Neoplastic diseases hold the second place of the most common causes of death worldwide. Available treatments include various combinations of surgery, chemotherapy, radiation, hormone therapy, immune therapy and targeted therapy. The emphasis is currently laid on nanomedicine, where new nanosized complexes are developed and applied for the targeted treatment and chemotherapy. The aim is to significantly improve the anticancer effect and decrease the damage to organism. In this thesis, ruthenium nanoparticles with a size of 12–14 nm were synthesized and their surface modified with polyvinylpyrrolidone. Furthermore these were subsequently modified with polyoxyethylene(40)stearate for binding of doxorubicin. These nanoparticles were tested on breast carcinoma cells (MDA-MB-231), ovarian carcinoma cells (A2780) and neuroblastoma cells (UKF-NB-4). Apoptosis and necrosis testing showed 60—64 % increase in apoptosis when comparing ruthenium nanoparticles modified with doxorubicin to nonmodified ruthenium nanoparticles. The modification increased level of oxidative stress in tumorous cells and slightly a genotoxicity to non-tumorous cells, nevertheless the hemocompatibility was significantly improved. Testing has proven with IC50 0.98 g/ml, 3.91 g/ml and 1.95 g/ml higher sensitivity to these cells and confirmed expected anticancer activity. Compared to one of the most common chemotherapeutic agents cisplatin the modified ruthenium nanoparticles are significantly more toxic to cell lines A2780 (IC50=21 µg/ml), MDA-MB-231 (IC50=9 µg/ml) and UKF-NB-4 (IC50=4 µg/ml).

Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both cytotoxic assessment assays showed an earlier cytotoxic effect in case of shorter NWs, with longer ones having a more marked toxicity, albeit with a delay in time. These findings demonstrate that different levels of biocompatibility can be obtained with specific doses and properties of Ni NWs and can serve as guideline for future experiments.

Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmacology and Toxicology Student: Anastasiya Shkut Supervisors: Mgr. Jana Mandíková, Virginia Motilva Ph.D. Title of diploma thesis: The role of melatonin in SIRT1 and p-AMPK regulation in HT-29 cell line. Sirituin 1 (SIRT1) is NAD+ dependent deacetylase present in wide range of organisms including mammals. It was found to extend life span in yeasts during calorie restriction (CR) conditions. SIRT1 deacetylates many regulator proteins and thus control metabolic status of cell as well as AMP-activated kinase (AMPK), which is also energy regulator enzyme depending on NAD+ levels in cell. Both of them play roles in cancer and could influence autophagy, although the exact mechanism remains unclear. We focused our study on hormone melatonin, which has anti-inflammatory and anti-cancer effects, to study its influence on human colon cancer cell line HT-29. If it has impact on SIRT1 and AMPK and what is hierarchic relationship between SIRT1 and AMPK. Also we observed its possible influence on autophagy. We used Western blotting (WB) technique and from our results it seems that melatonin has significant effect on SIRT1, which might activate AMPK as well as autophagy. Nevertheless some of results did not have sufficient...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Supervisor: Assoc. Prof. PharmD. Jan Zitko, PhD. Author: Marek Kerda Title of diploma thesis: Design, synthesis and biological evaluation of 2,3-disubstituted pyrazines This thesis deals with problem of tuberculosis. In a theoretical part are summarized information and knowledge about tuberculosis, nowadays epidemiology and drugs used in current treatment. There are also described drugs in the different stage of clinical trials and could be used for treatment of tuberculosis in the future. Searched information were used from accessible learning materials and in articles in online databases as Web of Science and PubMed. There are also summarized basic methods of computer design of new drugs. In practical part of this thesis was focus on novel inhibitor of prolyl-tRNA synthetase, which is based on structure of pyrazinamide. There was prepared in silico virtual library of pyrazine-based new potential ligands. Related docking to the structure of human prolyl t-RNA (pdb: 5VAD) and the bacterial version of this enzyme (pdb: 2J3M) and evaluation was performed in Molecular Operating Environment (Chemical Computing Group, Canada). From the results were predicted some of the relations between structure and activity. Virtual library of the...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Miškovčíková Supervisor: RNDr. Jana Maixnerová, PhD. Title of diploma thesis: Determination of organ toxicity of BRAF inhibitors in vitro. Malignant melanoma is one of the most serious skin diseases today. Therapy of advanced melanoma is difficult and often ineffective. BRAF inhibitors (dabrafenib and vemurafenib) have dramatically changed the results of melanoma treatment in the last few years. BRAF inhibitors are one of the most effective drugs against melanoma, but their clinical application is largely limited by drug resistance. Available clinical studies have shown an adverse nephrotoxic effect of BRAF inhibitors, but information on its mechanism is limited. Published studies further suggest that the toxic effect of BRAF inhibitors is primarily directed to podocytes located in the glomerular membrane. Thus, the aim of our study was to assess the cytotoxic effect of BRAF inhibitors on selected model renal cells in vitro in order to confirm the renal target toxicity. The main objective of the study was to analyse whether the nephrotoxic effect of BRAF inhibitors is specifically limited to podocytes or whether it can damage other renal cells. The experiments were performed on human cell lines...

