The general aim of this thesis was to optimise the current methodologies of oocyte cryopreservation in order to obtain live offspring from cryopreserved rabbit oocytes. In chapter 1, meiotic spindle configuration, cortical granules (CGs) distribution and oocyte developmental competence were evaluated after cryopreservation with the current slow-freezing and vitrification procedures. The meiotic spindle organisation was dramatically impaired regardless of the method used. Nevertheless, altered CG distribution is more evident in vitrified oocytes than in slow-frozen ones and the developmental rate to blastocyst stage after parthenogenetic activation was only obtained using slow-freezing method. From this chapter it may be concluded that both methodologies equally affect oocyte structure. However, slow-freezing method seems to be the recommended option for this species as a consequence of the sensitivity to high levels of cryoprotectants in this species. The aim of the following two chapters was the optimisation of cryopreservation procedures using different strategies to modify the oocytes in order to make them more cryoresistant. In chapter 2, Taxol and Cytochalasin B were employed to stabilise the cytoskeleton system during vitrification. The effect of these two molecules on the meiotic spindle and chromosome configuration and development to blastocyst stage after parthenogenesis activation were also evaluated. There were no significant differences in the structural configuration between vitrified groups. Regarding cleavage and blastocyst developmental rate, no statistical differences were found between vitrified-non-treated and Taxol-treated oocytes, but no oocytes treated with Cytochalasin B reached this stage. Therefore, structural configuration and blastocyst development were not improved by this pre-treatment. Moreover, Cytochalasin B pre-treatment seems to cause a deleterious effect on developmental ability to blastocyst stage of these oocytes. In chapter 3, oocytes were incubated with cholesterol-loaded methyl-ß- cyclodextrin (CLC) to increase the membrane fluidity and stability and improve their developmental ability after parthenogenetic activation or intracytoplasmic sperm injection (ICSI). Cholesterol incorporation and its presence after cryopreservation were evaluated using confocal microscopy. Results showed that cholesterol was incorporated into the oocyte and remained, albeit in a lesser amount after cryopreservation procedures. However, no improvements on developmental competence were obtained after parthenogenetic activation or intracytoplasmic sperm injection. In the last three chapters of this thesis, the main objective was to develop a reliable technique which would allow us to obtain live offspring from cryopreserved oocytes. For that purpose, in vivo fertilisation using intraoviductal oocyte transfer assisted by laparoscopy was considered a good alternative to bypass the inadequacy of conventional in vitro fertilisation in rabbit. In chapter 4, two recipient models (ovariectomised or oviduct ligated immediately after transfer) were used to compare the ability of fresh oocytes to fertilise in vivo. This first work showed that embryo recovery rates in all transferred groups decreased significantly, but ligated oviduct recipients provided significantly higher results compared to ovariectomised ones. For that reason, in the second experiment the ligated oviduct recipient model was used to generate live births. Results obtained in this chapter suggested that it was possible to obtain offspring from cryopreserved oocytes using this technique, but this kind of animal models compromised the use of the reproductive tract in a high percentage of females. For that reason, chapter 5 was focused on the development of another type of animal model as an alternative. First, the ability of cyanoacrylate tissue adhesive to block the oviducts before the ovulation would take place was evaluated. Then, in vivo fertilisation ability of fresh transferred oocytes after blocking the oviduct with the adhesive was also assessed. Finally, slow frozen oocytes were transferred to generate live birth. Results showed that cyanoacrylate tissue adhesive was effective in blocking the oviduct, as no embryos were recovered in the blocked oviduct six days after artificial insemination (AI). Moreover, this method could fertilise fresh and also slowfrozen oocytes with a higher live birth rate than the previous recipient models. This study showed that successful production of live offspring using slow-frozen oocytes in combination with in vivo fertilisation was possible, which suggested that in vivo environment could help improve the results of oocyte cryopreservation. Thus, this method was employed in the last chapter of this thesis to generate live offspring from vitrified rabbit oocytes for the first time. Results obtained revealed that there were no differences in the rate of birth between vitrified and slow-frozen transferred oocytes. Nevertheless, based on the results with fresh oocytes, further experiments are still needed if the efficiency of cryopreservation procedures are to be improved. / Jiménez Trigos, ME. (2014). Generation of offspring from cryopreserved rabbit (Oryctolagus cuniculus) oocytes [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37977

