Neuroblastoma is the most common extracranial solid tumor that occurs during infancy. Despite the great progress has been made in contemporary clinic medicine some forms of neuroblastoma disease are still found very difficult to treat . This work focuses on the effects of histone deacetylase inhibitors (HDAC) in the neuroblastoma cell lines. It is known that HDAC inhibitors may contribute to recurrence of the tumor cells by affecting the chromatin structure and thus increase the expression of critical tumor suppressor genes. These genes activate apoptotic pathways that may even be independent of caspases. We observed the efficiency of used HDAC inhibitors as under standard conditions an in hypoxia (1 % O2). Inadequate amount of oxygen supply is one of the characteristic features of tumors and it also may contribute to chemoresistance. With the hypoxia-induced chemoresistance of tumor cells, the influence of HIF-1α is expected. Some HDAC inhibitors reduce the amount of HIF-1α in hypoxia and thus HIF transcription factor activity. Thus, the first part of this study is concerned with the acquisition of suitable experimental arrangement for the monitoring of induction of cellular death in human neuroblastoma cell lines SK-N-AS and UKF-NB-3. Secondly, this paper provides the evaluation of the influence...

Faschingbauer J.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019. During the screening of biologically active secondary metabolites of plants carried out at the Department of Pharmaceutical Botany FAF UK, selected taxa of the genus Centaurea (Asteraceae) were investigated. This study is focused on a basic phytohemical research of extracts prepared from Centaurea cyanus, Centaurea jacea, Centaurea scabiosa, Centaurea pseudophrygia, Centuarea stoebe, Centaurea solstitialis a Centaurea benedicta. Extracts were prepared for evidence of the proof reactions of TLC and MS analysis (EI, ESI) to clarify a potential presence of alkaloids. EtOAc and ethanol extracts were evaluated for potential inhibitory activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) and cytotoxicity against selected 9 tumor lines. C. cyanus alkaloid extract had interesting cholinesterase activity which selectively inhibited BChE (IC50 BChE = 22.62 ± 3.62 μg / ml, IC50 AChE = 221.50 ± 44.56 g / ml). Other EtOAc extracts of selected Centaurea species were considered inactive (IC50 > 100 μg/ml)....

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Studentka: Lucie Vašková Školitel: RNDr. Jana Maixnerová, Ph.D. Název diplomové práce: Study on cytotoxicity of compounds in vitro The subject of this diploma thesis was to assess the effect of newly synthesized antimycobacterial substances on the viability of human hepatocellular carcinoma (HepG2) cells. The tested substances were esters (HE-nMe, HE-4PHOPH, HE-KARVA, HE-2NAFT, HE-METRO, HE-CH2PY, HE-8CHIN) and thioesters (HES-4H, HES- nETH) of antituberculotic isoniazid. Experiments performed with these substances have shown, that like isoniazid, the substances inhibit InhA enzyme in mycobacteria and therefore interfere with cell wall biosynthesis. Isoniazid is a drug standardly used in the first line of TB treatment. Together with other first-line antituberculotics, some hepatotoxic potential has been reported during treatment. To assess the possible cytotoxic effect of the tested isoniazid derivatives, the standard human hepatocyte cell line HepG2 was chosen as the cell model. Cell viability was assessed by a colorimetric method that measures the metabolic activity of cells based on the reduction of the tetrazolium compound MTS. Obtained values were quantitatively compared using the toxicological...

