Orientador: Ana Maria Aparecida Guaraldo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T21:54:16Z (GMT). No. of bitstreams: 1 Britto_MariaHelenaSeabraSoaresde_D.pdf: 1915996 bytes, checksum: 263b368c6fc66874e6b4cc046dda0817 (MD5) Previous issue date: 2007 / Resumo: O gênero Cryptosporidium compreende atualmente 16 espécies, que já foram encontradas parasitando mais de 150 espécies animais, incluindo o homem, no qual já foram referidas infecções causadas por sete delas. O Cryptosporidium parvum é o mais estudado porque é responsabilizado pelo caráter zoonótico da infecção. A espécie responsável pelo caráter antroponótico da criptosporidiose é o Cryptosporidium hominis. Embora já tenha sido descoberto há cem anos e mesmo com o avanço da genômica e biologia molecular, ainda existem muitas dúvidas sobre sua taxonomia, biologia e relação com seus variados hospedeiros. Dados na literatura apontam para a necessidade urgente da definição de modelos experimentais para cultivo de linhagens do parasito ¿in vivo¿ e ¿in vitro¿. As pesquisas ¿in vivo¿ esbarram na dificuldade da utilização de grande número de animais certificados do ponto de vista genético e sanitário e mantidos em ambientes controlados. Por outro lado, os métodos de cultivo ¿in vitro¿ têm-se mostrado de difícil reprodutibilidade, pois a grande variabilidade de linhagens celulares, com diferentes origens, cultivadas em diferentes meios, em condições ambientais diversas, tornam bastante controversos os resultados obtidos nessa área. Neste trabalho apresentamos o cultivo de uma cepa de origem humana de Cryptosporidium sp, de um único paciente portador de HIV/aids, com criptosporidiose, denominada de MMC/Uni, mantida pela infecção por tubagem intra-gástrica de 105 oocistos em camundongos neonatos livres de patógenos específicos (SPF) de linhagens, heterogênicas e isogênicas imunocompetentes e imunodeficientes , mantidos durante todo o experimento em isolador de PVC flexível. Oocistos da cepa MMC/Uni de Cryptosporidium sp foram purificados mediante gradiente de sacarose. Após excistação, foram cultivados em monocamadas da linhagem celular MDBK (Madin-Darby Bovine Kidney). A presença de merontes foi constatada após 90 horas de cultivo. A cepa MMC/Uni de Cryptosporidium sp, de origem humana, permaneceu infectante após 16 meses, sob refrigeração a 4°C, em solução de Dicromato de Potássio a 2,5% e manteve a infectividade de camundongos após 10 meses de congelamento em DMSO a - 70ºC / Abstract: Cryptosporidium covers actually 16 species, that have been found infecting more then 150 animal species, including humans, in wich have already been reported infections caused by seven of them. Cryptosporidium parvum is the most studied, due to be responsible by the zoonotic character of the infection. The specie responsible by the anthroponotic character of the criptosporidiosis is Cryptosporidium hominis. Although it has been discovered a hundred years ago, and even with the advances in genomics and molecular biology, still persists many doubts about its taxonomy, biology and the relations with its many different hosts. Data in literature point toward the urgent need in the definition of experimental models to cultivate strains of the parasite ¿in vivo¿ and ¿in vitro¿. The research ¿in vivo¿ faces the difficulty in the use of a great number of certificated animals, in the point of genetic and sanitary view, kept in controlled environment. In the other hand, ¿in vitro¿ culture methods have shown to be of difficult reproductibility, for the great variability of the cellular strains, with different origins, kept in different breeding media, in diverse environmental conditions, turn out of great controversy the results found in this area. Here we present a strain of Cryptosporidium sp, from human origin, taken from an unique HIV/aids patient with criptosporidiosis, named as MMC/Uni, kept by a intragastric tubage infection of 105 oocysts in Specific Pathogen-Free (SPF) neonates from outbred and inbred immunocompetent or immunodeficient mice strains, kept during the entire experiment in an isolator of flexible PVC. Oocists of MMC/Uni strain of Cryptosporidium sp were purified by sucrose gradient. After excistation, they were cultivated in MDBK (Madin-Darby Bovine Kidney) cells monolayers. The presence of meronts was observed after 90 hours of cultivation. The strain MMC/Uni of Cryptosporidium sp, from human origin, remained infectant after 16 months, under refrigeration at 4°C, in a 2.5% solution of Potassium Dichromate, and maintened infectiveness in mouse, after 10 months of freezing in DMSO at -70ºC / Doutorado / Doutor em Parasitologia