Les milieux de cryopréservation d'embryons contiennent généralement des produits d'origine animale, qui présentent des inconvénients majeurs : une composition variable et insuffisamment définie, et un risque de transmission d'agents pathogènes. La substitution de ces produits par des composés synthétiques chimiquement définis pourrait contribuer à l’amélioration des procédures de cryopréservation d’embryons, en réduisant la variabilité de composition des milieux, et le risque de contamination des ressources conservées.L’objectif de ce travail était d’évaluer l’effet du remplacement de l’albumine sérique bovine utilisée dans les milieux de congélation lente ou de vitrification d’embryons cunicoles et ovins par un milieu synthétique formulé à base d’acide hyaluronique (STEM ALPHA.Cryo3 – « CRYO3 »). Dans un premier temps, les propriétés thermodynamiques des substituts potentiels ont été évaluées à l’aide de la calorimétrie différentielle à balayage. Parallèlement, nous avons optimisé les différents outils expérimentaux dont nous avions besoin pour cette étude. Nous avons adapté un protocole d’évaluation de l'activité mitochondriale (JC-1) pour complémenter l'évaluation morphologique in vitro des embryons de lapin, et nous avons évalué l’efficacité de différents protocoles de superovulation sur la production d’embryons chez la brebis. Dans un second temps, nous avons procédé à la substitution de l’albumine sérique bovine (BSA) utilisée dans des milieux de congélation lente et de vitrification d’embryons cunicoles et ovins par du CRYO3. Une approche in vitro a été utilisée pour les protocoles de congélation et de vitrification, puis complétée par une approche in vivo pour les protocoles de vitrification.Nos résultats confirment que la BSA peut être efficacement remplacée par le CRYO3 dans des protocoles de cryoconservation d‘embryons de lapin et d’embryons ovins, qu’il s’agisse de congélation lente ou de vitrification / Embryo cryopreservation media usually contain animal-derived products, such as bovine serum albumin (BSA). These products present two major disadvantages: an undefined variable composition and a risk of pathogen transmission. The substitution of animal products of embryo cryopreservation media by synthetical products may improve procedure standardization (by avoiding variability in media composition) and avoid sanitary concerns inherent to animal-derived products.We aimed to evaluate the effect of replacing BSA in rabbit and ovine embryo slow freezing and vitrification media with a synthetic animal products free medium composed of synthetic hyaluronic acid: STEM ALPHA.Cryo3 (« CRYO3 »).During the first part, we evaluated the substitution candidates through a thermodynamic approach, using differential scanning calorimetry. In paralel, we adapted a mitochondrial activity evaluation protocol (JC-1) to rabbit embryo, which allowed us to complement morphological in vitro evaluation, and evaluated ewe superovulation protocols.During the second part, we used a biological approach to evaluate the replacement of BSA with synthetical products (containing CRYO3) in rabbit and ovine embryo slow freezing and vitrification media, using in vitro (slow freezing and vitrification) and in vivo (vitrification) evaluation methods.Our results seem to demonstrate that the chemically defined substitute CRYO3 can successfully replace BSA during rabbit embryo and ovine embryo cryopreservation (slow-freezing and vitrification)