Mining operations have led to an increase in polycyclic aromatic compound (PAC) concentrations in the Alberta oil sands area. However, the toxicity of most PACs and PAC mixtures is not well characterized. Some PACs and PAC mixtures are known to be mutagenic, though there is limited research on the genotoxicity of PACs from the Alberta oil sands to wildlife. This thesis tested the hypothesis that anthropogenic sources of PACs from the Alberta oil sands are mutagenic to wildlife. The objectives were: 1) to determine whether wildlife with increased exposure to PACs had increased mutations, and 2) to determine whether an anthropogenic source of PACs is mutagenic in a controlled lab setting. For the first objective, we used a single-molecule polymerase chain reaction (SM-PCR) assay to detect microsatellite mutations in river otters with differing liver tissue PAC concentrations in the Athabasca oil sand region (AOSR; Alberta, Canada). For objective two, an in vitro mammalian mutagenicity assay with the FE1 MutaMouse epithelial cell line (FE1) was used to determine the mutagenic potential of a bitumen extraction by-product, tailings pond bitumen. We found that PAC exposure in the AOSR was positively correlated with elevated microsatellite mutations in river otters. From the in vitro study, tailings pond bitumen extracts did not induce lacZ mutations in the FE1 cells. Differences in detection methods between the two assays and PAC profiles between the otter tissue and tailings pond bitumen are suspected reasons for contradictory results. Further investigation of the different sources and PAC profiles within the AOSR environment and wildlife food web will provide insights on what types of PACs are mutagenic. Cytotoxicity, observed following exposure to tailings pond bitumen extracts, also suggests other toxicity pathways should be considered when investigating the toxicity of bitumen from the AOSR. Overall, this thesis provided data on the potential mutagenicity of PACs in the AOSR, which can be used to elucidate potential molecular mechanisms of toxicity in wildlife exposed to oil processing contaminants.

The use of 3D printing in biological applications is a new field of study given that 3D printing technology has become more available and user friendly. Possible uses include using existing 3D printing polymers to use in extracorporeal or in vitro devices, like Lab-on-a-Chip, and the development of new biologically derived materials to print cell-containing constructs. The latter concept is what is more commonly known as bioprinting. Our research had the goal of developing a bioprinting system including the printer, a bioink, and a feedback system for printing parameter optimization which could be done cheaply and within the reach of nearly any research lab. To make the bioprinter, we were able to take a popular plastic 3D printer and convert it to a bioprinter with 3D printed parts and the addition of a new motherboard. This came with great contribution from Carnegie Melon University. We were also able to improve upon the original design and, along with the new bioprinting capabilities, maintain the original capabilities of the plastic 3D printer. A new bioink was developed to work in coordination with this bioprinting system. Our lab has the luxury of having access to decellularized tissue, which provided a unique material to create a bioink which is derived from the extra-cellular matrix of porcine hearts. The final bioink protocol allows the users to make their own bioink, from easily obtainable tissue and determine their own concentration of the extra-cellular matrix/collagen within a range. Lastly, a feedback system was developed using a Raspberry Pi and camera module to provide real-time visual feedback of the bioprinting process which is otherwise very difficult to see and optimize parameters from. A protocol was developed to sequentially optimize the parameters for an open-source slicing software which governs the resolution of the bioprinter itself. In related research, the cytotoxicity and cell adherence properties of a printing resin for a microfluidic 3D printer were evaluated for use in Lab-on-a-Chip applications. The existing resin was tested and determined to be cytotoxic to cells and therefore not suitable for biological applications. We showed that a simple ethanol washing step and plasma treatment pulled the cytotoxic elements out of the polymer and modified the surface such that cells could attach and proliferate on the printed resin. Another printed resin was also tested which was determined to have no natural cytotoxicity, but the same plasma treatment was needed to allow for cell adherence.

