Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom-up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies. / Ph. D.

Para que um produto de aplicação tópica apresente o efeito terapêutico é necessário que o fármaco seja liberado de seu veículo e atravesse a barreira do estrato córneo, atingindo seus sítios de ação nas camadas da pele nas quais o efeito é esperado. Os estudos de penetração cutânea in vitro, utilizando células de difusão de Franz modificadas, podem fornecer resultados úteis quanto à eficácia do produto. As dermatofitoses são micoses superficiais. São manifestações crônicas e resistentes ao tratamento, fato que se explica não só em decorrência da própria natureza destas afecções como também devido à baixa penetração de fármacos antimicóticos na pele. Dentre os antifúngicos disponíveis na terapêutica, ciclopirox olamina é uma substância que tem demonstrado elevada eficácia no tratamento de dermatofitoses. O presente trabalho teve como objetivo desenvolver formulações de uso tópico contendo 0,5% de ciclopirox olamina e avaliar a performance dessas formulações. Foi possível obter formulações estáveis fisicamente, contendo promotores de penetração cutânea. O sistema de amostragem automática utilizado para realização dos experimentos foi qualificado e apresentou-se em condições adequadas. / In order to present the pharmacological effect, a topical product must release the pharmaceutical active ingredient from its vehicle and this active must pass through the stratum corneum barrier, reaching the target layer of the skin, where its effect is desired. In vitro skin penetration studies, using modified diffusion Franz cells, can provide useful results regarding the product efficacy. Dermatophytosis is superficial mycosis resistant to treatment due to the disease nature and also to the low penetration profile of the antifungals into the skin. Among the antifungals available in the therapeutic arsenal, ciclopirox olamine has demonstrated a high efficacy in the treatment of dermatophytosis. The objectives of the present work were to develop topical formulations containing 0,5% of ciclopirox olamine and evaluate their performance. It was possible to obtain physically stable formulae, containing skin penetration enhancers. The automatic sampling system used in the experiments was qualified, showing adequate performance.

Para que um produto de aplicação tópica apresente o efeito terapêutico é necessário que o fármaco seja liberado de seu veículo e atravesse a barreira do estrato córneo, atingindo seus sítios de ação nas camadas da pele nas quais o efeito é esperado. Os estudos de penetração cutânea in vitro, utilizando células de difusão de Franz modificadas, podem fornecer resultados úteis quanto à eficácia do produto. As dermatofitoses são micoses superficiais. São manifestações crônicas e resistentes ao tratamento, fato que se explica não só em decorrência da própria natureza destas afecções como também devido à baixa penetração de fármacos antimicóticos na pele. Dentre os antifúngicos disponíveis na terapêutica, ciclopirox olamina é uma substância que tem demonstrado elevada eficácia no tratamento de dermatofitoses. O presente trabalho teve como objetivo desenvolver formulações de uso tópico contendo 0,5% de ciclopirox olamina e avaliar a performance dessas formulações. Foi possível obter formulações estáveis fisicamente, contendo promotores de penetração cutânea. O sistema de amostragem automática utilizado para realização dos experimentos foi qualificado e apresentou-se em condições adequadas. / In order to present the pharmacological effect, a topical product must release the pharmaceutical active ingredient from its vehicle and this active must pass through the stratum corneum barrier, reaching the target layer of the skin, where its effect is desired. In vitro skin penetration studies, using modified diffusion Franz cells, can provide useful results regarding the product efficacy. Dermatophytosis is superficial mycosis resistant to treatment due to the disease nature and also to the low penetration profile of the antifungals into the skin. Among the antifungals available in the therapeutic arsenal, ciclopirox olamine has demonstrated a high efficacy in the treatment of dermatophytosis. The objectives of the present work were to develop topical formulations containing 0,5% of ciclopirox olamine and evaluate their performance. It was possible to obtain physically stable formulae, containing skin penetration enhancers. The automatic sampling system used in the experiments was qualified, showing adequate performance.

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...

