This diploma work was assessed effects of different seed treatment (untreated flour from whole seed, flour from the uterus after soaking seeds, flour from the seeds sprouted cotyledon) by three species of legumes (Pisum sativum conv. Sativum L., Vicia faba L., Lupinus angustifolius L.) yield and composition of protein isolate obtained by isoelectric precipitation. It was found that the seeds of change most affects yield precipitated N (mg) and 45%. When determining the value of the yield of protein was affected by significant interactions (treatment and the type of legume seeds). The largest share was, however, precipitate in untreated and germinated lupine seeds, in which values are around 57%. Furthermore, the thesis was to reverse the precipitation of proteins, using which we obtained protein concentrates from different species of legumes. The yields of protein concentrates reach values in the range of 60-80%. Spectra of soluble proteins is clearly visible high concentration of isolated proteins. Variation of sprouted seeds and soaked that occurred during treatment of seeds for the collapse of proteins with higher molecular weight. Furthermore, these grafts are transferred protein extraction at pH 9.0 in the later produced protein isolates. After acid precipitation is observed on a spectrum that is re-soluble protein isolates.

O melhoramento do alho (Allium sativum L.) está limitado à seleção clonal de genótipos mutantes, uma vez que a quase totalidade do germoplasma da espécie é sexualmente estéril, não florescendo nas condições padrões de cultivo. A esterilidade do alho tem implicações não somente no melhoramento da espécie, mas também influi diretamente nos custos de produção. A necessidade de propagação vegetativa utilizando-se bulbilhos (\"dentes\") como material propagativo facilita a transmissão de doenças por vírus e pragas. A manipulação genética do alho com genes associados ao florescimento via Agrobacterium tumefaciens poderia permitir o desenvolvimento de um sistema mais eficaz para propagação da cultura e habilitar novas combinações genéticas. Portanto, o objetivo desse trabalho foi estabelecer protocolos de regeneração para cultivares comerciais brasileiras de alho, bem como de transformação genética mediada por A. tumefaciens de calos oriundos de segmentos radiculares. Foram utilizadas oito cultivares de alho, divididas em três grupos: semi-nobres de ciclo médio (\'Amarante-Embrapa\'; \'Roxinho 5063\'; \'IAC 75 - Gigante de Curitibanos\'; \'IAC 63 - Mexicano Br\'; e \'Lavínia 1632\'); comum, de ciclo médio (\'Cateto Roxo\') e de ciclo precoce (\'Cajuru 2315\'); e nobre vernalizado (\'Jonas\'). Para cada cultivar, foram isolados segmentos radiculares de bulbilhos cultivados in vitro. Esses explantes foram mantidos em cultura durante 60 dias em meio MS, adicionado de 4,5 \'mü\'M 2,4-D e 0,5 \'mü\'M iP para obtenção de calos. Os calos obtidos foram transferidos para meio MS suplementado com 8,8 \'mü\'M BAP e 0,1 \'mü\'M ANA, e avaliados quanto à freqüência de regeneração. Inicialmente, foram analisados diversos fatores durante a introdução dos bulbilhos e na obtenção de calo para cada cultivar. Esses resultados permitiram realizar testes para avaliar o potencial de regeneração dos calos obtidos, e a cultivar \'Jonas\' apresentou a melhor taxa de regeneração via organogênese indireta (84%), seguida da cultivar \'Amarante\' (52%), \'IAC 63\' e \'Cajuru\' (47%) e a cultivar que apresentou a menor taxa foi a \'Lavínia\' (30%). Foi analisada a dose letal de higromicina ou canamicina, testando concentrações crescentes visando identificar os níveis ótimos de uso como agentes seletivos para a transformação. As doses acima de 50 mg/L de higromicina ou 200 mg/L de canamicina foram letais ao cultivo de calos de todas cultivares. A cultivar \'Jonas\' foi utilizada para o estabelecimento do procedimento de transformação via A. tumefaciens LBA4404(pCAMBIA1303), empregando como repórter o gene uidA (\'beta\'-glucuronídase) para determinação da eficiência da transformação. Foi demonstrado que a cultivar foi a \'Jonas\' apresentou o melhor potencial de calogênese e de regeneração, sendo o meio de cultura proposto por Kondo, Hasegawa e Suzuki (2000) o melhor para regeneração das cultivares brasileiras. Além disso, as cultivares brasileiras de alho parecem ser passíveis de transformação via A. tumefaciens / Garlic (Allium sativum L.) breeding has been limited to clonal selection of mutant genotypes, since almost all the species germplasm is sterile, not flowering under standard culture conditions. Garlic sterility affects not only breeding but also directly influences production costs. Further, the