The ethanol extract of A. phylicoides was investigated for its antioxidant activity using the DPPH scavenging method. The extract showed good antioxidant results with a EC50 value of 10.64 ± 0.0842 µ/ml. The extract was also tested for antibacterial activity against microorganisms (Staphylococcus aureus, Enterococus faecalis, Bacillus cereus, Bacillus subtilis, Bacillus pumilus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia) commonly known to pose a threat in the wellbeing of man. All tested microorganisms were significantly inhibited by the extract with the MIC values ranging from 3.13 µg/ml to 6.25 µg/ml. Folin-Ciocalteu’s reagent method was used to determine total phenolic content of dried and freshly prepared crude extract of A. phylicoides. Higher total phenolic content (28.28 ± 0.019 mg GAC/100g) and antioxidant activity (EC50, 10.64 ± 0.084 µg/ml) was observed in the dried extract compared to the fresh extract with a TPC value of 23.04 ± 0.003 mg GAC/100g and EC50 of 13.97 ± 0.066 µg/ml. Bioassay-guided fractionation of ethanolic extract from aerial parts of Athrixia phylicoides using silica and sephadex column chromatography led to the isolation of four known flavanoids, 5-hydroxy-6,7,8,3’,4’,5’-hexamethoxyflavon-3-ol (1), 3-0- demethyldigicitrin (2), 5,6,7,8,3’,4’-hexamethoxyflavone (3) and Quecertin (4). Due to the low yield, no further tests were done on compound 3. A DPPH-scavenging assay was performed to evaluate the antioxidant activity of the isolated compounds. All the tested compounds showed potent antioxidant activity with EC50 values ranging from 1.27 to 3.41 µg/ml. Compound 4 showed a higher antioxidant activity (EC50, 1.27 µg/ml) than vitamin C (EC50, 2.66 µg/ml) used as a control. The MIC values of the isolated compounds against tested microorganisms varied from 20 to more than 40 µg/ml. All the tested compounds showed no activity against S. aureus, B. pumilus, K. pneumonia and P. aeruginosa at the highest concentration tested (40 µg/ml). These compounds together with the extract were further analyzed by XTT assay on Vero cells. The extract showed a low toxicity effect on the cells at lower concentrations exhibiting EC50 value of 107.8 ± 0.129 µg/ml. Compound 4 showed minimal toxicity effect on the cells with a EC50 value of 81.38±0.331 µg/ml, compared to Compound 1 and 2 which exhibited EC50 values of 27.91 ± 0.181 µg/ml and 28.92 ± 0.118 µg/ml respectively. The results obtained from this study provide a clear rationale for the medicinal uses of Athrixia phylicoides. / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

>Magister Scientiae - MSc / Silver nanoparticles (AgNPs) apart from being chemically significant, have shown a lot of health benefits, the most studied being their anti-bacterial and anti-inflammatory properties. These biological properties can be further enhanced by adding compounds with known medical properties giving rise to even more desired potent materials. Anti-bacterial and cytotoxicity studies show that these AgNPs can kill bacteria, prevent infections and regenerate skin cells. On the other hand, previous studies have reported dimethyl sulfoxide (DMSO) with attractive wound healing abilities specifically cell growth promotion. It was then envisaged that the combination of DMSO and AgNPs could lead to a potent wound healing agent. It is a well-known fact that non-healing wounds pose a socioeconomic threat to a large population worldwide. / 2023

Investigations of the molecular binding partners of the probiotic bacterium Bifidobacterium longum subspecies infantis (B. infantis) and the pathogen Salmonella enterica subspecies enterica serovar Typhimurium LT2 (Salmonella ser. Typhimurium) found that these two very different bacteria bind gangliosides. However, these organisms lead to completely different host health outcomes when present in the gut. B. infantis is the founding microbial population in the intestinal tract of breast-fed infants. S. typhimurium is the most important food-borne pathogen that results in humans. This study used an in vitro gut epithelial cell model to examine the host cellular response to adhesion of B. infantis, which led to an increase in intestinal epithelium survival. This observation led to a series of experiments to elucidate the pathway for host signaling initiated by adherence of B. infantis to the host membrane to explain the increase in host cell survival. B. infantis adhesion induced significant (q≤0.05) differential expression of 208 host genes. These genes were associated with increased broad mechanisms of cell survival that included BIRC3, TNFAIP3, and SERPINB9. We hypothesized that a biochemical link existed between the host membrane adhesion protein and the increase in cell survival, mediated via AKT. We tested this hypothesis to demonstrate that B. infantis interaction initiated signal transduction using G-proteins via phosphorylation of AKT and induced production of the BIRC3, TNFAIP3, and SERPINB9. This study discovered adhesion of B. infantis initiated activation of AKT via phosphorylation of both Ser473 and Thr308, which results in increased cell survival.