Il est présentement difficile de tester les médicaments anti-psoriasiques sur de nombreux échantillons de peaux pathologiques en raison de problèmes éthiques, de disponibilité et de variations interindividuelles. Ainsi, les formulations de médicaments proposées à la population atteinte de psoriasis ne sont pas bien adaptées à leur situation réelle. La pénétration cutanée de drogues est une méthode qui offre un grand potentiel et qui plus est particulièrement attrayante comparativement aux méthodes conventionnelles d'injection ou d'administration orale. Le principal objectif de notre étude est de présenter un nouveau modèle de substituts cutanés pathologiques faits à partir de cellules de patients psoriasiques et selon la méthode d'auto-assemblage, afin de tester la pénétration de différentes molécules et de vérifier si le substitut est similaire au tissu pathologique in vivo. En comparaison à la peau normale humaine et à la peau de souris, une fonction barrière efficiente a été observée pour les substituts de peau faits à partir de cellules de patients sains lorsque les molécules suivantes ont été appliquées: l'acide benzoïque, la caféine et l'hydrocortisone. Il est connu dans la littérature que les peaux de patients psoriasiques sont plus perméables. Cette même caractéristique a été observée pour les substituts cutanés préparés à partir de cellules de patients psoriasiques. Ces tests de pénétration cutanée sont un premier pas vers la validation d'un modèle de substituts cutanés pathologiques, précisément psoriasiques, en vue de pouvoir le considérer en tant qu'outil efficace pour des études dermopharmacologiques d'agents anti-psoriasiques.

L'usage d'animaux axéniques comme modèles a augmenté exponentiellement dans les dernières années en recherche expérimentale. Afin de conserver leur statut axénique, les animaux doivent être contenus dans des isolateurs traditionnels ou des isolateurs au niveau de la cage, incluant la cage scellée à pression positive de Tecniplast : l'Isocage. Cependant, le moyen le plus efficace de confirmer leur statut axénique des animaux reste incertain, particulier pour les isolateurs au niveau de la cage, comportant un plus grand risque de contamination que l'isolateur traditionnel en raison de leur contact plus fréquent avec l'environnement extérieur. Ce projet de maîtrise a pour buts de gérer le risque de contamination associé à l'Isocage en optimisant la désinfection par immersion et en déterminant l'efficacité des mesures de précautions axéniques, puis finalement de déterminer l'efficacité des mesures de détection à démontrer la présence de contaminants en cages axéniques Les travaux d'optimisation de la désinfection par immersion a permis de déterminer la charge microbienne résiduelle retrouvée à la surface des Isocages entre deux changements de cage, d'évaluer l'efficacité sporicide de deux désinfectants, et finalement de déterminer le temps optimal de désinfection pour l'immersion des cages axéniques lors d'un changement de cage selon la concentration du désinfectant utilisé. L'efficacité de mesures de précaution axéniques a été confirmée en comparant les flores microbiennes retrouvées à la surface de cages contenues dans des locaux axéniques et non axéniques. L'efficacité des mesures de détection à démontrer la présence de contaminants en Isocage a été déterminée en développant, validant et optimisant trois différentes méthodes, puis en testant in situ leur efficacité à détecter des contaminants artificiellement introduits dans des cages axéniques. Cet ouvrage constitue un apport important au domaine puisqu'il est l'un des premiers à aborder la gestion des risques et l'efficacité des méthodes de détection lors d'utilisation de l'Isocage et du bassin d'immersion annexé à la station de biosécurité avec des animaux axéniques. / The use of axenic animals as models have increased exponentially in recent years in experimental research. In order to maintain their axenic status, animals must be contained in traditional isolators or isolators at the cage level, which includes Tecniplast's sealed positive pressure Isocage. However, the most effective way to confirm animal axenic status remains uncertain, especially for cage-level isolators, which carry a greater risk of contamination than the traditional isolator due to their most frenquent contact with outer environment. This Master's project aims were to manage the risk of contamination associated with Isocage by optimizing disinfection by immersion and determining the effectiveness of axenic precautionary measures; then finally to determine the effectiveness of the detection measures to demonstrate the presence of microbial contaminants in axenic Isocages. The optimization of disinfection by immersion made it possible to determine the residual microbial load found on the surface of Isocages between two cage changes, to evaluate the sporicidal efficiency of two disinfectants, and finally to determine the optimal disinfection time for the immersion of the axenic cages during a change of cage depending on the concentration of disinfectants used. The effectiveness of axenic precautionary measures has been confirmed by comparing the microbial flora found on the surface of cages contained in axenic and non-axenic rooms. The effectiveness of detection measures to demonstrate the presence of contaminants in Isocage has been determined by developing, validating and optimizing three different methods, then by testing in situ their effectiveness to detect contaminants artificially introduced into axenic cages. This work constitutes an important contribution to the field since it is one of the first to discuss risk management and the effectiveness of detection methods when using Isocage and the immersion basin attached to the biosecurity station with axenic animals.