requirement of vegetative propagation using small bulbs as propagules increases the risk of pest and viral disease transmission. Genetic manipulation of garlic introducing genes associated with flowering by Agrobacterium tumefaciens would allow the development of an efficient system for propagation and enable new genetic recombination. Thus, the objective of this work was to establish an in vitro regeneration protocol for commercial Brazilian cultivars of garlic, as well as genetic transformation by A. tumefaciens using calli derived from root segments. Eight garlic cultivars were used, derived from three groups: medium cycle semi-noble (\'Amarante-Embrapa\'; \'Roxinho 5063\'; \'IAC 75 - Gigante de Curitibanos\'; \'IAC 63 - Mexicano Br\'; and \'Lavínia 1632\'); common garlic with medium (\'Cateto Roxo\'), or early cycle (\'Cajuru 2315\'); and from vernalized noble (\'Jonas\'). From each cultivar, root segments from small bulbs cultivated in vitro were isolated. These explantes were maintained in MS media supplemented with 4,5 \'mü\'M 2,4-D e 0,5 \'mü\'M iP for 60 days to develop callus. The calli were then transferred to fresh MS media containing 8,8 \'mü\'M BAP e 0,1 \'mü\'M ANA, and evaluated for regeneration frequency. Initially, diverse factors were analyzed for each cultivar during the introduction of the small bulbs and during calli development. These results enabled to conduct test of callus regeneration potential, with \'Jonas\' showing the best regeneration rate via indirect organogenesis (84%), followed by cultivar \'Amarante\' (52%), \'IAC 63\' and \'Cajuru\' (47%). The cultivar with less regeneration was \'Lavínia\' (30%). The lethal dosis of hygromycin or kanamycin were estimated, testing increasing concentrations to establish optimum levels to be adopted as selective agents for transformation. Doses above 50 mg/L hygromycin or 200 mg/L kanamycin were lethal to callus of all cultivars. Cultivar \'Jonas\' was chosen for the establishment of the genetic transformation protocol via A. tumefaciens LBA4404 (pCAMBIA1303), using uidA as a reporter gene (\'beta\'-glucuronídase) to determine the transformation efficiency. \'Jonas\' was the best cultivar in terms of callus and regeneration potential using the regeneration media proposed by Kondo, Hasegawa and Suzuki (2000). Moreover, the Brazilians garlic cultivars appeared to be amenable to transformation via A. tumefaciens

Made available in DSpace on 2015-05-14T13:00:01Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1293356 bytes, checksum: eb48e7877c4f4bdbd473d0f996411c16 (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / There are some strains of Escherichia coli that are pathogenic and often cause urinary tract infections, septicemia and meningitis in neonates. Some produce enzymes known as β-lactamases of extended spectrum (ESBL) that decrease the therapeutic options, thereby arousing interest in the discovery of new antibacterial products. Medicinal plants and their essential oils are especially rich in metabolites with antimicrobial properties as soon researches are developed in searches of these substances. Thus, the present study aimed to evaluate the effect of essential oil of Coriandrum sativum and its major phytochemicals against strains of E. coli ESBL. Was initially determined the sensitivity of the tested strains to conventional antibiotics, as well as screening of the antibacterial activity of the oils against these strains. Chosen C. sativum oil, its chemical composition was determined by gas chromatography coupled to mass spectrometer (GC/MS) and the antibacterial activity of both the oil as the major phytochemicals were evaluated by determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), the microdilution technique, the kinetics of microbial death oil was also analyzed. It has also played a modulating effect over the conventional antibiotics. It was found that the tested strains are producing ESBL. Among the phytochemicals, linalool (39.78%) presented itself as a major component, followed by linalool oxide (27.33%). The oil and linalool inhibited all strains having MIC and MBC of 256 μg/mL, 512 μg/mL and 1024 μg/mL, between 512 and 1024 μg/mL, respectively. Linalool oxide was inactive at the maximum test concentration of 1024 μg/mL. This oil has antibacterial activity dependent on its concentration and time of exposure in the micro-organism. Both oil and linalool modulates the action of the antibiotics ciprofloxacin and cefoxitin, synergistic interaction of oil and ciprofloxacin being observed. The combination with linalool showed synergistic effects with ciprofloxacin and norfloxacin. The results suggest