Rýdlová Kateřina: Alkaloids Narcissus 'Dutch Master' (Amaryllidaceae) and their biological activity III. Diploma thesis 2017. Charles university in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology. The aim of this work was isolation of compounds from the selected fraction ND 3 - 5 obtained by column chromatography of a Narcissus 'Dutch Master' alkaloid extract. Preparation of the extract and its column chromatography was performed by Mgr. Daniela Hulcová as a part of her doctoral study. Two substances NDM-1 and NDM-2 were isolated from fraction ND 3 - 5 by column chromatography and preparative TLC. The structures were determined as (+)-masonine and (+)-homolycorine on the basis of NMR, GC-MS analysis, optical rotation and their comparison with literature data. Isolated alkaloids were tested on inhibitory activity against human erythrocyte acetylcholinesterase, plasma butyrylcholinesterase and prolyloligopeptidase. Activity of alkaloids was expressed as IC50 values: (+)-masonine (IC50 AChE = 305 ± 34 μM, IC50 BuChE = 229 ± 24 μM, IC50 POP = 314 ± 34 μM), (+)-homolycorine (IC50 AChE = 63.7 ± 4.3 μM, IC50 BuChE = 151 ± 20 μM, IC50 POP = 173 ± 41 μM). In comparison with standards of galanthamine (IC50 AChE = 1.710 ± 0.1 μM, IC50 BuChE = 42.3 ± 1.3 μM),...

4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Dimitrios Vagiannis Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines Tyrosine kinases are important enzymes regulating crucial cellular processes including differentiation, proliferation, apoptosis, transcription, metabolism, and intercellular communication. Deregulation of these enzymes is the cause of various types of cancers. The blockade of their function by tyrosine kinase inhibitors (TKis) is considered a promising approach especially in antitumor pharmacotherapy. ATP-binding cassette (ABC) drug efflux transporters are a family of transmembrane proteins that pump a variety of structurally unrelated compounds out of the cell in an energy-dependent manner. They play an important role in pharmacokinetics (affect absorption, distribution, elimination) and, at the same time, can negatively influence efficacy of chemotherapy (participate in multidrug resistance phenomenon). In our research, we evaluated antiproliferative properties of four selected TKis, namely alectinib, brivanib, osimertinib and selumetinib, in MDCKII cell lines (parent one and those transduced with human...

The presented diploma thesis deals with the optimalisation of preparation and the preparation of combined nanostructured antimicrobial biomaterials itself. In the theoretical part, a review focused on used materials and consequently preparation of nanoparticles and nanofibers was elaborated. Furthermore, the used antimicrobial substances – curcumin and ampicillin, and the principle of cytotoxicity assay were described.In practical part the optimalisation process is described. Furthermore the safety of prepared materials and used antimicrobial substances on HaCaT cell line was tested, in order to confirm their possible further use in cosmetic and pharmaceutical industry. Great part of the thesis deals with evaluation of the antimicrobial activity of used substances and prepared combined nanomaterials on multiple microorganisms from grampositive bacteria, gramnegative bacteria and yeasts. Also the release speed of antimicrobial substances from prepared nanomaterials was determined by spectrophotometer. The amount of released ampicillin from prepared nanomaterials was determined by liquid chromatography.

The presented doctoral thesis is focused on preparation of nanoparticles and nanofibers with natural active ingredient and testing their biological effects. Modern types of application forms were prepared from biomaterials based on one or more natural polymers. Chitosan particles were prepared from cross-linked polymer using ultrasonication. A mixture of soy lecithin and cholesterol was used for preparation of liposomes. Poly-3-hydroxybutyrate was used for preparation of combined liposomes, too. All liposome particles were prepared by ultrasonication. Nanofibers were obtained from polyhydroxybutyrate using electrospinning. Mixtures of low-molecular antioxidants obtained by extraction from natural sources were used as active ingredients. Different types of teas, barks, herbs, spices, fruits and vegetables were selected as sources of natural antioxidants. Total phenolic and flavonoid content and total antioxidant activity of extracts were determined using spectrophotometrical methods. Obtained natural extracts were subsequently used for encapsulation. Prepared application forms were characterized in terms of their physicochemical properties. Particle size was monitored by dynamic light scattering. Colloidal stability of particles in suspension was determined using zeta potential. Spectrophotometry was used to evaluate the efficiency of encapsulation of active compounds into particles. The morphology of the new type of combined PHB liposomes was monitored by electron microscopy. Chromatography was used for quantification of individual components of particles. Morphology of nanofibers and incorporation of active agent into their structure were monitored using FTIR-ATR spectroscopy and electron microscopy. Afterwards, antimicrobial, cytotoxic and genotoxic effects of preparations were evaluated. It was found that the most suitable types of extracts for liposome preparation are aqueous and lipid extracts of natural antioxidants. Prepared particles showed excellent stability and good encapsulation efficiency. The study confirmed that incorporation of polydroxybutyrate into liposome structure does not reduce neither the colloidal stability of the particle, nor the efficiency of encapsulation process. Antimicrobial and antimycotic effect of preparations against model microorganisms Micrococcus lutues, Serratia marcescens and Candida glabrata was detected. It was found that process of encapsulation increases the inhibitory effect of natural extracts of antioxidants. The safety of preparations was assessed using two human cell cultures: epidermal keratinocytes and HaCaT cell line. Assays of cell viability and plasma membrane integrity were used to determine cytotoxicity of preparations. Low toxicity of liposome particles was confirmed by a series of cytotoxic tests. Obtained data showed that association of phospholipid with PHB polymer does not cause a significant increase in cytotoxicity in human skin cells. Genotoxicity testing on model procaryotic organism confirmed zero genotoxic potential of preparations. The new type of combined particles and polymeric fibers cant thus be used as a carrier for active ingredients, complex natural extracts, antimicrobial agents and many others.

