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Utilization of genome editing technology to knock out \kur{dnd1} gene in sturgeons
VU THI, Trang
In this study, for the first time we used CRISPR/Cas9 gene editing technology in sturgeons i.e., sterlets (Acipenser ruthenus). The sequences of sgRNA and primers were designed based on published dnd1 sterlet sequence. Each pair of sgRNA oligos after ligation ready duplex DNA fragment was cloned into vector pX330-U6-Chimeric_BB-CBh-hSpCas9 backbone and thereafter the transformation to competent cells Escherichia coli DH5 was done. The plasmid carried sgRNA was extracted for downstream applications. We diluted extracted plasmid with 10% of 2 M KCl and injection into animal pole of fertilized eggs of sterlets at one to four-cell-stage, 4 hours post fertilization (hpf). At the same time, second microinjection with 2.5% FITC-biotin-dextrans was injected into vegetal pole for labelling PGCs. In the control groups, the eggs were only injected by 2.5% FITC into vegetal pole. PGCs of sterlet were visualized and photographed using a uorescent stereo microscope Leica M165 FC. To confirm the presence or deletion/insertion occurring in the target gene, we used MCE-202 MultiNA microchip electrophoresis system for DNA analysis, in which the targeted gene after amplifying by PCR was analyzed. Mutations in both treated and control embryos of sterlet were further assessed by Sanger sequencing of the PCR product. In present study, we successfully established basic protocols such as preparation of competent cells, construction of vector carrying sgRNA and its transformation into competent cells to carry out the CRISPR/Cas9 technology in sturgeons. Less number of PGCs was observed in embryos that were treated with CRISPR/Cas9; however, sequencing did not provide us a reliable evidence for mutation of the targeted gene probably due to an unspecific PCR. Therefore, more authentication of dnd1 knockout should be done in future by more specific PCR and repeated sequencing.
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Optimization of air flow in hot-air sterilizer
Šťastná, Veronika ; Sekora, Jiří (referee) ; Provazník, Ivo (advisor)
The content of this work deals with the distribution of temperature, air flow and optimized airflow for medical and laboratory equipment. Especially in terms of using hot-air sterilizer chambers. The purpose of my bachelors thesis is the identification of the issues from the best theoretical description and explanation and my bachelors thesis includes picture with existing problematic . For this purpose I have dealt with the right choice literature and personally, I attended a business that is engaged in manufacturing various types of sterilizers.
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Proposal for a new system of continuous quality measurement pairs
Foltová, Anežka ; Sedlák, Petr (referee) ; Holcman, Vladimír (advisor)
This paper deals with proposal of new continual system for quality measurement of sterilization steam. At the begining the process of sterilization is introduced. Then the concept of saturated streams, parameters for sterilization and sensors for measurement are described. Then the current process of measurement of steam quality is described and in the last part the proposal of the new system is introduced. After the draft was the measurement data, analysis and evalution.
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Continuous measurement of the quality of steam
Rageh, Akram ; Sedlák, Petr (referee) ; Holcman, Vladimír (advisor)
Diplomová práce se zabývá měřením, analýzou a následným nalezením vhodných fyzikálních parametrů pro analýzu kvality syté sterilizační páry. Tato práce si klade za cíl určit, kritické parametry systému vyvíječe páry pro provoz dle norem ČSN. Dílčí částí projektu je popis fyzikální sterilizace, syté páry, metod a senzorů pro kontrolu sterilizační páry.
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Physical properties of steam sterilization
Kotlánová, Markéta ; Sedlák, Petr (referee) ; Holcman, Vladimír (advisor)
The master’s thesis deals with finding, measurement and evaluation of suitable physical parameters for quality analysis of saturated steam sterilization according to ČSN EN 285 + A2, which is the first step for the replacement of outdated processes of the analysis by modern automation systems. The other part of the project contains description of physical sterilization, saturated steam, methods and sensors for the monitoring of steam sterilization and the measurement and evaluation of these issues.
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Polymeric biocompatible materials
Matlák, Jiří ; Stránský, Lubomír (referee) ; Molliková, Eva (advisor)
The aim of the bachelor’s thesis is polymeric biocompatible materials. In the first part the problematic of the biocompatible materials is discussed and the basic terms are explained. In the next part are described polymeric materials and their chemical composition, mechanical and chemical properties and examples of applications.