that the essential oil of C. sativum can suppress the growth of species E. coli ESBL and its major phytochemical despite having weak antibacterial activity, may modulate the action of some antibiotics. / Existem algumas cepas de Escherichia coli que são patogênicas e frequentemente causam infecções urinárias, septicemias e meningites em neonatos. Algumas produzem enzimas conhecidas como β-lactamases de espectro estendido (ESBL) que diminuem as opções terapêuticas, despertando assim o interesse pela descoberta de novos produtos antibacterianos. As plantas medicinais e principalmente seus óleos essenciais são ricos em metabólitos com propriedades antimicrobianas, logo pesquisas são desenvolvidas em buscas destas substâncias. Assim, o presente estudo teve como objetivo avaliar o efeito do óleo essencial de Coriandrum sativum e seus fitoconstituintes majoritários contra cepas de E. coli ESBL. Inicialmente foi determinado o perfil de sensibilidade das cepas ensaiadas a antibióticos convencionais, bem como a triagem da atividade antibacteriana dos óleos contra estas cepas. Escolhido o óleo de C. sativum, a sua composição química foi determinada por Cromatografia Gasosa acoplada a Espectrômetro de Massa (CG/EM) e a atividade antibacteriana tanto do óleo quanto dos fitoconstituintes majoritários foram avaliadas pela determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM), pela técnica de microdiluição, a cinética de morte microbiana do óleo também foi analisada. Verificou-se também o efeito modulador desempenhado sobre os antibióticos convencionais. Constatou-se que as cepas ensaiadas são produtoras de ESBL. Entre os fitoconstituintes, o linalol (39,78%) apresentou-se como principal componente, seguido pelo óxido de linalol (27,33%). O óleo e o linalol inibiram todas as cepas apresentando CIM e CBM de, 256 μg/mL, 512 μg/mL e 1024 μg/mL, entre 512 e 1024 μg/mL, respectivamente. O óxido de linalol não apresentou atividade na concentração máxima ensaiada de 1024 μg/mL. Este óleo possui atividade antibacteriana dependente de sua concentração e do tempo de exposição no micro-organismo. Tanto o óleo quanto o linalol modulou a ação dos antibióticos ciprofloxacina e cefoxitina, sendo observada interação sinérgica do óleo e a ciprofloxacina.Já a combinação com linalol, apresentou efeitos sinérgicos com a ciprofloxacina e norfloxacina. Os resultados sugerem que o óleo essencial de C. sativum pode suprimir o crescimento de espécies de E. coli ESBL e que seu fitoconstituinte majoritário apesar de ter atividade antibacteriana fraca, pode modular a ação de alguns antibióticos

Made available in DSpace on 2017-07-10T17:48:09Z (GMT). No. of bitstreams: 1 Leticia_Angelica_Pereira.pdf: 691022 bytes, checksum: 4ea817fecf3a460a2a24e9762b62f93b (MD5) Previous issue date: 2015-10-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study aimed to evaluate the effect of garlic essential oil (Allium sativum) on the fertility rate and hatching of silver catfish eggs, Rhamdia quelen, artificially incubated. The research consisted of two experiments (I and II) at the Institute for Research in Environmental Aquaculture (InPAA), Toledo-PR, from october to december 2014. It used a randomly experimental design with the four treatments and five repetitions. The treatments consisted on the introduction of garlic essential oil on the incubators of fish eggs with the levels of 0; 5; 10 and 20 and 0; 1; 3 and 5 mg L-1 of oil, on experiments I and II respectively. The fertilization rate was determined after the closing of the blastopore, approximately 10 hours post-fertilization and the hatching rate was calculated 24 hours post-fertilization, when it was observed the larvae shedding egg. It used the standard morphological larvae to determine the normal rate, with the aid of a stereoscopic microscope. On the experiment I the dosage of 5 mg L-1 promoted the biggest (p<0.05) rates of fertilization and hatching of the eggs. At levels of 10 and 20 mg L-1 was death from all embryos within 10h after fertilization. In the second experiment the dosage of 5 mg L-1 promoted best fertilization rates (p<0.01) and hatching (p<0.05) compared to other essential oil concentrations of garlic. The fertilization and hatching rates ranged from 65.80 to 83.70% and 57.60 to 71.40%, respectively. The essential garlic oil levels showed linear effect on the fertilization rates (p<0.01) and hatching (p<0.05). Garlic oil did not influence the normal larvae (p<0.05). It is recommended to use 5 mg L-1 essential oil of garlic to promote better rates of fertilization and hatching of silver catfish eggs artificially incubated / A incubação artificial de ovos de peixes