Charles Univeristy in Prague, Faculty of Pharmacy in Hradec Králové Department Department of Pharmaceutical Chemistry and Drug Control Author: Marek Tauchman Supervisor: Prof. PharmDr. Martin Doležal, Ph.D. Title of Diploma Thesis: Pyrazine Derivatives as Potential Antituberculosis Drugs II. Tuberculosis still presents serious worldwide problem of today. The situation is complicated especially by increasing proportion of strains resistant to common antituberculotics. Therefore the need of a new compound active against mycobacterial causer of the disease is very actual. Synthesis of compounds derived from pyrazinamide, very effecitve anti-mycobacterial substance, is one of the perspective way of new drugs development. Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Králové, beside others, deals with this problem in a long term. There were synthesized hundreds of compounds containing pyrazine core and they were tested to antimycobacterial activity. The target of this thesis is join this effort and contribute to increase the number of compound that has been studied for antituberculosis activity. At the beginning of thesis, there is a summary of facts about tuberculosis, such as incidence, patogenesis and cure. Next, there are informations about newly developed...

Active compounds have been always originated from plants. Plants though, were able to produce only very low amounts of them and that was the reason for trying many alternative ways of production, one of them being plant tissue culture cultivation. This method is any fragment of living tissue or organ taken from an intact plant or an already existing explant culture, with the intention of growing an artificial growth medium. Even this method though, is not able to produce large amounts compared to extraction from field plants. Elicitation is considered a possible way to increase the production of secondary metabolites. This method used the plant's own defense system, in order to increase the production of secondary metabolites in vitro. The compound which is used to produce the effect is called elicitor. During our experimental work I used as an elicitor the compound Ethephon (2-Chloroethylphosphonic acid) upon callus and suspension cultures of Hypericum perforatum, with intention to observe its effect on flavonoid production. This experiment was based on three different concentrations and six different withdrawal times, being 6, 12, 24, 72, 168 hours. The maximum effect of elicitor was reached with concentration c1 (1mg/100ml) after 12 hours and with concentration c3 (100mg/100ml) after 72 hours.

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...

A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine Faculty of Health Sciences University of the Witwatersrand Johannesburg 2015 / Treatment of hormone-dependent breast tumours is typically conducted using hormone-therapy, with NK cell immunotherapy a novel modality. In this study, the effects of hormone-therapy and combined hormone-therapy with immunotherapy in MCF-7 breast cancer cells was investigated. A hormone pre-treatment of 17β-oestradiol and progesterone was performed to simulate the in vivo microenvironment. Subsequently cells were treated with the hormone-therapy drugs Anastrozole or RU486 alone, or with continued hormone simulation. Combined therapy was conducted with in vitro activated NK cells co-cultured with MCF-7 cells undergoing hormone-therapy. Biomarkers ERα, PR and MUC1 were immunolocalised and expression analysed qualitatively, and quantitatively using image analysis software. Hormone pre-treatment reduced biomarker expression, stressing the importance of hormone environment simulation for in vitro experimentation. Hormone-therapy increased cytoplasmic ERα and decreased PR expression. Anastrozole increased MUC1 and RU486 decreased nuclear MUC1. With continued hormone simulation, Anastrozole further decreased all biomarkers whereas RU486 decreased ERα, increased PR expression with variable effects on MUC1 expression. RU486 induced MUC1/PR and MUC1/ERα correlation, which, under continued hormone simulation, was maintained in the nucleus only. Anastrozole induced MUC1/PR correlation in the cytoplasm which was maintained under continued hormone simulation. Hormone-therapy also induced a decrease in apoptosis, with continued hormone simulation abrogating Anastrozole induced apoptosis. While hormone-therapy did not increase proliferation, the associated changes observed in biomarker expression are linked with tumour progression indicating that short-term treatment may be detrimental for overall survival. Combined NK cell immunotherapy resulted in decreased PR, while ERα and MUC1 expression increased in a hormone-dependent manner. Biomarker correlation was evident, albeit reduced with continued hormone simulation. Independently of hormone-therapy and hormone stimulation, immunotherapy reduced apoptosis, contrary to expectation. Proliferation was marginally reduced by immunotherapy. The results indicate that immune cell function is inhibited by interaction with tumour cells, an effect that hormone-therapy cannot abrogate. Furthermore, that this study shows treatment alters both nuclear and cytoplasmic expression of biomarkers, indicates that diagnostic procedures should consider both cellular compartments in tumour progression. It is further shown that qualitative analysis of biomarker expression is not always validated by quantitative analysis, with the latter proposed as a more objective and precise method to be used diagnostically.