Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucia Šinaľová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Testing of the cytotoxicity of potential drugs in the cell lines together with the measurement of their passage through the blood brain barrier by the PAMPA method This master thesis focuses on the prediction of tacrine derivatives permeability through blood- brain barrier and their cytotoxicity. We studied tacrine-benzothiazole and tacrine- thiaquinazoline derivates as potential drugs for treatment of Alzheimer's disease. The reason of testing new tacrine derivates was effort to find substances with same effect and lower toxicity, for which was the tacrine discarded from clinical practice. The probable permeability of the studied substances was determined in vitro by the PAMPA method. Based on the permeation coeficient values we have identified that tacrine-benzothiazole derivates A-D (Pe= 8,31- 16,9×10-6 cm/s) and tacrine-thiaquinazoline derivates 1, 3, 4 (Pe= 8,59-14,9×10-6 cm/s) were permeable through blood-brain barrier, tacrine-thiaquinazoline derivate 2 (Pe= 3,79×10-6 cm/s) had uncertain permeability and tacrine-thiaquinazoline derivate 5 (Pe= 2,0×10-6 cm/s) was not permeable. In the pre-clinical evaluation of...

Cyanobacteria are well-known producers of secondary metabolites of different chemical structures and a wide range of biological functions. In the present thesis, the cytotoxic activity of cyanobacteria, originating from different habitats, was studied in order to reveal whether cytotoxicity is an environmentally dependent characteristic. In addition, the data were compared with the toxicity of these extracts to the model invertebrate Artemia salina. The obtained data suggest that cytotoxic cyanobacteria are favoured under some conditions and thus more frequent in particular localities. The majority of the studied extracts and fractions exhibited cytotoxitity to the Sp/2 cell line not accompanied by toxicity to A. salina. Moreover, in most of the strains with both activities to A. salina and Sp/2 cells the toxic effect was caused by an identical fraction. This result suggests that the toxic effect of the cyanobacterial secondary metabolites is mostly affecting basal cell metabolism rather than targeting specific organisms. In one of studied strains, Cylindrospermum sp. C24/1989, novel lipopeptides puwainaphycin F and G have been detected, isolated and their structure and biological effect have been characterized. Both of these structures interfere with eucaryotic membranes and cause a Ca2+ leakage into the cell. Subsequently, an enhanced tyrosine phosohorylation and relocalization of f-actin in the cell was observed. Lastly, the correlation between metabolite production and the reconstructed phylogeny was studied in planktonic Dolichospermopsis strains. Most of the detected compounds were found to be randomly disspersed across the reconstructed phylogeny and thus cannot be considered as good chemotaxonomic markers. This result also hampers the possible detection of toxic cyanobacteria by morphological methods or molecular detection based on the 16SrDNA gene.

In past years, nanoparticles have been studied as possible platform to be used in biomedicine. In order to establish the application potential of nanoparticles, its impact to biological systems have to be determined. Herein, several silicon-based nanoparticles of different origins were studied in respect of their influence on metabolic activity of human cells, namely osteoblast cell line SAOS-2 and monocytic cell line THP-1. The obtained results proposed that the impact of nanoparticles on cells is highly dependent on cultivation conditions in which nanoparticles are administered to cells. Furthermore, microscopy experiments were implemented in order to localize the particles within cells, where conventional microscopy limitations are evident. Key words: silicon nanoparticles, quantum dots, cell-particle interaction, cytotoxicity

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kafuňková Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Evaluation of liver toxicity in vitro The subject of the diploma thesis is toxicity evaluation of newly synthesised substances on a cellular model representing a hepatocyte. The tested substances have been provided by the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University, as potential antifungal pharmaceuticals and medicine effective against Methicillin-resistant Staphylococcus aureus (MK-NO2-1, MK-NO2-2, DAB-5-K, PABA-Me-5, PABA-Et-5, MK-F-1, PABAN- 3, PABAN-5, MK-F-2, MK-CF3-1, MK-CF3- 2). In order to determine the toxicity we have implemented two methods. The first method is based upon measuring metabolic activity of cells by means of reducing tetrazolium to a colored product. The second method detects the amount of LDH released as a marker of toxicity. The human hepatoma cell line HepG2was used as a model. . The IC50 and EC50 parameters were used to assess the degree of viability and cytotoxicity. The final values obtained from the first method indicated all tested substances showed a certain level of toxicity to the hepatic tissue. MK-CF3-2 is the most...

