Dose limiting haematotoxicity has been associated with a range of therapeutic agents used for the treatment of a number of different conditions. Haematotoxicity is usually assessed as part of the preclinical safety studies in experimental animals, where changes in peripheral blood cell numbers and bone marrow cellularity are determined at the end of the study. Often no information on the mechanism of the haematotoxicity is revealed. This thesis demonstrates how in vitro bone marrow cultures can be utilized to assist in the assessment of haematotoxicity by two different approaches; firstly, in vitro bone marrow cultures can be used to assess the haematopoietic lineage specificity of vincristine sulphate, vinblastine sulphate, hydroxyurea and anagrelide hydrochloride using clonogenic cultures, enabling ranking of these compounds according to their haematotoxicity. Secondly, using in vitro assays only, elucidate the mechanism(s) of the megakaryocytic lineage specific inhibition of anagrelide hydrochloride. To this end both clonogenic cultures and LTBMC offer the ability to elucidate mechanisms of action on multipotent stem cells, lineage specific cells and effects on the bone marrow microenvironment following single and repeated administration. In addition, the combination of cell identification techniques flow cytometry and light microscopy was shown to provide a more detailed understanding of the different cell populations within the non-adherent cell layer. In vivo AN reduces platelet counts only, however, the mechanism of the megakaryocyte specific toxicity by AN is not understood. In these studies, the mechanism (s) of the megakaryocytic lineage haematotoxicity of AN was examined using the established human clonogenic and LTBMC. The action of AN was shown to be focused at a late stage in megakaryocyte (Mk) colony development. Ranking the potential mechanisms of action of AN by concentration at which they were noted, the inability to organize the microtubules appears to be secondary to 1) alteration in cell cycling, 2) surface receptor expression and 3) inhibition in achieving high (greater than 8N) ploidy number. However, identification of the primary mechanism based solely on concentration seems to be very crude and most probably reflects a limitation of in vitro systems. The inhibition of platelet production by AN is most likely a result from a combination of mechanisms; inhibition of cell cycling, disruption in the expression of cell surface receptors, inhibition of the ability of the cells to increase ploidy number and an associated inability to organize microtubules leading to a reduction in platelet release. This work also demonstrated the importance of the selection of the source of bone marrow used in the cultures. The concentration at which 50 percent of Mk colony growth was inhibited (IC50) by AN for murine cells was markedly (46 fold) different (88.6μM) compared to the IC50 with human cord blood (hCB) (1.92μM). This disparity is indicative of differences in species sensitivity possibly related to AN having a greater affinity towards the human c-mpl chrombopoietin (TPO) receptor than the equivalent murine receptor as suggested by McCarty et al (2006). This work highlights the utility of in vitro bone marrow cultures as a tool for investigating the lineage specific haematotoxicity by evaluating compounds used in the treatment of ET. In addition in vitro haematopoietic cultures can successfully be used as a tool to investigate potential mechanism(s) of haematotoxicity as demonstrated herein by providing an insight to mechanism of platelet count reduction by AN.

10 ABSTRACT The subject of this study was to evaluate the efect of abiotc elicitor on rutn producton in callus and suspension cultures of buckwheat. The cultvar of buckwheat used for this research was Fagopyrum esculentum Moench var. Bambi, cultvated in Murashige and Skoog nutrient medium with the additon of growth regulator 2,4-dichlorfenoxyacetc acid (2,4-D) in concentraton of 1 mg/l. The elicitor used in this study was a soluton of methylviologen, 1 ml of it was added to the cultures in three diferent concentratons: c1 = 100.0 mg/100 ml, c2 = 10.0 mg/100 ml and c3 = 1.0 mg/100 ml. The elicitor was afectng the cultures for 6, 12, 24, 48, 72 or 168 hours. Afer the defned period of tme, cultures were collected, dried out and stored for further analysis of rutn content. To control samples (without elicitor treatment) 1 ml of ethanol 96% was added and they were collected afer 6, 24, 72 or 168 hours. Releasing of rutn into the nutrient medium was also investgated. Rutn content in each sample of cultures and in each sample of nutrient medium was later determined by HPLC. Any signifcant increase in the producton of rutn was not observed in this study. The maximum amount of rutn detected was 0.1 mg/g DW, thus the lowest quantty detectable, and was found in suspension cultures in three cases, afer the additon of...