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Dominik Jandura Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: In vitro cultivation of tapeworm Hymenolepis diminuta - 2 Aim of this diploma thesis was to obtain cycticercoids of the rat tapeworm (Hymenolepis diminuta), excyst them and find out the conditions for the maximal in vitro incubation period. As the intermediate host mealworm beetle (Tenebrio molitor) infected by the rat feces containing tapeworm eggs was used. Excystment was done using L-cystein and sodium tauroglycocholate. Excysted larvae were cultured in vitro (37 řC, 5 % CO2) in RPMI 1640 medium enriched with other substances chosen according previously published methods. Mainly sheep, mouse or rat liver extracts eventually in combination with yeast extract and sheep bile were used. The effect of tested substances on the cultivation was evaluated by measuring of the tapeworm's growth. The best effect on the grow of the tapeworms was observed using medium containing serum, yeast extract and sheep liver extract where tapeworms achieved length of 1561 µm after 16 days of incubation. The further growth was limited by appearance of pathologic formations.

Made available in DSpace on 2016-02-26T12:59:12Z (GMT). No. of bitstreams: 1 JackelineSchultzSoares.pdf: 419520 bytes, checksum: 681b65601587b028c9aa1b2b88798f13 (MD5) Previous issue date: 2010-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Orchidaceae differs of most botanical families by its seeds did not have enough reservations for germination. The present work aimed: 1. to study the influence of plant growth regulators BAP (6-benzylaminopurine) e GA3 (gibberellic acid), at concentrations of 0.0; 2.0 and 5.0 mg L-1, as pre-germinative treatment in the process of initial development of Dendrobium nobile Lindl seedlings; 2. to determine the volume of coconut water (0; 50; 100; 150 and 200 mL L-1), that, added to culture media, promoted better germination and in vitro cultivation of D. nobile. Seeds of D. nobile that were produced by manual self-pollinating were used as material of study. After six months of sowing and keeping in germination and growth chamber with controlled temperature and photoperiod (12 hours and 23 ºC ±), seedlings were removed from pots and evaluated about number, percentage of germination, fresh mass, height, diameter and number of psedobulbs, number of leaves, number of roots and length of the greatest root. Completely randomized experimental design was used. Every variables and vegetal attributes were submitted to variance analysis and when they were significative, to regression analysis. D. nobile seeds germinated more in the absence of plant growth regulators and treatments with BAP or GA3 for imbibitions of seeds influenced statistically in vitro development of D. nobile. The addition of 200 mL L-1 of coconut water to culture media promoted the best results for plant heights, number or roots, number of pseudobulbs and fresh mass of D. nobile. / Orchidaceae diferencia-se da maioria das famílias botânicas por suas sementes não possuírem reservas suficientes para a germinação. O presente trabalho objetivou: 1- estudar a influência dos reguladores hormonais BAP (6-benzilaminopurina) e GA3 (ácido giberélico), nas concentrações de 0,0; 1,0; 2,0 e 5,0 mg L-1, como tratamento pré-germinativo no processo de germinação e desenvolvimento inicial de plântulas de Dendrobium nobile Lindl; 2- determinar o volume de água de coco (0; 50; 100; 150 e 200 mL L-1 ), que, acrescido ao meio de cultura proporcionou melhor germinação e cultivo in vitro de D. nobile. Foram utilizadas como material de estudo sementes de D. nobile produzidas mediante autopolinização manual Após seis meses do semeio e permanência em câmara de germinação e crescimento com temperatura e fotoperíodo controlados (12 horas e 23 ºC ± 2) as plântulas foram retiradas dos frascos e avaliadas quanto ao número, porcentagem de germinação, massa fresca, altura, diâmetro e número de pseudobulbos, número de folhas, número de raízes e comprimento da maior raiz. Foi utilizado o delineamento experimental inteiramente casualizado. Todas as variáveis e atributos vegetais foram submetidos à análise de variância e quando significativos à regressão. As sementes de D. nobile germinaram mais na ausência de reguladores vegetais, e os tratamentos com BAP ou GA na embebição das sementes influenciaram 3estatisticamente o desenvolvimento in vitro de D. nobile. A adição de 200 mL L-1 de água de coco ao meio de cultura propiciou os melhores resultados para altura de plantas, número de raízes, número de pseudobulbos e massa fresca de D. nobile.