The presented diploma thesis is focused on the study of composition and biological effects of some super-foods. Theoretical part deals with basic information about chosen superfoods and their bioactive substances. Further, theoretical part describes the overview of vesicular systems used for encapsulation and the most common methods of particle characterization. A brief review of cell cultures and cultivation of human cells is presented as well as methods for cytotoxicity a genotoxicity testing. In the experimental section, aqueous and ethanol extracts of super-foods were prepared. These extracts were then encapsulated into liposomal and combined PHB particles. Super-food extracts were characterized by spectrophotometrical methods in order to determine the content of polyphenols, flavonoids, anthocyanins, carotenes, chlorophyll, tannins, and antioxidant activity. The physico-chemical characteristics of prepared liposomal and combined particles were determined too. The particles with encapsulated extracts were further tested using the MTT assay and SOS chromotest to describe their potential cytotoxic and genotoxic effects.

The main goal of this thesis was to analyse the amount of beta glucans in selected mushroom species. The overall characterization of the composition and content of bioactive compounds in mushrooms was studied too. The theoretical part was focused on description of Fungi, especially the Basidiomycota phyla. The overview of fungal morphology and chemical composition was elaborated. Further, theoretical basis of analytical methods used was introduced. In the experimental part, extracts from selected species of mushrooms (shiitake, oyster mushroom, white champignon, brown champignon and jew’s ear) were prepared. Overall group parameters such as polyphenols, flavonoids, antioxidant activity were determined by spectrophotometry. The amount of beta-glucans in the fungi was measured after enzyme hydrolysis using commercial kit. The content and composition of fatty acids was measured by gas spectrometry. Using emission spectrometry (ICP/OES) the presence of heavy metals was analysed in fungal extracts. Cytotoxicity was tested by MTT test on human cell lines. In the last part the antimicrobial activity against Gram-positive bacterium Micrococcus luteus, Gram-negative Serratia marcescens and yeast Candida labrata was tested. In the thesis, a complex characterization of composition and biological effect of some mushrooms was given.

Tato práce se zabývá syntézou sférických zlatých nanočástic pro biomedicínské aplikace. Zlaté nanočástice byly syntetizovány ekologicky nezávadnými metodami, kterým je věnována i značná část rešerše. Cílem bylo nasyntetizovat stabilní koloidní zlaté nanočástice vhodné pro různé biomedicínké aplikace zejména, pro in vivo a in vitro zobrazovací metody, kterých přehled je take obsažen v teoretické části práce. Také byl proveden test cytotoxicity, jelikož částice mají být použitelné pro in vivo aplikace. Částice byly dale charakterizovány metodami SEM, DLS a UV-VIS.

The diploma thesis deals with synthesis of gold nanoparticles and their surface functionalization suitable for in vitro imaging. In this view there are requirements for optical properties such as stability, monodispersity and no presence of cytotocxicity. In order to fulfill these demands the synthesis by Turkevich method with surface modification by glutathione and polytethylenglycol was performed. Analytical methods such as DLS, SEM and Zeta potential measurement were utilized to characterize the physical and chemical properties of synthesized gold nanoparticles. Finally, MTT assay was performed to evaluate toxicity of gold nanoparticles using HEK 293 cell line.

The application of nanoparticles in the field of theranostics requires knowledge of the specific interaction of nanoparticles with the immune system. One of the first cells with which nanoparticles interact when given to the body are cells of the mononuclear phagocytic system. The aim of this diploma thesis is to prepare an in vitro study that describes the effect of two types of gold and three types of silicon ultra-small nanoparticles on immune cells. Immune cells are presented in the form of primary PBMCs isolated from whole blood , and cells of monocytic cell line THP-1 in the form of monocytes and differentiated macrophages. During the experiments with primary cells, emphasis is placed on maintaining the concept of personalized protein corona. After characterization of the immune cells used, cells are subsequently stimulated with ultra-small nanoparticles and the influence of these nanoparticles on cell metabolism, viability, degree of differentiation and secretion of pro- and antiinflammatory cytokines is monitored. The outcome takes into account further use of the tested nanoparticles in the field of biomedicine. Key words: primary monocytes, cell lines, differentiation, macrophages, cytokines, cytotoxicity

Miklová V.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus II. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2020. This Diploma Thesis was carried out at the Department of Pharmaceutical Botany FAF UK and it is a part of a screening of biologically active secondary metabolites of selected on taxa of the genera Centaurea L. and Psephellus L. in the family Asteraceae. Secondary metabolites are responsible for various effects on the human body. The study is focused on the phytochemical research of extracts prepared from aerial parts (cauloms with leaves) of Centaurea cyanus L., Centaurea stoebe L., Cyanus montanus (L.) Hill, Centaurea benedicta L., Centaurea jacea L., Centaurea macrocephala Muss. Puschk. ex. Willd, Centaurea solstitialis L., Centaurea nigra L., Centaurea scabiosa L., Psephellus bellus (Trautv.) Wagenitz, Centaurea pannonica (Heuff.) Hayek and Psephellus dealbatus (Willd) K. Koch. Both ethyl acetate and summary ethanolic extracts were prepared for detection of individual groups of secondary metabolites by thin-layer chromatohraphy. Consistently with previously published results, sesquiterpenes, flavonoids, coumarins, phenolic compounds,...