflora were defined. These patterns were detected using three methods of seed germination (in vitro culture, filter papers and potting soil) and morphometric approaches. A significant differentiation among populations' patterns of molecular, cytogenetic and phenotypic variation was detected within the A. grandiflora species complex. Presence of two closely related cytotypes (diploids 2n=2x=22 and tetraploids 2n=4x=44) was detected using both classical and more recent methods (chromosome number count and flow cytometry respectively). The species complex of A. grandiflora exhibits high variation in 2C-DNA value, the genome size ranges from 2.11 ± 0.74 pg to 2.70 ± 0.11 pg for the diploid populations and from 4.30 ± 1.51 pg to 5.27 ± 0.14 pg for the tetraploid populations. Moreover, the seeds of tetraploids germinate well and in high proportion than the seeds of the diploid ones. In addition, both acicular and linear leaves from the diploid populations differ significantly within the diploids and with the lanceolate leaves of the tetraploid ones. New protocol of seed germination for the tetraploids by in vitro culture after scarifying was described for th first time. The affected factors on seed germination percentages were determinated by an explanatory model of six predictors (altitude, longitude, latitude, ploidy levls, both period and condition of seed storage). Consequently, all these findings are fundamental for the determination of the evolutionarily significant units (ESUs) within A. grandiflora species complex and thus the definition of efficient restoration plans in the future. This study would consider as the preliminary signal for necessary revision for the intraspecific taxonomic keys problematic for this species complex.

La brûlure foliaire causée par Alternaria dauci est la maladie foliaire la plus dommageable pour les cultures de carottes, entravant la récolte mécanique. Seuls des cultivars partiellement résistants sont connus et commercialisés, mais leurs niveaux de résistance sont insuffisants. Les mécanismes de la résistance quantitative des plantes aux agents pathogènes sont mal caractérisés. Nous avons choisi d'étudier ces mécanismes dans l'interaction A. dauci-carotte. Auparavant, plusieurs résultats expérimentaux convergents ont montré que la résistance aux toxines fongiques entre en jeu dans cette interaction. Les tests de toxicité effectués avec des suspensions cellulaires de carotte ont révélé une corrélation entre la résistance des carottes à A.dauci et la résistance des cellules de carotte aux exsudats du champignon. Ces résultats nous ont incités à étudier les toxines impliquées dans le pouvoir pathogène d'A. dauci et afin de pouvoir étudier la réponse de la plante à celles –ci. En utilisant les profils HPLC de la phase organique d'exsudats de différentes souches de champignons, nous avons découvert une corrélation entre la production de toxines et l’agressivité de ces souches suggérant que la production de toxines joue un rôle majeur dans l’interaction A. dauci-carotte. Nous avons effectué l'extraction, la purification et la caractérisation de l'une des molécules candidates que nous avons nommé aldaulactone. Nous avons démontré sa toxicité grâce à un nouveau protocole de quantification de cellules mortes et vivantes. Un transcriptome d’A. dauci et une étude de l’expression des gènes en fonction de la production d’aldaulactone ont été utilisées pour étudier sa voie de biosynthèse. / Alternaria leaf blight, caused by the necrotrophic fungus Alternaria dauci, is the most damaging foliar disease of carrots, especially because it hampers leaf-pull harvesting. Only partially – and insufficiently – resistant cultivars exist. In general, partial resistance mechanisms are poorly understood, so we chose to study them in this interaction. Previous results obtained in the lab highlighted a correlation between plant resistance to the fungus and plant cell resistance toward fungal toxins. It was also shown using carrot cell suspensions that fungal exudates’ toxicity was only present in the organic phase. These results led us to better characterize the toxins produced by A. dauci, in order to get a deeper understanding of carrot cell resistance mechanisms toward those toxins. HPLC analysis of the exudates from different fungal strain uncovered a correlation between toxin production and the aggressiveness of the fungal strains, suggesting that toxin production is an important component of said aggressiveness. We extracted, purified and characterize one of these candidates, and named it aldaulactone. Using a new image analysis protocol, we demonstrated the toxicity of Aldaulactone on carrot cell suspensions. Transcriptomic data from Alternaria dauci were used to explore the biosynthesis pathway of Aldaulactone. Candidate Genes were selected and their level of expression compared with aldaulactone production in various A. dauci cultures.