Elicitation is one of the methods used for increasing the production of secondary metabolites in vitro cultures. The aim of this work is to evaluate the effect of abiotic elicitor methylviologen (paraquat) on the production of flavonoids in callus and suspension cultures of Fagopyrum esculentum Moench., variety Pyra. The cultures were cultivated in Murashige and Skoog nutrient medium with addition of 2,4-D in concentration of 1 mg/l as a growth regulator. Elicitor was added as a solution in three different concentrations (c1 = 2.1929 · 10-4 mol/l, c2 = 2.1929 · 10-3 mol/l and c3 = 2.1929 · 10-2 mol/l). The effect of elicitation on rutin production was monitored in six time intervals: 6, 12, 24, 48, 72 and 168 hours. The rutin content was determined by HPLC analysis. No rutin was produced in callus and suspension cultures without the presence of elicitor. Even after the elicitation, there was no statistically significant increase in the production of rutin. The maximum rutin content was detected in the suspension culture after 12 hours of elicitor treatment in c2 concentration, the content was 0.1 mg/g DW. The release of rutin into the nutrient medium was also not observed.

Plants are an important source of secondary metabolites, which are a valuable natural substance used in many fields. One way to increase their production is by the elicitation method. In this paper the effect of abiotic elicitor methylviologene in three different concentrations was studied on the rutin production in callus and suspension culture of Fagopyrum esculentum Moench variety Spačinki. The cultivation was carried out on Murashige and Skoog nutrient medium with the addition of a 1 mg/l 2,4- dichlorophenoxyacetic acid as growth regulator. Samples were taken at regular time intervals after 6, 12, 24, 48, 72 and 168 hours of elicitation. The rutin content was analyzed by HPLC. The maximum rutin production (0.3 mg/g DW) was recorded in the callus culture after 48 hours of methylviologene treatment at a concentration of 2.1929.10-4 mol/l. No increase in rutin content after methylviologene elicitation was observed in the suspension culture. The study also included monitoring of the rutin release into nutrient medium, but this was not demonstrated.

Elicitation is one of possible methods for increasing the secondary metabolites production in plant cell cultures. This paper explores the potential effect of methylviologene on isoflavonoids production in Genista tinctoria L. suspension and callus cultures. Schenk and Hildebrandt nutrient medium was used with the addition of growth regulators 2,4- D in concentration of 5 mg/L and kinetin of 1 mg /L of medium. Elicitor was added in the form of ethanol solution at concentration of 2.19·10-2 mol l-1 , 2.19·10-3 mol l-1 , 2.19·10-4 mol l-1 . Samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor exposure. Control samples were collected at 6, 24, 72 and 168 hours. The isoflavonoid content in suspension and callus cultures and in nutrient medium was evaluated by high performance liquid chromatography. The biggest increase of isoflavonoid content in callus culture was reached in daidzein content after 48 hours elicitor treatment in concentration of 2.19·10-3 mol l-1 (8.5 mg/g DW) and 2.19·10-4 mol l-1 (1.6 mg/g DW). The production of genistin, genistein and formononetin was slightly increased or zero compared to controls. Biochanin A was almost absent in the samples. The highest level of isoflavonoids was measured in the suspension culture after 48 hours elicitor application in the...

