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Temoporfin-conjugated upconversion nanoparticles for NIR-induced photodynamic therapy of pancreatic cancer
Shapoval, Oleksandr ; Větvička, D. ; Kabešová, M. ; Engstová, Hana ; Horák, Daniel
Photodynamic therapy (PDT), a clinically approved cancer treatment strategy, has the potential to cure pancreatic cancer with minimal side effects. PDT primarily uses visible wavelengths to directly activate hydrophobic photosensitizers, which may be insufficient for deep-seated cancer cells in clinical practice due to poor penetration. Upconversion nanoparticles (UCNPs) serve as an indirect excitation source to activate photosensitizers (PSs) in the NIR region, overcoming the limitations of molecular PSs such as hydrophobicity, non-specificity, and excitation in the UV/Vis region. Here, monodisperse upconversion NaYF4:Yb3+, Er3+, Fe2+ nanoparticles (UCNPs) have been surface-engineered with poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) and temoporfin (mTHPC), a clinically used PDT prodrug, for near-infrared (NIR) light-triggered PDT of pancreatic cancer. The incorporation of Fe2+ ions into the particles increased the fluorescence intensity in the red region matching the activation wavelength of mTHPC. Covalent binding of mTHPC to the surface of UCNP@PMVEMA particles provided colloidally stable conjugates enabling generation of singlet oxygen. In vitro cytotoxicity and photodynamic activity of the particles were evaluated using INS-1E rat insulinoma and Capan-2 and PANC-01 human pancreatic adenocarcinoma cell lines. The PDT efficacy of UCNP@PMVEMA-mTHPC conjugates after irradiation with 980 nm NIR light was tested in vivo in a pilot study on Capan-2 human pancreatic adenocarcinoma growing subcutaneously in athymic nude mice. The intratumoral administration of the nanoconjugates significantly hindered tumor growth and demonstrated promising PDT efficacy against human pancreatic cancer.
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Magnetic nanoparticles with antibacterial properties: Synthesis, characterization and biological applications
Shatan, Anastasiia-Bohdana ; Horák, Daniel (advisor) ; Kaman, Ondřej (referee) ; Matějíček, Pavel (referee)
In response to the escalating global threat of antibiotic resistance, innovative strategies are imperative. This thesis focuses on surface-engineered magnetic nanoparticles (MNPs) with potent antibacterial properties, aiming to combat antibiotic resistance effectively. Specifically, uniform 16-nm Fe3O4 nanoparticles were synthesized via oleic acid-stabilized thermal decomposition of Fe(III) oleate in a high-boiling organic solvent. Optionally, 8-nm γ-Fe2O3 particles were obtained by coprecipitation of Fe2+ and Fe3+ salts in a basic medium. For the application of antibacterial MNPs in biological media, water-dispersible nanoparticles were required. Hence, original magnetic particles containing hydrophobic oleic acid (OA) coating were modified with silica using a water-in-oil reverse microemulsion. Subsequent modification with (3-mercaptopropyl)trimethoxysilane and decoration with silver nanoclusters yielded Fe3O4@SiO2-Ag nanoparticles. Additionally, neat Fe3O4 particles were coated with Sipomer PAM-200 containing both phosphate and methacrylic groups, facilitating attachment to the iron oxide and enabling (co)polymerization with 2-(dimethylamino)ethyl methacrylate and/or 2- tert-butylaminoethyl methacrylate. Furthermore, γ-Fe2O3 nanoparticles were rendered antimicrobial through modification with...
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Automated system for installing and configuring GNU/Linux virtual hosts
Horák, Daniel ; Polívka, Michal (referee) ; Pelka, Tomáš (advisor)
This work deals with possibilities of administration of virtual machines (mainly from the developer's point of view) and available methods for automatic installation of GNU/Linux distributions. First part contains the information about virtualisation technologies and possibilities to configure them with a libvirt library. The definition and specification of each part of virtualisation environment (virtual machines, disks, networks) using XML is described. Further more the possibilities of automatic installation of Linux distributions based on RedHat and Debian considering the virtualisation environment are discussed. The possibility of copying an existing disk with installed system is also mentioned. In a practical part a server application for control and management of virtual machines including automatic installation of operating system is proposed and realised.
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The use of magnetic microparticles for DNA isolation
Jelínek, Zdeněk ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.
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OpenSource SW for data traffic inspection
Horák, Daniel ; Stančík, Peter (referee) ; Hajný, Jan (advisor)
This work deals with a usage of free and open software for filtering of network traffic and its installation in network gateway in small and middle-sized networks. Main focus is on filtering the http protocol on the level of a application layer. At the start a problems of free and open software is analyzed along with its advantages that result from its usage.There are as well given reasons for choosing the Debian GNU/Linux distribution. Further there are discussed possibilities of filtering network traffic on packets level and status filter along with the soft- ware iptables. Main part focuses on a filtering of http traffic on the application layer level using program Squid and DansGuardian. In a practical part the installation and configuration of operating system Debian GNU/Linux is presented including base services (ssh, DNS and DHCP server). Further more there are installations and configurations of softwares Squid and DansGuardian demonstrated. For DansGuardian there is a web configuration interface created.