Made available in DSpace on 2016-02-26T12:59:35Z (GMT). No. of bitstreams: 1 JackelineSchultzSoares.pdf: 419520 bytes, checksum: 681b65601587b028c9aa1b2b88798f13 (MD5) Previous issue date: 2010-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Orchidaceae differs of most botanical families by its seeds did not have enough reservations for germination. The present work aimed: 1. to study the influence of plant growth regulators BAP (6-benzylaminopurine) e GA3 (gibberellic acid), at concentrations of 0.0; 2.0 and 5.0 mg L-1, as pre-germinative treatment in the process of initial development of Dendrobium nobile Lindl seedlings; 2. to determine the volume of coconut water (0; 50; 100; 150 and 200 mL L-1), that, added to culture media, promoted better germination and in vitro cultivation of D. nobile. Seeds of D. nobile that were produced by manual self-pollinating were used as material of study. After six months of sowing and keeping in germination and growth chamber with controlled temperature and photoperiod (12 hours and 23 ºC ±), seedlings were removed from pots and evaluated about number, percentage of germination, fresh mass, height, diameter and number of psedobulbs, number of leaves, number of roots and length of the greatest root. Completely randomized experimental design was used. Every variables and vegetal attributes were submitted to variance analysis and when they were significative, to regression analysis. D. nobile seeds germinated more in the absence of plant growth regulators and treatments with BAP or GA3 for imbibitions of seeds influenced statistically in vitro development of D. nobile. The addition of 200 mL L-1 of coconut water to culture media promoted the best results for plant heights, number or roots, number of pseudobulbs and fresh mass of D. nobile. / Orchidaceae diferencia-se da maioria das famílias botânicas por suas sementes não possuírem reservas suficientes para a germinação. O presente trabalho objetivou: 1- estudar a influência dos reguladores hormonais BAP (6-benzilaminopurina) e GA3 (ácido giberélico), nas concentrações de 0,0; 1,0; 2,0 e 5,0 mg L-1, como tratamento pré-germinativo no processo de germinação e desenvolvimento inicial de plântulas de Dendrobium nobile Lindl; 2- determinar o volume de água de coco (0; 50; 100; 150 e 200 mL L-1 ), que, acrescido ao meio de cultura proporcionou melhor germinação e cultivo in vitro de D. nobile. Foram utilizadas como material de estudo sementes de D. nobile produzidas mediante autopolinização manual Após seis meses do semeio e permanência em câmara de germinação e crescimento com temperatura e fotoperíodo controlados (12 horas e 23 ºC ± 2) as plântulas foram retiradas dos frascos e avaliadas quanto ao número, porcentagem de germinação, massa fresca, altura, diâmetro e número de pseudobulbos, número de folhas, número de raízes e comprimento da maior raiz. Foi utilizado o delineamento experimental inteiramente casualizado. Todas as variáveis e atributos vegetais foram submetidos à análise de variância e quando significativos à regressão. As sementes de D. nobile germinaram mais na ausência de reguladores vegetais, e os tratamentos com BAP ou GA na embebição das sementes influenciaram 3estatisticamente o desenvolvimento in vitro de D. nobile. A adição de 200 mL L-1 de água de coco ao meio de cultura propiciou os melhores resultados para altura de plantas, número de raízes, número de pseudobulbos e massa fresca de D. nobile.