Lors de cette étude, il a été étudié l'influence de l'ajout de microparticules de polymère réticulé (CPM) dans des formulations d'époxy-amine, sur la cinétique, la morphologie et les propriétés thermo-mécaniques des réseaux finaux obtenus. Tout d'abord, un protocole simple, robuste et bien contrôlé a été développé afin d’ obtenir une large gamme de taille de CPM, de Tg et de fonctionnalité amine. Ce protocole de polymérisation par précipitation, basé sur les phénomènes de séparation de phases, a également été appliqué à différentes compositions chimiques et différents monomères époxy hydrosolubles, ceci montrant les grandes possibilités de cette méthode. Une bonne interface entre les CPMs et la matrice a été recherchée en synthétisant les CPMs en excès de groupes amines. La quantification de ces groupes amines réactifs sur les CPMS était d'un grand intérêt et a donc été étudiée en profondeur. Le titrage des amines de surface a été réalisé en mettant au point un nouveau protocole qui a permis la quantification des amines primaires et secondaires sur les CPMs. Il a ensuite été mis en évidence que, bien que ces microparticules réticulées ne soient pas poreuses, des fonctions amines sont disponibles au cœur des particules et peuvent réagir avec d'autres molécules qui sont capables de diffuser dans la CPM. Il a été montré que lorsque les CPM ont été dispersées dans des mélanges d'époxy- amine, la diffusion des monomères dans le cœur de la CPM s'est produite mais différemment selon le procédé de dispersion. En effet, en utilisant le tétrahydrofurane comme solvant pour aider à la dispersion, la diffusion de la DGEBA est amplifiée et modifie les propriétés thermo-mécaniques du réseau final en modifiant le rapport stœchiométrique de la matrice. Le même phénomène a été observé mais moins amplifié lorsque les microparticules sont uniquement dispersées mécaniquement. En dispersant les CPMs dans l'amine qui est l'agent réticulant, on observe l'absorption complète de l'amine au coeur des CPMs, conduisant ainsi à la désorption de celle-ci dans une deuxième étape, permettant de créer le réseau. Ainsi, un comportement très complexe des CPM a été mis en évidence en présence des monomères et/ou solvant : le gonflement et les phénomènes de diffusion qui dépendent d'un certain nombre de paramètres tels que la température, la densité de réticulation des CPM, les paramètres de solubilité, etc. L'intensité du phénomène de diffusion conduit à une variété de comportements lorsque les CPMs sont ajoutées dans une formulation d'époxy-amine tels que: (a) une légère diminution du temps de gélification et l'augmentation de la conversion, (b) la modification de la température de transition vitreuse de la matrice. / Throughout this work, the influence of the addition of cross-linked polymer microparticles (CPMs) in epoxy-amine formulations on the kinetics, morphology and thermo-mechanical properties of the final networks have been investigated. First, an easy, robust and well-controlled protocol was developed to obtain a large range of CPM size, Tg and amine functionality. This protocol based on reaction induced phase separation via precipitation polymerization was also applied to different chemistries and water soluble epoxy pre-polymers showing the large possibilities of this method. The capacity of obtaining a good compatibility between the CPMs and the matrix was ensure by synthesizing the CPMs in excess of amino groups. The study of the remaining reactive amino groups on the CPMS was of great interest and therefore deeply investigated. The titration of the surface amine was performed by developing a new protocol that enabled the quantification of primary and secondary amines on CPMs. It was then highlighted that even though these cross-linked microparticles were not porous, amino groups are available into the core and can react with other molecules that are able to diffuse into the CPM core. It was shown that when CPMs were dispersed into epoxy-amine blends, the diffusion of monomers into the CPM core occurred but differently depending on the dispersion process. Indeed, using tetrahydrofuran as solvent to help for the dispersion increased the diffusion of DGEBA into the CPM core and changed the thermo-mechanical properties of the final network by modifying the stoichiometric ratio of the matrix. Same phenomenon was observed but less amplified when CPMs were mechanically dispersed in DGEBA. Regarding the dispersion of CPMs in the amine cross-linker, IPD, its complete absorption could be observed into the CPMs, leading then to the desorption of IPD to create the network. Thus, a very complex behavior of CPMs was highlighted in presence of monomers or/and solvent: swelling and diffusion phenomena that are dependent on a number of parameters such as temperature, CPM cross-link density, solubility parameters, etc. The intensity of those phenomena leads to a variety of behaviors when CPMs are added into an epoxy-amine formulation: (a) slight decrease of gel times and increase of conversion, (b) modification of glass transition temperature of the matrix.

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.