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Plasmid DNAs interactions with lanthanoide compounds
Budko, Kateryna ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Recently much attention is given to lanthanides and their complexes as excellent catalysts for cleavage of nucleic acids. The thesis has been focused on the cleavage of plasmid and bacterial DNA by ions Nd3+ and Y3+ and by different carriers containing the lanthanide compounds. The creation of single-stranded nicks and double-stranded ones in the plasmid DNA molecules was studied by agarose gel electrophoresis. Verification of the cleavage of bacterial DNA was made by polymerase chain reaction using primers specific for the domain Bacteria and genus and species-specific primers. The results will be used in the development of the method that will allow perfect carriers`s coverage verification with the magnetic perovskit nucleus and other carriers with the lanthanide compounds.
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Software Design of Single Purpose Machine for Visual Inspection
Horák, Daniel ; Krejsa, Jiří (referee) ; Štarha, Pavel (advisor)
This master’s thesis deals with the fundamentals of machine vision application and its practical implementation. The research part is focused on the basic possibilities of image acquisition and image processing in different dimensions. The practical part describes the design of the dimension control algorithm using a 3D camera. This algorithm is then implemented in a single-purpose machine for optical dimension control.
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Preparation and Characterization of Magnetic Carriers from Hypercrosslinked Polystyrene Microspheres and their Application in a Biosensor.
Šálek, Petr ; Šňupárek, Jaromír (referee) ; Šafařík,, Ivo (referee) ; Horák, Daniel (advisor)
With the aim to develop and characterize a functionalized highly magnetic polymer carrier of micrometer size and of a narrow particle size distribution that will be suitable for biological application, hypercrosslinked microspheres were prepared. Simultaneously, the relation between structure and properties of product was observed. Condition of dispersion polymerization were optimized to obtain starting monodisperse poly(styrene-co-divinylbenzene) [P(St-DVB)] microspheres. The P(St-DVB) microspheres of different degree of crosslinking were prepared and effect of some polymerization parameters such as type of solvent, initiator, concentration and mode of DVB addition on morphology, size and particle size distribution were investigated. The starting microspheres were hypercrosslinked to obtain microporous inner structure. Hyperosslinked particles had very large specific surface area (> 1000 m2/g) and a high content of micropores (ca. 0.6 ml/g). First, P(St-DVB) microspheres were chloromethylated using three different chloromethylation agents to regulate their porous properties. Hypercrosslinking was achieved by the addition of stannic chloride as a catalyst and by increasing a temperature. The hypercrosslinked microspheres were then functionalized with sulfo- or aminogroups. The functional groups captured precipitated iron oxide inside the porous structure of the microspheres and also served as a reactive site for intended immobilization of the protein. A solution of ferrous and ferric chloride was imbibed under vacuum into the porous structure and the iron oxide was precipitated by an aqueous ammonia solution. Finally, the magnetic functionalized hypercrosslinked micropsheres were integrated into a biosensor for qualitative detection of ovalbumin.
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Isolation of bacterial DNA from foods using magnetic carriers
Bubeníková, Lucia ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was the selective isolation of bacterial DNA with help of magnetic carriers covered by streptavidine (PGMA-NH2-STV, MPG® Streptavidin). Conditions of functionalisation of carriers using two biotinylated probes were optimized: the amount of carrier, the amount of probe, binding of biotinyled probe to streptavidine. Purified DNA Lactobacillus was used for hybridization. DNA binding to the probe (DNA/DNA hybridization) and nospecific adsorption of DNA to the carrier were tested. Target DNA eluted from the carrier was identified using PCR with primers R16-1 and LbLMA1-rev and with primers P_eub and F_eub. The amount of probe bound to the carrier was estimated using UV spectrophotometry. It was estimated that biotinyled probe can be used for functionalisation in concentration 5 pmol/µl added to the carrier in the ration carrier : probe 1:1. It was shown that nonspecific DNA adsorption to the MPG® Streptavidin is significantly lower than to the carrier PGMA-NH2-STV.Using DNA/DNA hybridization and the MPG® Streptavidin, DNA from pure culture Lactobacillus was isolated. Procedure was applicated for DNA isolation from milk products.
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Identification of probiotic Bifidobacterium strains in dairy products
Zovčáková, Monika ; Horák, Daniel (referee) ; Španová, Alena (advisor)
Lactobacilli are dominant bacteria of the vaginal flora. Lactobacillus-containing probiotics products are used for the treatement and profylaxis of bacterial urogenital infections. This work is focused on DNA identification and species identification of probiotic bacteria in 5 different vaginal tablets using molecular-genetic methods. Total DNA isolated from complex matrix of vaginal tablets was used for amplification in polymerase chain reaction. DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA) and by method of phenol extraction. Identification of species of probiotic bacteria was verified using genus-specific and species-specific PCRs. Results of bacterial identification obtained by PCR were compared with declared specification given by producers. Bacteria of genus Lactobacillus were proved in all tablets whereus species identification was in accordance with the stated composition in 1 tablet only.
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