proporciona maiores quantidades de alevinos em relação às condições naturais. Porém, um dos procedimentos para que esta técnica reprodutiva obtenha bons resultados é a utilização de produtos antimicrobianos na água de incubação. Objetivou-se avaliar o efeito da utilização do óleo essencial de alho (Allium sativum) sobre as taxas de fertilidade, eclosão e normalidade das larvas de ovos de jundiá, Rhamdia quelen, incubados artificialmente. A pesquisa consistiu de dois experimentos (I e II) realizados no Instituto de Pesquisas em Aquicultura Ambiental (InPAA), Toledo, PR, no período de outubro a dezembro de 2014. Foi utilizado delineamento experimental inteiramente casualizado com quatro tratamentos e cinco repetições. Os tratamentos consistiram na introdução de óleo essencial de alho nas incubadoras, utilizando um sistema fechado de recirculação de água, com os níveis de 0; 5; 10 e 20 e 0; 1; 3 e 5 mg L-1 de óleo essencial de alho, respectivamente para o experimento I e II. A taxa de fertilização foi determinada após o fechamento do blastóporo, aproximadamente 10 horas pós-fertilização e a taxa de eclosão foi calculada com 24 h pós-fertilização, quando observado as larvas se desprendo do ovo. Utilizou-se do padrão morfológico de larvas para determinar a taxa de normalidade, com o auxílio de um microscópio estereoscópio. No experimento I a dosagem 5 mg L-1 promoveu maiores (p<0,05) taxas de fertilização e eclosão dos ovos. Nos níveis 10 e 20 mg L-1 houve morte de todos os embriões nas primeiras 10h após a fecundação. No experimento II a dosagem de 5 mg L-1 promoveu melhores taxas de fertilização (p<0,01) e eclosão (p<0,05) em relação às demais concentrações de óleo essencial de alho. As taxas de fertilização e eclosão variaram de 65,80 a 83,70% e 57,60 a 71,40%, respectivamente. Os níveis de óleo essencial de alho apresentaram efeito linear sobre as taxas de fertilização (p<0,01) e eclosão (p<0,05). O óleo de alho não influenciou na normalidade das larvas (p>0,05). Recomenda-se utilizar 5 mg L-1 de óleo essencial de alho para promover melhores taxas de fertilização e eclosão de ovos de jundiá incubados artificialmente

Made available in DSpace on 2014-08-20T13:59:08Z (GMT). No. of bitstreams: 1 dissertacao_marcio_espinosa_de_farias.pdf: 3645960 bytes, checksum: 2a09be33a4c426dea1aef53c4073f253 (MD5) Previous issue date: 2014-02-17 / Photosynthesis plays a fundamental role in the processes of growth and development of plants. It occurs in the chloroplasts of leaf mesophyll cells and consists of two steps, a photochemical phase that converts light energy into chemical energy, and other biochemical phase, which uses the energy of the first step for fixing CO2. The study aimed to evaluate, in detached leaves of pea (Pisum sativum L.), the behavior of the electron transport chain in the presence of inhibitors of specific points. The plants were grown in a greenhouse. Two to four weeks after sowing, when the leaves had become fully expanded, they were detached and the petioles were immersed in solutions containing 0, 25, 50, 100, 250, and 500 μM of DCMU, atrazine, DBMIB and methyl viologen. To DCMU, atrazine and methyl viologen the leaves remained for 2 h in contact with the solutions, to DBMIB was 5h. Measurements of transient fluorescence of chlorophyll, delayed fluorescence and modulated reflection at 820nm were made using M-PEA fluorometer. The leaves were dark adapted for 30 minutes, before a saturating pulse emission, and the fluorescence intensities were measured for 60 seconds. Pea leaves treated with 500 μM concentration of DCMU and atrazine showed to be sensitive to this dose of inhibitors, in analyzing the results of transient fluorescence, delayed fluorescence and modulated reflection at 820 nm. The leaves treated with 500 μM of methyl viologen showed differences in parameters related to the mechanism of action of the inhibitor.The same concentration used to DBMIB, had no significant difference in many results compared to the control, only in those involved in the reduction of end acceptors of photosystem I. Therefore, a detailed analysis of the transient fluorescence, delayed fluorescence and modulated reflection at 820nm, allows us to collect and correlate a series of information about the whole electron transport chain. / A fotossíntese exerce um papel fundamental para os processos de crescimento e desenvolvimento das plantas. Ela ocorre nos cloroplastos das células do mesofilo foliar e consiste de duas etapas, uma fase fotoquímica, que converte energia luminosa em energia química e outra fase bioquímica, que utiliza a energia da primeira etapa pra a fixação do CO2. O trabalho teve como