A vitrificação de ovócitos pode ser de grande auxílio na manutenção genética após a morte e na pesquisa científica preservando ovócitos em diferentes estágios de maturação. Esta técnica é uma biotecnologia pouco utilizada na espécie equina devido às baixas taxas de viabilidade celular após a criopreservação. O objetivo deste estudo foi determinar a expressão de genes ligados a viabilidade celular e apoptose em complexos cúmulus ovócitos (CCOs) antes e após à vitrificação. Com este propósito, folículos entre 5 a 30 mm foram aspirados e 240 CCOs imaturos foram recuperados em abatedouro localizado em São Gabriel, RS, durante a estação reprodutiva. Os CCOs foram divididos em dois grupos: não vitrificados (n=120) e vitrificados (n=120). O protocolo de vitrificação foi realizado de acordo com método comercial EquiPro-VitKit (Minitube-Alemanha). Após vitrificação, CCOs foram mantidos em meio de maturação in vitro durante 6 horas e avaliados imediatamente. Os genes utilizados para análise de viabilidade dos CCOs foram: “bone morphogenetic protein” 15 (BMP 15); bcl-2- “like protein” 4 (BAX); Caspase 3 (CASP 3) e GAPDH (controle). A análise gênica foi realizada através do método de real-time PCR quantitativo (qRT-PCR). Foi observada diferença na expressão de mRNA do gene BAX (0.85 ± 0.08) em CCOs não vitrificados em comparação com vitrificados (2.05 ± 0.47; P = 0.03). Não foi observada diferença na expressão de mRNA do BMP15 (1.55 ± 0.73) em CCOs não vitrificados em comparação com vitrificados (2.84 ± 2.20; P > 0.05). Não foi observada diferença na expressão de mRNA do CASP3 (0.63 ± 0.20) em CCOs não vitrificados em comparação com vitrificados (0.64 ± 0.01; P > 0.05). Não foi observada diferença na expressão de mRNA do CASP3 (0.63 ± 0.20) em CCOs não vitrificados em comparação com vitrificados (0.64 ± 0.01; P > 0.05). Em conclusão, os resultados observados na expressão de BMP15 sugerem a permanência da viabilidade celular em CCOs equinos, porém, BAX sugere a ativação do processo de apoptose nas células expostas ao processo de vitrificação. / Oocyte vitrification is a modern technique that can be used to ensure genetic maintenance after animal death and to improve oocyte cryopreservation at different stages of maturation. Equine oocyte vitrification is a biotechnology rarely used in horses due to the low post-cryopreservation cell viability. The aim of this study was to determine the expression of genes linked to cell viability and apoptosis in equine cumulus-oocyte complexes (COCs) prior and after vitrification. With this purpose, 5 to 30 mm ovarian follicles were aspirated and 240 COCs were collected from a slaughterhouse in Southern Brazil during breeding season. The COCs were divided into two groups: non-vitrified control COCs (CON, n=120) and vitrified oocytes (VIT, n=120). Vitrification protocol was provided according to a commercially available method from EquiPro-VitKit. After vitrification, COCs were in vitro matured for 6 hours and immediately evaluated. Genes product abundance were: Bone morphogenetic protein 15 (Bmp 15); Bcl-2-associated X protein (Bax); Caspase 3 (Casp 3); and GAPDH (control). The gene expression analysis was performed by quantitative real-time PCR (qRT-PCR). Difference was observed in COCs mRNA abundance for Bax gene from non-vitrified (0.85 ± 0.08) compared to vitrified (2.05 ± 0.47; P = 0.03). No difference was observed in the COCs mRNA abundance for Bmp 15 from non-vitrified (1.55 ± 0.73) compared to vitrified (2.84 ± 2.20; P > 0.05). There was no difference in mRNA abundance for Casp 3 in non-vitrified (0.63 ± 0.20) compared to vitrified (0.64 ± 0.01; P > 0.05). In conclusion, results demonstrate that Bmp 15 expression in vitrified COCs indicate cell viability; however, Bax suggest the activation of apoptosis cascade in cells exposed to vitrification process.

