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Study of DNA-doxorubicine interaction by electrochemical methods on nanostructured electrodes
Přibyl, Jakub ; Fohlerová, Zdenka (referee) ; Kynclová, Hana (advisor)
Cancer diseases are one of the leading causes of mortality worldwide, for this reason, great attention anticancer drugs. Doxorubicin falls into the category of the most widely used cancer drugs. The method of electrochemical impedance spectroscopy were investigated properties of golds electrodes without deposit nanoparticles and electrodes with deposit gold nanoparticles. Using voltammetric measurements we detected doxorubicin and investigated the interaction of doxorubicin with oligonucleotides on gold electrodes without nanoparticles and electrodes modified with gold nanoparticles.
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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Měsíčková, Klára ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
The isolation of plasmid DNA is an important and often used method in microbiology. The isolation itself is preceded by preparation of bacterial competent cells and by amplification of the plasmids. In this stage, plasmids CHR2, ASAP1, ASAP-3, ASAP-5 and Kir2.1. are first amplified in E.Coli bacteria of the DH5 strain and then isolated through the method of phenol-chloroform extraction. Gel electrophoresis and transfection to cellular line HEK293 are used for determining the correctness of the isolation.
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Study of mesenchymal stem cell migration in the extracellular matrix based on principles of chemotaxis
Scholasterová, Viktorie ; Fohlerová, Zdenka (referee) ; Chmelíková, Larisa (advisor)
This thesis engages in a study of mesenchymal stem cell migration in extracellular matrix based on principles of chemotaxis. First, attention is focused on a theoretical part associated with a clarification of basic terms such as extracellular matrix, migration, confocal microscopy, mesenchymal stem cells or chemotaxis. There is also included a list and a description of some basic methods for monitoring cell migration and a more detailed description of a method called transwell assay, which has been chosen for an experiment in a practical part of this thesis. This part includes protocols of individual steps for the preparation of the experiment, the procedure of data processing obtained by scanning cells with a confocal microscope and a description of the resulting confluence values.
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Ruthenium-based nanoparticles and testing of their anticancer activity
Žáková, Eliška ; Fohlerová, Zdenka (referee) ; Heger,, Zbyněk (advisor)
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).
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Detection of single photon-upconversion nanoparticles by luminescence microcopy
Polachová, Natálie ; Kolář, Radim (referee) ; Fohlerová, Zdenka (advisor)
This bachelor thesis deals with the detection of photon-upconversion nanoparticles using the U-net convolutional neural network, by using epiluminescence microscopy. The theoretical part contains an introduction to the issue of photon-upconversion, description and use of photon-upconversion nanoparticles. Furthermore, the thesis deals with the functioning of basic and convolutional neural networks. In the practical part, we prepared samples of nanoparticles with subsequent acquisition of images by epiluminescence microscopy. The convolutional neural network U-net was designed, which further serves for the detection of nanoparticles bz using H-maxima morphological operations. In the end, everything was summarized and statistically evaluated..
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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Skala, Kateřina ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
DNA isolation is one of the basic methods in molecular biology. There are several methods of DNA amplification and isolation. In this paper phenol-chloroform extraction of three plasmid types - Channelrhodopsin-2, ASAP1 and Kir 2.1 is used. Six plasmids were isolated in total. These plasmids are then validated using gel electrophoresis. Successfully isolated plasmids are then transfected to HEK293 cells and images taken on confocal microscope 24, 48 and 72 hours after transfection.
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The influence of modified TiO2 nanotubes on biointerfacial interaction
Bílek, Ondřej ; Kočí,, Kamila (referee) ; Hlaváček,, Antonín (referee) ; Fohlerová, Zdenka (advisor)
Nanotrubičky oxidu titaničitého v průběhu posledních let nabyly na významu v poli biomedicíny. Jakožto biokompatibilní nanostrukturovaný povrch nachází potenciál pro své uplatnění především v oblasti implantačních aplikací. Teoretická část této práce je tak věnována různým přístupům pro syntézu TiO2 nanotrubiček, jejich modifikacím a aplikacím v biomedicíně. Experimentální část pak pojednává o nanotrubičkách oxidu titaničitého, které jsou připraveny z titanu metodou jednokrokové anodické oxidace v organickém elektrolytu. Jako výchozí materiály jsou používány křemíkové disky s naprášenou vrstvou titanu a titanové folie. Zprvu amorfní nanotrubičky jsou žíháním převedeny na svou krystalickou podobu, a následně modifikovány selenovými a stříbrnými nanočásticemi. Připravené struktury jsou zkoumány z hlediska povrchových vlastností a biologických interakcí s vybranými tkáňovými kulturami (MG-63, NIH-3T3) a bakteriemi (E. coli, P. aeruginosa, S. aureus). V závěru experimentální práce jsou stručně porovnány výsledky selenových a stříbrných nanočástic. Hlavním cílem této práce je rozšířit znalosti týkající se bio-rozhraní tvořeným adherentními buněčnými liniemi, bakteriálními buňkami a nanostrukturovaným povrchem tvořeným TiO2 nanotrubičkami dekorovanými selenovými a stříbrnými nanočásticemi.
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Fabrication and characterisation of silver decorated TiO2 nanotubes for biomedical applications
Bílek, Ondřej ; Horynová, Miroslava (referee) ; Fohlerová, Zdenka (advisor)
This diploma thesis summarizes knowledge from the fields of synthesis, characterization and application of titanium dioxide nanotubes and its combination with silver nanoparticles for biomedical purposes. Basic protocols of working with cell cultures and bacteria are also included. Experimental part of this diploma thesis focuses mainly on the synthesis of tubular structures made of titanium dioxide via anodic oxidation of 500nm titanium layer and their subsequent decoration with silver by electrodeposition. Last section of the experimental part is devoted to testing of antibacterial properties of the new material and examining the effect of different silver concentrations on the adhesion of MG-63 cells. All results are compared to reference samples consisting of titanium dioxide nanotubes without silver.
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Study of disassembly/reassembly mechanisms of ferritin protein cages and their utilization in nanomedicine
Krausová, Kateřina ; Fohlerová, Zdenka (referee) ; Heger, Zbyněk (advisor)
Diploma thesis deals with the study of dissociation and reassociation of ferritin protein cages and their use in nanomedicine. Most studies that are focused on targeted transport of pharmaceuticals using ferritin cages work with horse spleen ferritin. It is, however, its origin, which leads to increasingly frequent questions about possible immunogenicity in the patient's organism, which also provides the main motivation to test the possibility of encapsulation of low-molecular drugs into ferritins originating from alternative organisms. In the practical part the method for the study of dissociation was experimentally designed. Native polyacrylamide gel electrophoresis was used to study dissociation of equine ferritin composed of different subunit, human ferritin, and archeal Pyrococcus furiosus ferritin. The obtained subunit dissociation results were used to encapsulate the low molecular chemotherapeutic drug doxorubicin and for further characterization of the ferritin-doxorubicin complex. The efficacy of the designed nanoformulations has been verified in the treatment of malignant breast cancer. Human ferritin proves to be the optimal one. Its composition of heavy subunits corresponds to a lower protein stability, thus a more efficient opening of the structure and consequent encapsulation of the cytostatics occurs. With its 60% encapsulation efficiency of doxorubicin, low polydispersity index, effective cytotoxicity of ferritin-doxorubicin complex and minimal risk of immune response to the patient's organism, human ferritin achieves better results than commonly used horse spleen ferritin.
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