Aim of this thesis is voltammetric study influence of liposomal platinum cytostatics on cancer cells. One of the goals is summarize available informations about influence of cisplatine on cancer cells, its encapsulation into liposome and affection of this cytostatic cisplatin encapsulated in liposome on cancer cell lines. In literary recherche is detail description of these issues. Than is there specification of voltammetric methods, which serve to electrochemical detection of cisplatin. Based on literary recherche was chosen the best method for detection and subsequently the method was optimalized and than was applied to measuring itself.

Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12) Abstract Natural killer (NK) cells constitute the subpopulation of large granular lymphocytes which mediate spontaneous immune response against infected, transformed or allogeneic cells and thus represent an important component of the innate immunity. NK cells express a wide repertoir of surface receptors which can be either activating or inhibitory and which mediate NK cell recognition and regulation of cytolytic activity. NKR-P1 and Ly49 receptor families belong to the most important murine NK receptors. Both NKR-P1 and Ly49 families are members of C-type lectin-like superfamily of receptors encoded by natural killer gene complex (NKC) on chromosome 6 and include both activating and inhibitory members. The aim of this diploma thesis was to elucidate the impact of Nkr-p1c gene divergence on Ly49 receptors expression and to find out whether the Ly49 and Nkr-p1 gene clusters (which are localized on opposite ends of NKC) are inherited independently or whether the NKC domain is inherited as a complex. The second research interest was to illustrate the influence of the above mentioned divergence on cytotoxic activity of NK cells and tumor growth. In this study, inbred mouse strains C57BL/6 and Balb/c...

(english) Natural Killers - NK cells play an important role in immune surveilance and regulation either by direct cytotoxicity towards infected, transformed or otherwise damaged cells, or by production of cytokines and chemokines. The resulting response of NK cells is given by the sum of stimulating and inhibiting signals, tranduced by a wide array of receptors. Killer Ig-like receptors KIR2DL4 and LILRB1, which recognize self HLA-G molecules in pregnancy, as well as NKR-P1 receptors, which differ in the number of isotypes, are species-dependent and reduced during phylogenesis. NKG2D, reacting to stress-inducible proteins, and adenosine receptors (AR), which supress the inflamatory reaction, remain evolutionary conserved. The aim of this work was to study the involvement of NK cells and their receptors in several immune disorders and in various species, to provide new insights into their function and posisible immune modulation. We have shown here, that the choice of species in the study of NK cell effector functions may be crucial in some cases. The reaction to glycans, using synthetic GlcNAc-terminated glycomimetics GN8P, exerted opposing effects on NK cell function in humans and C57Bl/6 mice. In humans, the glycomimetic decreased cytotoxic activity of high NKR-P1A expressing NK cells, while in...