objetivo avaliar, em folhas destacadas de ervilha (Pisum sativum L.), o comportamento da cadeia de transporte de elétrons na presença de inibidores de pontos específicos. As plantas foram cultivadas em casa de vegetação. Duas a quatro semanas após semeadura, quando as folhas apresentavam-se completamente expandidas, elas foram destacadas e os pecíolos foram imersos em soluções contendo 0, 25, 50, 100, 250 e 500 μM de DCMU, atrazina, DBMIB e metil viologênio. Para DCMU, atrazina e metil viologênio as folhas permaneceram por 2h em contato com as soluções, para o DBMIB foi de 5h. As medidas da fluorescência transiente da clorofila, da decaída de fluorescência e da reflexão modulada à 820nm, foram feitas utilizando fluorômetro M-PEA. As folhas foram adaptadas ao escuro por 30 minutos, antes da emissão de um pulso saturante, e as intensidades de fluorescência foram medidas por 60 segundos. As folhas de ervilha tratadas com a concentração de 500 μM de DCMU e atrazina demonstraram ser sensíveis a essa dose dos inibidores, ao analisar os resultados da fluorescência transiente, decaída de fluorescência e da reflexão modulada a 820 nm. As folhas tratadas com 500 μM de metil viologênio apresentaram diferenças nos parâmetros relacionados com o mecanismo de ação do inibidor. A mesma concentração utilizada para DBMIB, não teve diferença em muitos resultados em relação ao controle, apenas naqueles envolvidos com a redução dos aceptores finais do fotossistema I. Dessa maneira, uma análise detalhada da fluorescência transiente, da decaída da fluorescência e da reflexão modulada a 820 nm, permite coletar e correlacionar uma série de informações sobre toda a cadeia de transporte de elétrons.

La connaissance des effets et modes d’action des QTL (Quantitative Trait Loci) de résistance des plantes aux maladies est un enjeu majeur pour l’exploitation durable des résistances génétiques en agriculture. Dans le pathosystème pois/Aphanomyces euteiches, présentant une importance économique majeure, l’objectif de la thèse a consisté à valider les effets de QTL de résistance, seuls ou en combinaison, et à connaitre leurs modes d’action sur les étapes du cycle de l’agent pathogène. Un total de 157 NILs (Near Isogenic Lines) de pois issues d’un programme de Back-cross Assisté par Marqueurs, porteuses d’aucun, un, deux ou trois des sept principaux QTL de résistance préalablement identifiés, a été génotypé à l’aide d’une puce 15K SNPs et évalué pour la résistance. Les NILs porteuses de QTL à effets majeurs, seuls ou en combinaison avec des QTL à effets mineurs,ont présenté des niveaux accrus de résistance partielle en conditions contrôlées et au champ par rapport aux NILs dépourvues de QTL, dans différents fonds génétiques. Certaines NILs comportant des QTL individuels ou combinés à effets mineurs ont également montré un niveau réduit de sévérité de la maladie dans l’une ou les deux conditions de test. La plupart des QTL a présenté des effets significatifs sur le ralentissement de l’infection et/ou de la quantité d’ADN pathogène ayant colonisé la racine pendant sept jours après inoculation. Cette thèse fournit des outils et éléments de choix de QTL à combiner en sélection pour augmenter l’efficacité de la résistance partielle à A. euteiches dans les futures variétés de pois / Knowledge of the effects and action modes of resistance QTL to plant diseases is a major challenge for the durable use of genetic resistances in agriculture. In the pea/Aphanomyces euteiches pathosystem, which has a major economic importance, the aim of this study was to validate the single or combined effects of main resistance QTL, and study their action modes on steps of the pathogen life cycle. A total of 157 pea NILs (Near Isogenic Lines) created by Marker-Assisted Back-crossing and carrying no, one, two or three of the seven main resistance QTL previously identified, was genotyped using a 15K SNPs array and evaluated for resistance. The NILs carrying major-effect QTL, individually or in combination with minor-effect QTL, had increased levels of partial resistance in controlledconditions and in the field compared to NILs without QTL, in different genetic backgrounds. Several NILs carrying single or multiple minor-effect QTL also showed reduced levels of disease severity in one or the two test conditions. Most of the QTL had significant effects on slowing down infection and/or pathogen DNA quantity which had colonized the root for seven days after inoculation. This study gives tools and information for the choice of resistance QTL to use in pyramiding breeding strategies for increasing partial levels of resistance to A. euteiches in future pea varieties.