Made available in DSpace on 2014-08-20T14:22:09Z (GMT). No. of bitstreams: 1 Tese_Juliana_Magalhaes_Bandeira.pdf: 1698906 bytes, checksum: 40fa28ef99ce7cee9d0955c4f8688cdf (MD5) Previous issue date: 2010-04-09 / The Japanese plum (Prunus salicina Lindl.) has an important role in the horticulture worldwide. It has great importance in Brazilian market and is gaining fans for its production, especially in Rio Grande do Sul and Santa Catarina where winter conditions are propicious to its cultivation. However, there are some problems faced by farmers and breeders, which limit the increase in domestic production of plum trees, among these, there are the gametophytic self-incompatibility, due to the presence of a codominant multi-site (containing the so-called S-alleles) and susceptibility to Xillela fastidiosa Wells, which affects quality of propagation material. In face of these problems, the objective of this work was to identify physiologically Salleles of six cultivars of Japanese plum and develop a micropropagation protocol for the cv. America. Thus, experiments of controlled pollination were carried out in experimental fields at Embrapa Clima Tempreado (Pelotas/RS) and the in vivo pollination were carried out in the laboratory of six cup cultivars of Japanese plum (America, Rosa Mineira, Pluma 7, Amarelinha, Reubennel and Santa Rosa), to evaluate the fruit set and pollen tube growth (CTP), respectively. Additionaly, were also performed experiments of micropropagation in the Laboratory of Plant Tissue Culture (UFPel/RS) to identify an appropriate protocol for micropropagation of cv. America. In studies of reproductive compatibility, the fruit set was very low for all crosses, with the larger value had occur in the autopollination of 'Reubennel' (8.41%). 'America' only formed fruit when pollinated with the cvs. Rosa Mineira, Amarelinha, Santa Rosa and Reubennel, these last one had mutual compatibility with cv. America. 'Amarelinha' and 'Rosa Mineira' proved to be compatible with the pollen of 'Rosa Mineira' and 'Reubennel'. There was significant interaction for CTP level and pollen used to pollinate each female parents, and the crosses between 'America' x 'Pluma 7' and 'Rosa Mineira' x 'Santa Rosa' were incompatible while cv. America is self-incompatible. Some crosses that showed no pollination and fruiting in the field, hit the egg (level 6) or ovary (level 5) during in vivo pollination, such as 'Reubennel' x 'Rosa Mineira', 'Rosa Mineira' and 'Amarelinha' x 'America' and 'Amarelinha' and 'Pluma 7', and 'Santa Rosa' x 'America'. These results, together with the molecular data of S-alleles, allow a better understanding of the reproductive biology of P. salicina. In the micropropagation experiments we found that the in vitro establishment of basal 11 explants of cv. America in MS medium is higher than the apical explants. BAP concentrations between 0.25 and 0.50mg dm-3 are more appropriate for the multiplication while MS medium supplemented with 1.0mg dm-3 GA3 and 2.0g dm-3 of vegetal charcoal is the most appropriate to in vitro elongation of their shoots. Half of concentration of salts and vitamins of MS medium with the addition of 1.0mg dm-3 of IBA is the most efficient for the in vitro rooting of Japanese plum, cv. America, during periods of less than 20 days, reaching 88% survival after 30 days of acclimatization. / A ameixeira japonesa (Prunus salicina Lindl.) tem papel de destaque na fruticultura mundial. Possui grande importância no mercado brasileiro e vem ganhando adeptos à sua produção, principalmente no Rio Grande do Sul e Santa Catarina onde as condições de inverno favorecem o seu cultivo. Porém, existem alguns problemas enfrentados pelos produtores e melhoristas que limitam o aumento da produção nacional de ameixeiras, entre esses, destacam-se a autoincompatibilidade gametofítica, devido à presença de um loco multialélico (contendo os denominados alelos-S) e a suscetibilidade à Xillela fastidiosa Wells, que afeta a qualidade do material propagativo. Frente a esses problemas, o objetivo da realização deste trabalho foi identificar fisiologicamente os alelos-S de seis cultivares de ameixeira japonesa e desenvolver um protocolo de micropropagação para a cv. América. Desta forma, foram realizados experimentos de polinização controlada nos campos experimentais da Embrapa Clima Temperado (Pelotas/RS) e polinização in vivo, em laboratório, de seis cultivares copa de ameixeira japonesa (América, Rosa Mineira, Pluma 7, Amarelinha, Reubennel e Santa Rosa), onde foram avaliados a frutificação efetiva e o crescimento do tubo polínico (CTP), respectivamente. Também foram realizados experimentos no