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Study of the sterilization effect of temperature and UV light present in dielectric barrier discharge
Kramárová, Petra ; Grossmannová, Hana (referee) ; Kozáková, Zdenka (advisor)
The main theme of this bachelor thesis is to study the effect of temperature and UV radiation to the total sterilizing effect of dielectric barrier discharge. Sterilization is a process, which can eliminate all forms of life. The presented bachelor thesis discusses plasma sterilization, which is one of the method, which are suitable for sterilization of heat and chemical sensitive materials. This sterilization method is effective on the wide spektrum of procaryotic and eucaryotic microorganisms. Basically, the main inactivation factors for cells exposed to plasma are heat, UV radiation and various reactive species. In my thesis a dielectric barrier discharge (DBD) operating at atmospheric pressure was used for the sterilization of the samples. Plasma was generated in argon and nitrogen. According to the previous measurement the best sterilization results were observed using plasma power input 2 562,96 mW•cm-3 (argon) a 2 044,44 mW•cm-3 (nitrogen), therefore the same plasma power input was applied during our measurement. Fungi spores of Aspergillus niger were used as model organisms. Whatman paper No. 1st was used as the carrying medium. Each sample series was exposed to plasma for 5, 10, 20, 40, 60, 120 and 180 s. In order to separate the effect of UV radiation generated by DBD the quartz glass transmitting UV radiation was used. During the plasma exposition one of the sample was covered with the quartz window and the other sample was directly exposed to the plasma, afterwards the results were compared. The microbial abatement observed for the samples covered by quartz window was much lower than for the the samples directly exposed to the plasma. In first case the UV radiation and temperature is the main inactivation mechanism, while in the latter one the synergistic effect of UV radiation, temperature and active species is employed. Furthermore the effect of plasma sterilization increases with increasing the plasma exposition time. While comparing results observed for the argon and nitrogen, better results were achieved in argon. Effects of UV radiation and temperature were studied separately. The temperature between the DBD electrodes was measured by means of thermocouple. Afterwards the samples were placed in an oven and exposed to the same temperature as was measured between the electrodes. By comparing the results of heat treated samples and plasma treated samples it can be assumed that the influence of the temperature during the sterilization process in DBD was negligeable. The discharge parameters were studied by means of the optical emission spectroscopy.
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Study of sterilization effect of diaphragm discharge in liquids
Holíková, Lenka ; Slámová, Jitka (referee) ; Kozáková, Zdenka (advisor)
The main objective of this thesis is to study the sterilizing effect of diaphragm discharge in liquids. Sterilization is a process, which can eliminate all forms of life. Generally, sterilization is divided into physical and chemical methods. Plasma sterilization is ranked among physical methods although the action of chemical processes participates in the decontamination as well. Effects of UV radiation, free radicals and temperature are utilized in these methods. Fungi spores of Aspergillus niger F8189 and bacteria spores of Bacillus subtilis are selected as model organisms. Aspergillus niger microorganism is suitable for its resistance to changes in pH and it is viable in a wide range of pH values. Bacillus subtilis is chosen because of a good heat resistance. Diaphragm discharge is one of the possible types of electrical discharges in generated liquids. It is a low-temperature plasma, which is generated using a high DC voltage. In the created plasma streamers various physical and chemical processes rise. The chemical processes are mainly the generation of active species and particles that initiate subsequent chemical reactions and attack mould spores and bacteria in liquids. The physical processes include shock waves, strong electric field and ultraviolet radiation. Experiments were conducted in a reactor with separate cathode and anode space. In the dielectric barrier PET diaphragm with a pin-hole (initial diameter of 0.4 mm) was attached. Enlargement of the hole was observed as a result of degradation of material at the edge of holes due to the discharge. Degradation of spores was observed, depending on the time and on the input power. Other parameters measured were pH, conductivity and temperature. Experiments did not show any difference of discharge activity in the cathode and anode area. The main factor in the removal of spores Aspergillus niger was probably the temperature because no demonstrable effect of sterilization by the diaphragm discharge operation had been observed when heat resistant Bacillus subtilis was used. Also, it was made an attempt, which has been studied only the temperature effect of the fungi spores of Aspergillus niger. The samples were placed in a thermoregulator, where it was simulated the same temperature increase as at the discharge. The experiment had a similar course as an attempt at the discharge.
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Voluntary sterilization of women in Europe: Study 5.314
Lovětínský, Vojtěch ; Mylková, Petra
Práce se zabývá komparací právních úprav vybraných evropských států týkajících se možností provedení lékařského zákroku sterilizace na žádost pacientky jako formy antikoncepční metody. Zhruba od 70. let dochází k postupnému přijímání zákonů, které umožňují podstoupit sterilizaci na základě dobrovolné a svobodné vůle pacientky. Komparace srovnává zejména podmínky, na jejichž splnění právní úpravy vážou možnost provedení tohoto zákroku. Právní úprava v České republice v porovnání s úpravami ostatních zpracovaných zemí zakotvuje poměrně přísné požadavky a omezení, za nichž může být sterilizační zákrok proveden, zatímco jiné, zejména severské země, volí výrazně liberálnější přístup a ponechávají toto rozhodnutí na pacientce samotné. Společným znakem většiny úprav je pak zvýšená ochrana mladistvých či právně nezpůsobilých pacientek při podávání žádostí o provedení sterilizačního zákroku.
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