Les plantes cultivées peuvent modifier la disponibilité en ressources et les perturbations perçues par les communautés d’organismes présentes dans les agroécosystèmes et en conséquence, influencer les fonctions écosystémiques utiles à la production agricole telles que la minéralisation de N et de S ou la compétition culture-adventices. Notre objectif était d’évaluer l’effet du pois (Pisum sativum L.), introduit dans une succession de cultures, sur les communautés d’adventices et les communautés microbiennes impliquées dans la minéralisation de N et S. Pour ce faire, nous avons mené des expérimentations en conditions contrôlées et utilisé les données d’une expérimentation au champ conduite pendant 5 ans. En conditions contrôlées, nous avons montré que le pois influence via ses traits racinaires certaines activités enzymatiques liées à la minéralisation de N. En revanche au champ, les variables climatiques influencent principalement le fonctionnement des communautés microbiennes impliquées dans la minéralisation de N et S. Par ailleurs, nous avons observé que le pois sélectionnait des communautés d’adventices fonctionnellement différentes de celles d’autres cultures, comme le colza, mais cet effet du pois ne persistait pas sous la culture suivante. En conclusion, les effets du pois sur les communautés microbiennes impliquées dans la minéralisation de N et S sont faibles comparés à ceux d’autres facteurs comme les conditions climatiques. En revanche l’effet du pois sur les communautés d’adventices est fort mais limité dans le temps. Ces résultats montrent la nécessité de mener les expérimentations au champ sur plusieurs années afin de prendre en compte la variabilité climatique. / Crops species modify resources availability and perturbation regimes perceived by the communities of organisms residing in the agroecosystems and, as a consequence, can lead to alteration of ecosystem functions useful to crop production, such as nitrogen and sulfur mineralization or crop-weeds competition, realized by weeds or microorganisms. Our objective was to study the effect of a legume, pea, when introduced in a crop succession, on weed communities and microbial communities involved in protein decomposition and sulfate ester mineralization, which are the main forms of N and S, respectively, in agricultural soils. To do so, we performed experiments in controlled conditions used data from a 5-years field experiment. In controlled conditions, we showed that pea during its development modify through its root functional traits several enzymatic activities involved in N decomposition/mineralization. However, in the field experiment, climatic factors were the main drivers of the microbial communities involved in N and S decomposition/mineralization. Moreover, pea selected weed communities that were functionally different from weed communities growing in other crops such as oilseed rape. However, this effect did not persist under the following crop. As a conclusion, our results suggest that pea modifications of microbial communities are weak compared to other factors such as climatic factors. However, pea effects on weed communities are strong but short lived. Those results highlight the complementarity of microcosm and field experiments, and the necessity of long term field experiments to take into account climate variability.