Laboratório de Cultura de Tecidos de Plantas (UFPel/RS), visando identificar um protocolo adequado para a micropropagação da cv. América. Nos estudos de compatibilidade reprodutiva, a frutificação efetiva foi muito baixa para todos os cruzamentos, sendo observado maior valor na autopolinização da Reubennel (8,41%). América somente apresentou frutificação quando polinizada com as cvs. Rosa Mineira, Amarelinha, Santa Rosa e Reubennel, sendo que essa última apresentou compatibilidade mútua com a cv. América. Amarelinha e Rosa Mineira mostraram-se compatíveis com o pólen da Rosa Mineira e Reubennel . Houve interação significativa para o grau de CTP e o pólen utilizado para polinizar cada um dos genitores femininos, sendo que os cruzamentos entre América x Pluma 7 e Rosa Mineira x Santa Rosa foram incompatíveis enquanto que a cv. América apresentou-se autoincompatível. Alguns cruzamentos que não apresentaram polinização e frutificação no campo, atingiram o óvulo (grau 6) ou o ovário (grau 5) durante a polinização in vivo, tais como Reubennel x Rosa Mineira ; 9 Rosa Mineira e Amarelinha x América e Amarelinha e Pluma 7 , e Santa Rosa x América . Estes resultados, juntamente com os dados moleculares dos alelos-S, permitem um melhor entendimento a respeito da biologia reprodutiva de P. salicina. Nos experimentos de micropropagação verificou-se que o estabelecimento in vitro de explantes basais da cv. América em meio MS, é superior ao de explantes apicais. Concentrações de BAP entre 0,25 e 0,50mg dm-3 são mais adequadas para sua multiplicação enquanto que o meio MS suplementado com 1,0mg dm-3 de AG3 e 2,0g dm-3 de carvão vegetal é o mais adequado para o alongamento in vitro de suas brotações. A metade da concentração dos sais e vitaminas do meio MS com a adição de 1,0mg dm-3 de AIB é o mais eficiente para o enraizamento in vitro de brotações de ameixeira japonesa, cv. América, por períodos inferiores a 20 dias, atingindo 88% de sobrevivência após 30 dias de aclimatização.

As bromélias são plantas que podem utilizar o nitrogênio em suas formas inorgânica e orgânica. Algumas delas apresentam comportamento heteroblástico, isto é, na fase adulta apresentam a sobreposição das folhas, formando um tanque, o qual pode armazenar água e nutrientes. Já na fase juvenil, também chamada de atmosférica, o tanque inexiste. Plantas adultas da espécie hetereblástica Guzmania monostachia quando cultivadas na natureza parecer preferir o nitrogênio na forma amoniacal ou ureídica; no entanto, para as atmosféricas não existem ainda estudos a respeito. Quanto à fotossíntese, plantas adultas com tanque de G. monostachia apresentam um sistema facultativo, isto é, numa condição hídrica favorável realizam a fotossíntese C3, já sob seca, podem alterar para o metabolismo ácido das crassuláceas (CAM). Por outro lado, as plantas atmosféricas dessa espécie expressam o CAM constitutivamente. Em resposta às adversidades ambientais, como a seca temporária ou a indisponibilidade de nutrientes, um aumento na produção de espécies reativas de oxigênio (ROS) pode ocorrer e, dependendo da concentração alcançada nos tecidos, pode causar injúrias. Entretanto, as ROS também podem estar envolvidas com processos de sinalização celular, desde que em baixas concentrações. Folhas destacadas de plantas adultas de Guzmania monostachia, submetidas a um déficit hídrico e cultivadas na presença de diferentes fontes nitrogenadas, mostraram diferentes intensidades de CAM e teores consideráveis de ROS no ápice foliar. Entretanto, pouco se conhece, até o momento, sobre a modulação do CAM e a produção de ROS em plantas da fase juvenil. Os principais objetivos desta pesquisa foram 1) investigar quais seriam as fontes nitrogenadas mais propícias ao crescimento e desenvolvimento de plantas atmosféricas de Guzmania monostachia cultivadas in vitro na presença isolada de nitrato, amônio, glutamina ou ureia, na concentração total de N de 5mM. 2) analisar os efeitos do cultivo prévio de plantas atmosféricas em diferentes fontes de nitrogênio (mesmas condições acima) submetidas, em seguida, a um período de 7 dias de déficit hídrico provocado por PEG adicionado ao meio de cultura, sobre a modulação da intensidade do CAM, produção de ROS e atividades das enzimas antioxidantes. Foram realizadas análises morfométricas, anatômicas, além das fisiológicas, como conteúdo de pigmentos fotossintéticos (capítulo 1). Além disso, analisaram-se a suculência foliar, o acúmulo noturno de ácidos orgânicos, conteúdo de H2O2, atividade das enzimas PEPC e das relacionadas ao sistema antioxidante (capítulo 2). Os resultados de cultivo in vitro apontam que diferentes fontes nitrogenadas modificam