62 10 ABSTRACT The object of this study was the influence of abiotic elicitor on the production of rutin in suspension culture of Fagopyrum esculentum Moench. The culture was cultivated in Murashigeho and Skoog nutritive medium with growth regulator: 2,4−dichlorfenoxyacetic acid (1 ml/l). The ultrasound was used as abiotic elicitor (0,1 W/cm3 , 35 kHz) for time period of 1, 2, 3, 4 and 5 min. The samples were taken 0, 6, 12, 24, 48, 72 and 168 hours after elicitation. The kontrol samples (without the influence of ultrasound) were taken 24 and 168 hours after elicitation. The amount of rutin was analyzed by HPLC. Suspension culture of Fagopyrum esculentum Moench. didn't produce any rutin under the influence of ultrasound. No release of rutin into the nutritive medium was observed during this study.

Medicinal plant cultures in vitro - XVI The subject of this diploma thesis is the evaluation of secondary metabolites production in Silybum marianum, (L.) Gaertn. cultures in vitro after elicitor treatment. The aim of the study was to find if an abiotic elicitor 5-tert-butyl-N-(4- chlorbenzyl)pyrazine-2-carboxamide increases the flavonolignan production Silybum marianum cultures in vitro. Experiment was carried out in callus and suspension cultures of Silybum marianum using Murashige - Skoog nutrient medium supplemented with 10 mg/l α-naphthylacetic acid. The elicitor was added in the form of solution in three different concentrations (C1 = 3. 292.10-3 mol/l, C2 = 3. 292.10-4 mol/l and C3 = 3. 292.10-5 mol/l) and it was affecting 6, 12, 24, 48, 72 and 168 hours. The content of flavonolignans was determined by HPLC. The maximum flavonolignan production (0. 280 mg.g-1 DW) in callus culture was observed after 24 hours of elicitor application in concentration of C2 = 3. 292.10-4 mol/l, when the highest content of silychristin was detected. The second significant increase in flavonolignan production (0. 271 mg.g-1 DW) in callus culture was noticeable after 12 hours of elicitor treatment in concentration of C3 = 3. 292.10-5 mol/l, when the highest increase in silydianin and silybin B accumulation was found. The...

The subject of this study is the evaluation of secondary metabolites production in Hypericum perforatum L. cultures in vitro after elicitor treatment. The aim was to find if orthosilicic acid as abiotic elicitor increases the flavonoid and hypericin production in Hypericum perforatum L. cultures in vitro. Experiment was carried out in callus and suspension cultures of H. perforatum using Murashige - Skoog nutrient medium78 supplemented with 10 mg. ml-1 α-naphtylacetic acid as growth regulator. The elicitor was added in the form of solution in 3 different concentrations (C1 = 10.4047∙10-3 mol l-1 , C2 = 10.4047∙10-4 mol l-1 , C3 = 10.4047∙10-5 mol l-1 ), it was affecting 6, 12, 24, 48, 72 and 168 hours. The content of flavonoids and hypericin was determined by HPLC. Secondary metabolites release into nutrient medium was also a part of this study. The increasing flavonoid and hypericin production in callus cultures after elicitor application at any concentrations was not observed. The maximum flavonoid content (0.04 mg g-1 DW) in suspension culture was detected after 72 h of elicitor treatment in concentration of C1 where the maximum hyperoside production was observed. The maximum hypericin production (0.21 mg g-1 DW) in suspension culture was detected after 12 h of elicitor application in...