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Call number: LD2668 .T4 1962 B73

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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.

Affecting tissue cultures with precursors is one of methods that increases the production of secondary metabolites in vitro. This thesis deals with precursors affecting production of mescaline in suspension tissue cultures of Trichocereus pachanoi. As precursors dopamine, D, L-tyrosine, casein hydrolysate and shikimic acid were used. Three concentrations of these precursors were prepared. Suspension cultures with different concentrations of precursors were analysed by HPLC method after 48 and 168 hours. Murashige and Skoog medium was used for cultivation. Mescaline biosynthesis was most affected by dopamine. The largest amount of mescaline was produced in suspension culture with the highest concentration of dopamine 50 mg/100 ml.The culture was cultivated for 168 hours. Suspension cultures that were cultivated for 48 hours produced the highest amount of mescaline with the highest concentration of dopamin 50 mg/100 ml. The biosynthesis of mescaline was inhibited by other precursores (D, L-tyrosine, casein hydrolysate, shikimic acid).

Production of hypericine in explantate cultures of Hypericum perforatum Pavel Sazama Diploma thesis Charles university in Prague, Faculty of Pharmacy in Hradec Králové, Pharmacy Key words: St. John's Wort, precursors, hypericine, flavonoids, acetate, cinnamic acid, cinnamate, tyrosine, shikimic acid The goal of this work was to affect the production of hypericin (naphto-dianthrone), hyperoside and quercitrin (flavonoids) in the suspensional Hypericum perforatum explantate cell cultures. The method of precursor feeding was used. The precursors of naphtodianthrones and flavonoids were added into the medium in final concentrations 10 mg.l-1, 50 mg.l-1 and 100 mg.l-1. Samples were taken after 72 and 168 hours after adding the precursor. Potassium acetate, cinnamic acid, sodium cinnamate, tyrosine and shikimic acid were used as precursors. Cultures were cultivated on the Murashige and Skoog medium with the addition of the growth stimulator α-NAA. Concentration of hypericin, hyperosid and quercitrin was measured by HPLC analysis. The highest influence on the production of hypericin in cultures was detected after adding of tyrosine. The concetration of hypericin in cells raised in this experiment from 0,003% (control culture) to 0,03% (culture with tyrosine concen-tration of 100 mg.l-1). This...

In vitro cultures of medicinal plants - XI. Elicitation is one of the few strategies that can be used in enhancement of secondary metabolites production from explant cultures. The effect of abiotic elicitor (silver nitrate) on flavonolignan and flavonoid taxifolin production in suspension culture of Silybum marianum L. (Gaertn.) and on isoflavones production in suspension culture of Genista tinctoria L. was tested. Silver nitrate in various concentrations (5.887.10-3 mol/l; 5.887.10-4 mol/l; 5.887.10-5 mol/l) was used as elicitor. Content of secondary metabolites in suspension cultures was determinated by high performance liquid chromatography (HPLC). The samples were taken after 6, 12, 24, 48, 72 and 168 hours after elicitor treatment. The highest content of taxifolin production (0.02 %) in suspension culture of Silybum marianum L. (Gaertn.) after silver nitrate (5.887.10-4 mol/l) treatment and 72 h sampling was detected. The highest content of genistin (0.05 %) in suspension culture of Genista tinctoria L. was found after silver nitrate (5.887.10-4 mol/l) treatment and 48 h sampling. The highest content of daidzein (0.09 %) was detected after elicitor (5.887.10-4 mol/l) treatment and 168 h sampling.