Avaliou-se neste estudo o efeito do tratamento com somatotrofina recombinante bovina (bST) na população folicular, na taxa de recuperação e qualidade dos oócitos e na produção in vitro de embriões bubalinos. A hipótese foi de que o bST aumenta o número de folículos recrutados e a qualidade oocitária, melhorando a eficiência da técnica de aspiração folicular em fêmeas bubalinas. Foram utilizadas 10 novilhas bubalinas, sendo que o grupo bST (n=5) recebeu 500mg de bST em intervalos regulares, enquanto o grupo Controle (n=5) não recebeu tratamento adicional. Ambos os grupos foram submetidos a 10 sessões de aspiração duas vezes por semana. Foram quantificados o número total de folículos e o tamanho destes a cada sessão. Os oócitos recuperados foram quantificados e classificados de acordo com a sua qualidade (A, B, C, D, E), sendo os A+B+C considerados de boa qualidade. Os embriões produzidos foram vitrificados, e uma parte destes, transferidos em tempo fixo. Foram colhidas amostras de sangue para mensuração de IGF-I plasmático durante o estudo. O bST aumentou o número de folículos observados (12,2 vs. 8,7; p<0,05) e o número de oócitos recuperados (5,2 vs. 4,1; p=0,07), no entanto não afetou a qualidade destes, bem como a sua capacidade de desenvolvimento (p>0,05). Observou-se efeito significativo (p<0,05) de sessão de aspiração no número de folículos observados, de folículos aspirados e de oócitos recuperados. As concentrações plasmáticas de IGF-I não apresentaram efeito de tratamento (p>0,05), no entanto sofreram efeitos de sessão e também da interação entres sessão e tratamento (p<0,05). Obteve-se taxa de concepção de 10,53 % (2/19) após a inovulação dos embriões, gerando o nascimento de dois bezerros normais. Os resultados são sugestivos de que o tratamento com bST aumenta o número de folículos recrutados por onda de crescimento folicular, demonstrando seu potencial em aumentar a eficiência da técnica de aspiração folicular na espécie bubalina / The aim of this experiment was evaluated the effect of recombinant bovine somatotropin (bST) on follicular population, oocyte recovery rates and quality, and on in vitro buffaloes embryo production. The hypothesis was that bST improves the number of follicles per follicular growth wave and oocyte quality, enhancing the results in buffalos females submitted to ovum pick-up programs. A total of ten heifers were assigned in two experimental groups (bST Group that received 500mg of bST in regular intervals and Control Group that did not received any additional treatment). Both groups were submitted to 10 OPU sessions twice weekly (every 3 or 4 days). The number of follicles and its diameters were recoded in all OPU. The oocytes harvest were counted and classified in five categories (A, B, C, D, E). The A+B+C categories were considered as good quality oocyte. The embryos produced were vitrified and some of these embryos were transferred of fixed time. Blood samples for IGF-I were obtained every once weekly. The bST improved the follicular population (12.2 vs. 8.7; p<0.05) and the number of oocytes per session (5.2 vs. 4.1; p=0.07), however the treatment did not affect the oocyte quality and its in vitro development capacity (p>0.05). A significant effect (p<0.05) of OPU session was observed in follicular population, number of aspirated follicles and number of oocyte recovered. The plasma IGF-I was not affected (p>0.05) by treatment, however presented a significant effect of OPU session and also of the treatment by OPU session interaction (p<0.05). The conception rate was 10.5% (2/19) and two healthy and normal calves were born from transferred embryo. These results indicate the viability of bST treatment to improve the follicular recruitment and its potential application in buffaloes donors submitted to OPU programs

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-14T14:53:53Z No. of bitstreams: 1 Emanoel Cabral de Medeiros.pdf: 910531 bytes, checksum: a49d49dbcc2fa0475cf92eaff69ab023 (MD5) / Made available in DSpace on 2017-02-14T14:53:53Z (GMT). No. of bitstreams: 1 Emanoel Cabral de Medeiros.pdf: 910531 bytes, checksum: a49d49dbcc2fa0475cf92eaff69ab023 (MD5) Previous issue date: 2011-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Opuntia stricta Haw, cactus of great economic importance as forage in northeastern Brazil, was subjected to different in vitro systems and their performance was assessed by physiological and biochemical variables. We established three different cropping systems: temporary immersion system (TIS), with low frequency of immersion (immersion of one minute every 48 hours) and high-frequency TIS (immersion for five minutes every three