Valeriana glechomifolia é uma espécie vegetal endêmica da região sul do Brasil. Ela acumula valepotriatos em todos os seus órgãos, que são os possíveis componentes sedativos das espécies de Valeriana utilizadas farmaceuticamente. Foi comparado o crescimento in vitro de V. glechomifolia em meios de cultura sólidos Murashige e Skoog completo (MS), com 75% dos nutrientes inorgânicos (MS 75) ou em uma formulação modificada (M ) em culturas mantidas a longo prazo, por até 9 meses sem subcultura. Alterações da biomassa, do desenvolvimento de raízes e partes aéreas, bem como a produção dos valepotriatos acevaltrato, valtrato e diidrovaltrato foram avaliadas mensalmente. O maior aumento de biomassa e desenvolvimento foliar foi detectado em plantas cultivadas em meio MS, e o melhor desenvolvimento radicular foi observado em plantas cultivadas em meio MS modificado (M ) durante o cultivo. A análise por Cromatografia Líquida de Alta Eficiência mostrou que o máximo de rendimento de valtrato e diidrovaltrato foi após os seis meses de cultivo em plantas em meio M , enquanto a maior concentração de acevaltrato foi encontrada em plântulas cultivadas em meio MS 75, após sete meses de cultivo. Os resultados sugerem uma relação direta entre crescimento e acúmulo de valepotriatos, e um efeito positivo do aumento da quantidade de micronutrientes e de mesoinositol nos rendimentos valepotriatos em plantas mantidas em longo período de cultivo. Também foi analisado o efeito neurocomportamental de um extrato contendo uma mistura de valepotriatos (EV) de V. glechomifolia. Camundongos adultos foram tratados com doses de 1, 3 e 10 mg/kg de EV ou veículo, 30 minutos antes dos testes. Durante a exploração no campo aberto, os camundongos tratados com 10 mg/kg mostraram redução na locomoção e no comportamento exploratório (número de rearings) em comparação aos animais controle, e o EV não induziu alteração na ansiedade. Todos os grupos realizaram normalmente a tarefa de memória de reconhecimento de novo objeto, exceto o grupo que recebeu 3 mg/kg, que apresentou piora na memória de reconhecimento do novo objeto. Os resultados indicaram que os camundongos tratados com valepotriatos não apresentaram déficits de memória aversiva de longa duração, e apenas a dose de 3 mg/kg apresentou um prejuízo na tarefa de memória de reconhecimento de novo objeto, além de uma possível propriedade sedativa na dose de 10 mg/kg. / Valeriana glechomifolia is a plant species endemic to southern Brazil. It accumulates the terpene derivatives valepotriates, the presumed sedative components of the pharmaceutically used species of Valeriana, in all of its organs. In vitro growth of V. glechomifolia on solid Murashige and Skoog (MS) without phytohormones at full, 75% (MS 75) or on a modified formulation (M ) was compared in long term stock cultures kept for up to 9 months without subculture. Changes in biomass accumulation, development of roots and shoots, as well as the production of valepotriates acevaltrate, valtrate and didrovaltrate were monthly evaluated. The best root development was observed in plants grown on modified MS medium (M ∆ ), whereas highest biomass accumulation and leaf development were detected in MS medium grown plants throughout the period. High Performance Liquid Chromatography analysis showed maximal valtrate and didrovaltrate yields on M ∆ grown plants harvested after six months of culture, whereas acevaltrate concentration was highest on MS 75 grown plants after seven months of culture. The overall results suggest a direct relationship between growth and valepotriate accumulation, and a positive effect of increases in micronutrient and myo-inositol amounts on valepotriate yields of long-term stock-cultures. An extract containing a mixture of valepotriates (EV) of V. glechomifolia was evaluated in relation to neurobehavioral parameters. Adult mice were treated with doses of 1, 3 and 10 mg/kg of EV or vehicle, 30 minutes before tests. During exploration of an open field, mice treated with 10 mg/kg showed reduced locomotion and reduced exploratory behavior (number of rearings) compared to control animals, and the EV did not induce alterations in anxiety. All groups performed normally the task of novel object recognition memory, except the group receiving 3 mg/kg dose, which showed decrease in novel object recognition memory. The results indicated that mice treated with valepotriates presented no deficits in long-term memory for aversive training and presented an impairment in novel object recognition memory task only at 3 mg/kg, as well as a possible sedative proprieties at 10 mg/kg.