metabolicamente e estruturalmente a bromélia Guzmania monostachia na fase atmosférica, sendo a fonte nítrica a que propiciou o surgimento de maiores proporções dos parênquimas aquífero e clorofiliano, aumentando a possibilidade de estocagem de água e de produção de clorofilas e carotenoides. Diferentemente das outras fontes de N, o nitrato foi a que propiciou o maior acúmulo noturno de ácidos orgânicos nas plantas submetidas à deficiência hídrica. Houve também uma maior suculência dos tecidos no tratamento com nitrato, mesmo após a imposição dos 7 dias de deficiência hídrica, indicando que um aumento na capacidade de armazenar água possa ser uma condição estrutural importante para se incrementar a expressão do CAM. O conteúdo de peróxido de hidrogênio aumentou quando as plantas previamente cultivadas em nitrato foram submetidas ao déficit hídrico, sugerindo que essa ROS poderia atuar como um sinalizador na modulação positiva do CAM; já que as atividades das enzimas antioxidantes diminuíram em relação ao controle (SOD e GR) ou permaneceram iguais estatisticamente (CAT, APX, GPOX). Em conclusão, as plantas atmosféricas de Guzmania monostachia, cresceram e se desenvolveram mais adequadamente na presença de nitrato e esta forma de N propiciou um aumento da suculência e da intensidade do CAM, quando houve a posterior imposição de um período de deficiência hídrica, sugerindo um aumento da tolerância à falta de água. Ao contrário, trabalhos anteriores do grupo mostraram que as plantas adultas com tanque de G. monostachia intensificaram o CAM na presença de amônio, indicando, portanto, que plantas de diferentes fases de desenvolvimento possuem, além de diferenças morfológicas, comportamentos fisiológicos diferentes / Bromeliads are plants that can use inorganic and organic nitrogen forms. Some of them feature the heteroblastic behavior which in adulthood have an overlapping of leaves forming a tank that can store water and nutrients. On the other hand, the juvenile phase, also called atmospheric, does not form the tank. Adult plants of the bromeliad species Guzmania monostachia are heteroblastic when growing in the wild and seem to prefer ammonium and urea. However, to the best of our knowledge, there are no studies with the atmospheric plants. Considering the photosynthetic metabolism, G. monostachia adult plants have a facultative system where the plant performs the C3 metabolism in good water conditions and change to the Crassulacean acid metabolism (CAM) under drought. Furthermore, plants of this species can express CAM constitutively. In response to environmental adversities, such as drought or variation in nutrient availability, an increase in the production of reactive oxygen species (ROS) can occur and, depending on the concentration achieved in their tissues, can cause injury. However, the ROS can also be involved in the processes of signaling of the cells when at low concentrations. Leaves that were withdrawn from adult plants of G. monostachia and were subjected to treatments of water deficit and different nitrogen sources showed different intensities of CAM and considerable levels of ROS in the foliar apex. However, little is known about the modulation of the CAM metabolism and the production of ROS in the juvenile G. monostachia bromeliads. The aims of this research were to 1) investigate which nitrogen source contribute more to the growth and development of atmospheric plants cultivated in vitro with isolated nitrate, ammonium, glutamine or urea, in a total N concentration of 5 mm; 2) examine the effects of the previous cultivation of atmospheric plants in different nitrogen sources on the modulation of the intensity of the CAM, ROS production and activities of antioxidant enzymes when the plants where submitted to in a period of seven days of water deficit caused by PEG added to the culture medium. Morphometric, physiological, anatomical and the photosynthetic pigments were analyzed (Chapter 1). In addition, foliar succulence the accumulation of nocturnal organic acids, H2O2 content, the activity of PEPC enzymes and the antioxidant system related were analyzed, (Chapter 2). The results of in vitro cultivation indicated that different nitrogen sources modified the metabolism and the tissue structure of the atmospheric Guzmania monostachia, as the nitrate provided the emergence of the larger proportions of the aquifer and chlorophyll parenchyma, increasing the possibility of water storage and production of chlorophylls and carotenoids. Unlike other sources of N, nitrate was the only that provided the greatest accumulation of nocturnal organic acids in plants submitted to water deficiency. There was also