hours); static culture in semi-solid medium (6.0 g.L-1 agar); static culture in liquid medium. The middle for inducing proliferation in vitro consisted of Murashige and Skoog (1962) salts and vitamins plus 30.0 g.L-1 sucrose and 0.0, 0.25, 0.5 or 1.0 mg.L-1 6-benzilaminpurine (BAP). Cultures were maintained in a growth room with 25 ± 2 º C under cool white light (40 mol.m-2.s-1), with 16 hours photoperiod. The experimental design was completely randomized factorial arrangement with 4 x 4 (cropping systems x BAP concentrations in the middle). After cultivation in vitro shoots of each treatment were acclimatized. Biochemical and biometrics analysis were performed on the material in vitro and biometrics on the acclimatized material. The static culture on solid medium, provided more stability since the multiplication by the acclimatization of the seedlings, while the systems with liquid presented limitations in some stages of micropropagation. The vitrification and the presence of betacyanin can be used as quality indicators physiological of the Elephant Ear Mexicana and its potential for acclimatization. / A Opuntia stricta Haw, cactácea de grande importância econômica como forrageira na região Nordeste do Brasil, foi submetida a distintos sistemas de cultivo in vitro e seu desempenho foi avaliado mediante variáveis fisiológicas e bioquímicas. Foram estabelecidos quatro diferentes sistemas de cultivo: sistema de imersão temporária (SIT), com baixa freqüência de imersões (imersão de um minuto a cada 48 horas); alta freqüência (imersão de cinco minutos a cada três horas); cultivo estático em meio semi-sólido (6,0 g.L-1 de ágar); cultivo estático em meio líquido. Os meios para indução de multiplicação in vitro constaram dos sais e vitaminas de Murashige e Skoog (1962) acrescidos de 30,0 g.L-1 de sacarose e 0,0; 0,25; 0,5 ou 1,0 mg.L-1 de 6 benzilaminopurina (BAP). As culturas foram mantidas em sala de crescimento com 25±2ºC, sob luz branca fria (40 μmol.m-2.s-1), com 16 horas de fotoperíodo. O desenho experimental foi o inteiramente casualizado com arranjo fatorial 4 x 4 (sistemas de cultivo x concentrações de BAP no meio). Após o cultivo in vitro, brotações de cada tratamento foram aclimatizadas. Análises bioquímicas e biométricas foram realizadas no material in vitro e biométricas no material aclimatizado. O cultivo estático em meio sólido, proporcionou mais estabilidade desde a multiplicação até a aclimatização das mudas, enquanto os sistemas com meio líquido apresentaram limitações em alguma das fases da micropropagação. A hiperidricidade e a presença de betacianina podem ser usadas como indicadores da qualidade fisiológica das mudas micropropagadas da Orelha de Elefante Mexicana e do seu potencial para aclimatização.

La cryopréservation par vitrification est une technique qui devrait permettre de rallonger considérablement les durées de conservation des greffons en abaissant leur température de stockage jusqu'à – 196°C. Elle nécessite l'utilisation de solutions cryoprotectrices (contenant des antigels biocompatibles) pour empêcher la cristallisation de glace dans les tissus, ainsi que des vitesses de variation de la température relativement rapides. La calorimétrie différentielle à balayage permet de déterminer les vitesses requises pour la vitrification des solutions cryoprotectrices. L'exemple de l'éthylène glycol est présenté, complétant les informations connues sur ce cryoprotecteur dans la plage des concentrations concernant la vitrification. Au niveau de la procédure de vitrification, la méthode du recuit a permis d'éviter l'apparition de fractures dans des volumes importants de solution cryoprotectrice. Le cas particulier de la solution VM3 (21st Century Medicine) est détaillé. Il permet de montrer que la stabilité de l'état amorphe ainsi que la quantité de glace cristallisée au réchauffement sont influencées par la température et la durée du recuit. Une analyse sur l'influence du type et de la forme du container porte-échantillon par rapport à la qualité du verre fabriqué a été commencée en parallèle, mais les limites du dispositif cryogénique utilisé nous ont poussé à développer un nouveau cryostat automatisé, permettant de faire des études systématiques dans des conditions reproductibles. Ce cryostat, entièrement conçu et construit au CRTBT, représente le prototype de ce que nous souhaitons proposer à des médecins pour les aider dans leur étude clinique de la cryopreservation des systèmes biologiques. Sa précision de mesure et sa facilité d'utilisation nous a pour le moment permis de mettre en évidence différents événements ayant lieu pendant la vitrification et le réchauffement de nos échantillons.