Valeriana glechomifolia é uma espécie vegetal endêmica da região sul do Brasil. Ela acumula valepotriatos em todos os seus órgãos, que são os possíveis componentes sedativos das espécies de Valeriana utilizadas farmaceuticamente. Foi comparado o crescimento in vitro de V. glechomifolia em meios de cultura sólidos Murashige e Skoog completo (MS), com 75% dos nutrientes inorgânicos (MS 75) ou em uma formulação modificada (M ) em culturas mantidas a longo prazo, por até 9 meses sem subcultura. Alterações da biomassa, do desenvolvimento de raízes e partes aéreas, bem como a produção dos valepotriatos acevaltrato, valtrato e diidrovaltrato foram avaliadas mensalmente. O maior aumento de biomassa e desenvolvimento foliar foi detectado em plantas cultivadas em meio MS, e o melhor desenvolvimento radicular foi observado em plantas cultivadas em meio MS modificado (M ) durante o cultivo. A análise por Cromatografia Líquida de Alta Eficiência mostrou que o máximo de rendimento de valtrato e diidrovaltrato foi após os seis meses de cultivo em plantas em meio M , enquanto a maior concentração de acevaltrato foi encontrada em plântulas cultivadas em meio MS 75, após sete meses de cultivo. Os resultados sugerem uma relação direta entre crescimento e acúmulo de valepotriatos, e um efeito positivo do aumento da quantidade de micronutrientes e de mesoinositol nos rendimentos valepotriatos em plantas mantidas em longo período de cultivo. Também foi analisado o efeito neurocomportamental de um extrato contendo uma mistura de valepotriatos (EV) de V. glechomifolia. Camundongos adultos foram tratados com doses de 1, 3 e 10 mg/kg de EV ou veículo, 30 minutos antes dos testes. Durante a exploração no campo aberto, os camundongos tratados com 10 mg/kg mostraram redução na locomoção e no comportamento exploratório (número de rearings) em comparação aos animais controle, e o EV não induziu alteração na ansiedade. Todos os grupos realizaram normalmente a tarefa de memória de reconhecimento de novo objeto, exceto o grupo que recebeu 3 mg/kg, que apresentou piora na memória de reconhecimento do novo objeto. Os resultados indicaram que os camundongos tratados com valepotriatos não apresentaram déficits de memória aversiva de longa duração, e apenas a dose de 3 mg/kg apresentou um prejuízo na tarefa de memória de reconhecimento de novo objeto, além de uma possível propriedade sedativa na dose de 10 mg/kg. / Valeriana glechomifolia is a plant species endemic to southern Brazil. It accumulates the terpene derivatives valepotriates, the presumed sedative components of the pharmaceutically used species of Valeriana, in all of its organs. In vitro growth of V. glechomifolia on solid Murashige and Skoog (MS) without phytohormones at full, 75% (MS 75) or on a modified formulation (M ) was compared in long term stock cultures kept for up to 9 months without subculture. Changes in biomass accumulation, development of roots and shoots, as well as the production of valepotriates acevaltrate, valtrate and didrovaltrate were monthly evaluated. The best root development was observed in plants grown on modified MS medium (M ∆ ), whereas highest biomass accumulation and leaf development were detected in MS medium grown plants throughout the period. High Performance Liquid Chromatography analysis showed maximal valtrate and didrovaltrate yields on M ∆ grown plants harvested after six months of culture, whereas acevaltrate concentration was highest on MS 75 grown plants after seven months of culture. The overall results suggest a direct relationship between growth and valepotriate accumulation, and a positive effect of increases in micronutrient and myo-inositol amounts on valepotriate yields of long-term stock-cultures. An extract containing a mixture of valepotriates (EV) of V. glechomifolia was evaluated in relation to neurobehavioral parameters. Adult mice were treated with doses of 1, 3 and 10 mg/kg of EV or vehicle, 30 minutes before tests. During exploration of an open field, mice treated with 10 mg/kg showed reduced locomotion and reduced exploratory behavior (number of rearings) compared to control animals, and the EV did not induce alterations in anxiety. All groups performed normally the task of novel object recognition memory, except the group receiving 3 mg/kg dose, which showed decrease in novel object recognition memory. The results indicated that mice treated with valepotriates presented no deficits in long-term memory for aversive training and presented an impairment in novel object recognition memory task only at 3 mg/kg, as well as a possible sedative proprieties at 10 mg/kg.