<p style="text-align: justify;">Glavni cilj ove disertacije koji obuhvata dobijanje i hemijsku karakterizaciju ekstrakata i etarskih ulja rtanjskog čaja (Satureja montana L.), bosiljka (Ocimum basilicum L.) i korijandera (Coriandrum sativum L.), kao i njihove sme&scaron;e, je određivanje biolo&scaron;ke aktivnosti dobijenih ekstrakata u in vitro uslovima pradenjem sposobnosti neutralizacije slobodnih radikala i antiproliferativne aktivnosti.<br />Prvo je izvr&scaron;ena karakterizacija biljnih sirovina (rtanjski čaj, bosiljak i korijander), kao i njihove sme&scaron;e jednakih masenih udela (CBS) u pogledu sadržaja vlage, srednjeg prečnika čestica i sadržaja etarskog ulja. U cilju dobijanja etarskih ulja i ekstrakata iz ispitivanih sirovina su primenjeni hidrodestilacija (konvencionalni postupak) i superkritična ekstrakcija (savremena tehnika). GC-MS analizom je određen kvalitativni sastav i relativni udeo pojedinačnih monoterpenskih ugljovodonika, oksidovanih monoterpena, seskviterpena i ostalih jedinjenja. Dalje je GC-FID analizom kvantitativno određen sadržaj i prinos pojedinačnih terpena (&alpha;-pinen, &beta;-pinen, d-limonen, &gamma;-terpinen, eukaliptol, linalool, kamfor, &alpha;-terpineol, geraniol, metil-havikol, karvakrol i eugenol) i izvr&scaron;eno je poređenje uzoraka dobijenih tradicionalnim i savremenim postupcima.<br />Etarskim uljima i lipofilnim ekstraktima je određena biolo&scaron;ka aktivnost ispitivanjem antioksidativne i antiproliferativne aktivnosti. In vitro anti-oksidativna aktivnost superkritičnih ekstrakata i etarskih ulja je određena DPPH testom, dok je antiproliferativna aktivnost određena u in vitro uslovima na tri linije tumorskih delija (HeLa, MDA-MB-453, K562) i na normalne humane fibroblaste (MRC-5).<br />U cilju boljeg određivanja sličnosti između etarskih ulja i lipofilnih ekstrakata rtanjskog čaja, bosiljka, korijandera i njihove sme&scaron;e (CBS) su primenjene hemometrijska tehnike: analiza glavnih komponenata (Principal component analysis) i hijerarhijska klaster analiza (Hierarchical cluster analysis).</p> / <p>The main aim of this dissertation was recovery and chemical characterization of extracts and essential oils of winter savory (Satureja montana L.), basil (Ocimum basilicum L.) and coriander (Coriandrum sativum L.), as well as their mixture, and determination biological activity of extracts in in vitro conditions by capacity towards neutralization of free radicals and antiproliferative activity.<br />Plant materials (winter savory, basil and coriander) and their mixture (CBS) were characterized in terms of moisture content, mean particle size and essential oil content. In order to obtain essential oil and extracts from investigated plants, hydrodistillation (conventional procedure) and supercritical fluid extraction (novel technique) were applied. GC-MS analysis was used for determination of qualitative content and relative abundance of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes and other volatile compounds. Furthermore, GC-FID analysis was used for quantitative determination of content and yield of terpene compounds (&alpha;-pinene, &beta;-pinene, d-limonene, &gamma;-terpinene, eucalyptol, linalool, camphor, &alpha;-terpineol, geraniol, methyl-chavicol, carvacrol i eugenol) and samples obtained by traditional and novel techniques were compared. Biological potential of essential oil and lipophilic extracts was determined by antioxidant and antiproliferative activity. In vitro antioxidant activity of supercritical extracts and essential oils was determined by DPPH assay, while antiproliferative activity was determined in in vitro conditions on three cancer cell lines (HeLa, MDA-MB-453, K562) and on normal human fibroblasts (MRC-5).<br />Chemometric techniques such as principal component analysis (PCA) i hierarchical cluster analysis (HCA) were applied for better understanding of similarities between essential oils and lipid extracts.</p>

Darbo tikslas - įvertinti teršalų poveikį sėjamajai pipirnei (Lepidium sativum L.), augintai ant magelaninio kimino (Sphagnum magellanicum), pasyviai kaupusio gamyklų oro teršalus, terpės. Darbo metodika. Kiminų maišeliai buvo eksponuojami 5 savaites prie Lietuvos svarbiausių ir daugiausiai teršalų į aplinką išmetančių gamyklų – AB “Achema”, AB „Akmenės cementas“ ir AB „Mažeikių nafta“. Po ekspozicijos kiminai laboratorijoje buvo išdžiovinti +20º C ir sumalti. Iš sumaltų kiminų Petri lėkštelėse, įpylus po vienodą kiekį kiminų ir distiliuoto vandens buvo paruoštas biotestinio augalo – sėjamosios pipirnės - auginimui substratas. Į kiekvieną lėkštėlę buvo pasėta po 25 sėjamosios pipirnės sėklas. Lėkštelės antrą augimo parą buvo atidengtos, pipirnė buvo auginama 4 paras, 5 parą buvo atlikti sėjamosios pipirnės daigų morfometriniai matavimai. Tyrimo rezultatai. Lyginti atskirų transektų skirtingos vietos, mažiausi sėjamosios pipirnės daigeliai užaugo prie AB „Achema“ eksponuotuose kiminuose: viso augalo aukštis – 27,9 mm, stiebo aukštis – 20,0 mm, šaknies ilgis – 7,9 mm. Prie AB „Mažeikių nafta“ eksponuotų kiminų substrate išaugo šiek tiek aukštesni daigeliai, kurių atitinkami ilgiai (33,3 mm, 24,2 mm ir 8,1mm). Aukščiausi augalai (visas augalas – 39,9 mm, stiebas – 29,8 mm, šaknis – 10,1mm) išaugo prie AB „Akmenės cementas“ eksponuotuose kiminų substratuose. / Goal of the research: evaluate the influence of air pollution on cress (Lepidium sativum L.), that grows on sphagnum (Sphagnum magellanicum). Sphagnum (Sphagnum magellanicum) passively store pollutants extracted from factories. Methodology of the research: The bags of sphagnum were displayed near the most important and biggest Lithuanian factories (JS ‘Achema’, JS ‘Akmenes cementas’ and JS ‘Mazeikiu nafta’) for five weeks. Sphagnum were collected, dried at 20 º C temperature, and grinded in the laboratory. Grinded sphagnum were placed in the Petri dishes in equal parts with distil water and was prepared substratum for biological test. In each dish there were planted 25 seeds of cress. After two days the dishes were opened. The morthometric tests were made on five day after planting. Results. During the test different parts of cress were measured. The smallest sprouts of cress were measured in the area of JS ‘Achema’. The height of plants were – 27,9 mm, length of stems – 20,0 mm, length of roots – 7.9 mm. In the area of JS ‘Mazeikiu nafta’ area displayed sphagnum substratum cress plants were a little bit higher, appropriate the height of plants were - 33.3 mm, length of stems – 24,2 mm and length of roots – 8,1 mm. The highest cress plants were measured in the sphagnum substratum exposed in the areas of JS ‘Akmenes cementas’. The heights of plants were – 39,9 mm, length of stems – 29.8 mm, length of roots – 10,1 mm.

Allium sativum has a long tradition in medicine. While much is known about its potential healthy effects, nearly nothing is known about wild garlic (allium sativum, ramson), which is very common in the area of Leipzig and has been used as a herbal remedy since centuries. The goal of the present study was to assess a potential anti-platelet activity of these two allium species and to try to identify the chemical active principle. For that purpose various extracts (hydrophilic and lipophilic) were prepared from Allium sativum and Allium ursinum, and analysed using thin layer chromatography and HPLC. After identifying an active, i.e. antiaggregatory extract (see below), this was fractionated and the active fraction was further sub-fractionated for subsequent chemical analysis by mass spectroscopy, ESI (Electrospray ionization), and COSY (Correlation effect spectroscopy), and functional testing. Anti-platelet activity was assessed in human platelets (platelet rich plasma) using a classical turbidimetric method. Platelets were stimulated with various agonists (arachidonic acid, ADP, epinephrine, collagen, A23187) with and without the addition of the extracts or the fractions /sub-fractions. Both Allium Ursinum and Allium sativum extracts exert antiaggregatory effects with EC50 values around 0.1 mg/ml. The garlic extracts are acting by inhibition of the ADP pathway comparable as known from the clinically used drug clopidegrol.The pharmacological active antiaggregatory component of the extracts appears to be lipophilic rather than hydrophilic. This is the first report on an antiplatelet activity of Allium Ursinum. One final structure determined by HPLC, MS, ESI and COSY which exerts the antiplatelets inhibitory effect is β-sitosterol-3-O-β-D-glucoside of the fraction 7-14 crystals. It is considered that about three up to five grams of dried leaves might be enough to exert antiaggregatory effects (comment: in pharmacy normally dried plant material is used in therapy). The second compound with antiaggregatory activity was identified as 1-β-D-galactopyranoside-2, 3-bis-linolenic glycerate. The problem of loosing the active volatile oily components by drying the leaves in future studies looking for the clinical use may be solved by looking for a raw or a refined extract which would be the form of a real phytomedical drug; for example capsules about 120 to 200 micrograms of an alcoholic or better an heptane / oily extract gained from wood garlic leaves would be an useful drug formulation to reach respective concentrations in blood. However, we have to admit that since our investigations were in-vitro, the in-vivo situation is somewhat different due to the metabolism, which is nearly unknown. Nevertheless, this study shows for the first time that allium ursinum does exert anti-platelet activity and that both allium species can unfold antiaggregatory effects which are worth to be investigated in subsequent in-vivo studies. β-sitosterol-3-O-β-D-glucoside and 1-β-D-galactopyranoside-2, 3-bis-linolenic glycerate could be identified as active antiaggregatory principals.