Cryopreservation of adherent cells may be advantageous for cell types that are difficult to preserve in suspension or when it is necessary to preserve characteristics of the adherent cultured cells. Vitrification is a promising procedure for the preservation of adherent cells that prevents ice crystal formation and the resulting dissociation and morphological damage. To successfully vitrify adherent cells, high concentrations of CPA are required which increases the likelihood of osmotic and toxic damage. In this dissertation, we describe a rational design strategy that predicts mathematically optimized CPA addition and removal procedures based on the minimization of a toxicity cost function. These rationally designed procedures rely on the accurate knowledge of cell biophysical parameters. We validate an in situ calcein fluorescence quenching method for the determination of membrane permeability parameters for adherent cells. We also describe the determination of osmotic tolerance limits for adherent cells. We use rational design strategies to determine CPA addition and removal procedures for adherent endothelial cells, neuronal cells, and induced pluripotent stem cells as well as oocytes. Also, we provide experimental support for the feasibility of these methods using adherent endothelial cells. The mathematical methods and experimental procedures outlined in this dissertation are important tools for the design of addition and removal procedures for concentrated CPA solutions. This dissertation is an important step toward successful design and implementation of vitrification strategies for adherent cells and tissues. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Feb. 14, 2013 - Feb. 14, 2015

Resumo: A maioria das indústrias do Pólo de Santa Gertrudes utiliza as rochas sedimentares da Formação Corumbataí como única matéria-prima para a fabricação de placas cerâmicas para revestimento com absorção de água entre 6,0 e 10,0%. Os minerais geralmente encontrados nessas rochas são illita, albita, quartzo, hematita e, em níveis localizados, carbonatos. A produção de placas de baixa porosidade é complicada principalmente devido aos problemas de instabilidade dimensional causados pela deformação piroplástica. A illita, hematita e carbonatos aumentam a susceptibilidade a esse fenômeno. Este trabalho estudou minuciosamente as características de alguns litotipos da formação e analisou detalhadamente a influência da adição de outras matérias-primas sobre um litotipo rico em feldspato. A adição de diabásio, alumina e feldspato potássico não aumenta a estabilidade dimensional. A adição de caulim reduz a susceptibilidade à deformação piroplástica. A composição formada pelo litotipo feldspático, caulim, feldspato e quartzo mostrou-se mais estável dimensionalmente, porém as peças dessa mistura ficaram fracas. A massa composta pelo litotipo feldspático, diabásio e caulim é menos suscetível à deformação piroplástica. A influência da granulação e da compacidade foi testada em algumas amostras. Massas com distribuição granulométrica próximas às praticadas no Pólo são mais suscetíveis à deformação piroplástica. E uma maior compacidade não necessariamente implica em uma maior estabilidade dimensional. / Abstract: The majority of Santa Gertrudes Pole's factories use the sedimentary rocks from Corumbataí Formation as the unique raw material for the manufacture of ceramic tiles with the water absorption between 6,0 and 10,0%. The formation generaily is composed by ulite, albite, quartz, hematite and, in specific beds, by carbonates. The production of iow porosity tiles is complicated mainly due to the larger occurrence of dimensional instability problems occasioned by pyroplastic deformation. ulite, hematite and carbonates increase susceptibility to these phenomena. This work studied the characteristics of some formation iithotypes and anaiyzed the influence of other raw materiais over a feldspar rich uithotype. The addition of diabase, alumina and feldspar-K doesn't increase dimensional stability. Kaolin's addition decreases the susceptibility to pyropiastic deformation. The mass composed of feidspar rich lithotype, kaolin, feldspar and quartz generated more dimensional stability, but the tiies were weaker. The blending composed of feldspar rich lithotype, diabase and kaolin is less susceptible to pyroplastic deformation. The influence of particle size distribution and pressing density was experimented in some samples. Blendings with particle size distribution next to those practiced in the Pole are more susceptibie to pyropiastic deformation. A larger pressing density not necessarily implies a greater dimensional stability. / Orientador: Antenor Zanardo / Coorientador: Ana Paula Margarido Menegazzo / Banca: Anselmo Ortega Boschi / Banca: Márcio Raymundo Morelli / Banca: José Francisco Marciano Motta / Banca: Maria Margarita Torres Moreno / Doutor