a greater succulence in the tissues submitted to the nitrate treatment, even after the exposition of seven days of water deficit, indicating an increase in the capacity of storing water that can be an important structural condition to increase the expression of CAM. The H2O2 content increased when the plants previously cultivated in nitrate were submitted to water deficit, suggesting that ROS could act as a signaling in the modulation of the CAM metabolism as the activities of antioxidant enzymes decreased compared to the control treatments (SOD and GR) or remained the statistically same (CAT, APX, GPOX). In conclusion, the atmospheric plants grew and developed more in the presence of nitrate and this form of N provided an increment in leaf succulence and intensification of CAM when there was the subsequent imposition of a period of water deficit, suggesting an increment of the tolerance for water deficit conditions. In contrast, previous researches showed that the adult plants of G. monostachia with tank intensified the CAM in the presence of ammonium, indicating that plants of different stages of development have, in addition to morphological differences, differences in their physiological behaviors

Cell cultures are grown in special incubators ensuring appropriate culture conditions for cell survival. The introduction of the diploma thesis is dedicated to the cultivation of cells in vitro and description of sensors, especially designated for the sensing of gas concentration. The main essence of this work is a project and realization of the monoclimatic incubation chamber for the incubation of cell cultures with the opportunity to observe them under a confocal microscope. Another part of this work is dealing with the project and application of autonomous sensing system and regulation of gas concentration (O2 and CO2) in the chamber. The nal part of thesis is dedicated to the presentation of the project correctness verication of the incubation chamber.

Plants are exposed to many adverse factors during their life cycles. Abiotic stresses are significantly limiting plant growth and development. Abiotic stress response mechanisms involve compatible solute synthesis (e.g. sugars, sugar alcohols and amino acids). The aim of this study was to characterise the responses of plants from Plantago genus to different abiotic stresses (drought, salinity, cold and stress combination). The complete plants grew under in vitro conditions. I compared morphological parameters, selected metabolic parameters (carbohydrates balance and proline accumulation) under optimal conditions and stress exposure. This study was focused on plants from genus Plantago, because they differ from each other in their tolerance to the salinity. Both, the glycophyte and the halophyte species are described within this genus. Plantains produce besides widespread soluble carbohydrates (sucrose, glucose, fructose) also sugar alcohol sorbitol, which has been reported as a significant component of the stress response. In addition, the reaction of plants to different carbon and energy sources was tested. More severe growth inhibition of the glycophyte Plantago lanceolata compared to the halophyte P. maritima was observed under salt treatment. Significant accumulation of sorbitol was observed...

Plants are exposed to the stress of the environment throughout their entire live. They therefore had to develop sophisticated mechanisms to avoid or tolerate stress. In some plants, repeated exposure to stress increases tolerance, which is known as stress memory. The aim of this study is to assess stress memory in terms of oxidative stress level. Content of malondialdehyde (MDA), reactive oxygen species (ROS) and the electrolyte leakage were chosen as markers of oxidative stress. Fresh weight, plant height and water content were also evaluated. The level of oxidative stress was assessed in several genotypes of Solanum tuberosum subspecies, Andigena and Tuberosum. In vitro culture did not work well for the evaluation of stress memory, ex vitro partially did. The content of MDA in 66B was significantly lower than in cv. Lada (Tuberosum) in root (in vitro) and shoot (ex vitro) in control groups as well as during water shortage. Treatment of acclimation-inducing cold before the stress-inducing cold had a variable effect on growth and electrolyte leakage according to the culture conditions. In vitro, the effect on electrolyte leakage in roots of cv. Lada was positive, in leaves of 66B (Andigena) it was negative. In ex vitro, a positive effect was found on electrolyte leakage in 66B leaves, but at the...

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.