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Research and development of surface modifications of bearing steels by means of ion nitridation
Klimčáková, Kateřina ; Klakurková, Lenka (referee) ; Doležal, Pavel (advisor)
Basic requirements placed on rolling bearing components include the high fatigue limit, the wear resistance and the corrosion resistance. As full production of bearings from materials with these attributes would be quite expensive, surface modifications are used to improve mechanical characteristics. This thesis deals with application of layers to the bearing steel by means of ion nitridation. It studies effects of input conditions of the ion nitridation and of the surface condition on tribological characteristics of the bearing steel with the above mentioned finishing. The study employed the interaction light microscopy, the scanning electron microscopy equipped by energy dispersive chemical microanalysis, the hardness measurement by Vickers, the glow discharge optical emission spectroscopy, and methods of the phase analysis by means of the X-ray diffraction were used.
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Multi-element Systems of Biomaterials Based on Magnesium and Zinc
Hasoňová, Michaela ; Nový, František (referee) ; Vojtěch, Dalibor (referee) ; Doležal, Pavel (advisor)
Dissertation thesis deals with basic research in the field of materials from pure Zn powders and Mg, Zn, and Ca binary mixtures prepared by powder metallurgy. General powder metallurgy principles and methods, a brief description of Mg, Zn, and Ca structure and properties, and the latest research in the field of bulk materials preparation from these elements via powder metallurgy are summarized in the theoretical part of the thesis. The experimental part focuses on the preparation of materials from finer and coarser Zn powder particles by hot pressing at 300 and 400 °C using the pressure of 100, 200, 300, 400, and 500 MPa. Binary mixtures based on Mg with the addition of Zn or Ca were prepared by hot pressing in the solid-state (300 °C) and hot pressing in the semi-solid state (400 °C, 450 °C in the case of Mg-Ca system) using the pressure of 500 MPa. Binary mixtures based on Zn with the addition of Mg or Ca were prepared by hot pressing in the semi-solid state (400 °C) using the pressure of 500 MPa. The prepared materials were evaluated in terms of microstructure, elemental and phase composition, microhardness, flexural strength, and fractography. The results showed that in the case of processed from pure Zn powders, a better combination of the flexural strength and displacement was achieved in the case of the finer Zn powder, namely in the material prepared at a temperature of 400 °C and a pressure of 500 MPa. In the case of mixtures, the best connection between the powder particles was achieved in the case of a material based on finer Zn powder with 0.5 wt.% of Mg, which had a significant effect on the achieved values of flexural strength and displacement. The amount of minor powder in the mixture had a significant effect on the prepared material structure and phase composition, while the processing conditions influenced the reached strength characteristics and fracture mechanism.
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Nanofibrous membranes as drug delivery systems II
Stránská, Denisa ; Doležal, Pavel (advisor) ; Dittrich, Milan (referee) ; Maťha, Vladimír (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Pharmaceutical technology Candidate: Ing. Denisa Stránská Supervisor: Doc. RNDr. Pavel Doležal, CSc. Title of Doctoral Thesis: Nanofibrous membranes as drug delivery systems Nanofibres are material structures of unique properties that are well-useable in many exciting areas, including medical products. In the field of air filtration, different types of nanofibre filters are produced industrially. On the other hand, in the area of medicated nanofibres, there is still a major barrier for production, and hence the commercial use of nanofibres, especially due to the low weight and required content uniformity of the final products. Electrospinning technology is the most promising in this context, however, no results on the evaluation of nanofibrous products intended for the systemic administration of drugs obtained by certified manufacturing process have been published. In this doctoral thesis the individual steps of the nanofibrous products production are described, experimentally evaluated in compliance with good manufacturing practice and documented with an emphasis on the validation procedures and thus on the desired repeatability of all steps leading to the production of the final product. Validation processes included...
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Mechanisms of iron-sulfur cluster biogenesis in eukaryotes
Temešinko, Tomáš ; Doležal, Pavel (advisor) ; Malych, Ronald (referee)
Many essential cellular proteins use iron-sulfur (Fe-S) clusters as cofactors. These proteins often serve as enzymes, components of the electron-transport chain or as intracellular sensors. Prior to the use of the cluster in a protein, it needs to be formed or created de novo. In total, four different mechanisms of Fe-S cluster biogenesis can be used by the eukaryotic cell - ISC, CIA, SUF and NIF. All of these pathways include a specific targeting system for delivering the cluster to its acceptor protein. Errors in biosynthesis ofFe-Sclustersaremostlylethalandcanleadtofailureindevelopmentofmulticellularorganisms.Despite this a better characterization of these mechanisms is needed as research is currently still in progress. This bachelor's thesis provides current information regarding the mechanisms of Fe-S clusters biogenesis in eukaryotes acquired mostly from mammalian cells, including humans, and from well-known model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana, and parasitic protist Giardia intestinalis.
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Conserved mechanism for targeting of Tail-anchored proteins in eukaryotes
Najdrová, Vladimíra ; Doležal, Pavel (advisor) ; Borgese, Nica (referee) ; Svärd, Staffan (referee)
Approximately one-fourth of all cellular proteins represent integral membrane proteins (IMPs) that are transported through the cytosol across or into the organellar or plasma membrane. Transport of IMPs requires precise timing which needs to be precisely regulated for them to reach their final destination. Tail-anchored (TA) proteins represent specific class of membrane proteins that lack the N-terminal signal peptide, which targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for the co-translational transport. Instead, they possess single C-terminal transmembrane domain (TMD) that serves as their targeting signal. Therefore, TA proteins are transported only post-translationally when the C-terminal TMD appears from the ribosome. The Guided Entry of Tail-anchored proteins (GET) pathway is the dominant way of how TA proteins find their way into the ER membrane. It is a multistep process that is mediated by six (Sgt2, Get1-Get5) proteins in yeast and seven (plus Bag6) proteins in human, which involves recognition of a TA protein, its targeting to the ER membrane and the actual membrane insertion. In addition to the model cell systems, some of GET pathway components were studied in plants and recently in Plasmodium falciparum, which makes our knowledge on the distribution and the...
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Biogenesis, structure and physiological functions of mitochondrial ATP synthase
Eliáš, Jan ; Mráček, Tomáš (advisor) ; Doležal, Pavel (referee)
Mammalian mitochondrial ATP synthase is an enzyme composed of 18 protein subunits, which is localised in the inner mitochondrial membrane. Its main function is to utilise proton gradient, produced by respiratory chain complexes (RCC), for the synthesis of ATP. Aside from the creation of ATP it is known that its dimers contribute to the correct mitochondrial morphology through the formation of cristae apexes. Furthermore, ATP synthase was proposed to have a role in the mitochondrial permeability transition phenomenon, which is important for regulation of programmed cell death. Over the recent years, our understanding of mammalian ATP synthase biogenesis has been tremendously improved. Its assembly process is now clarified, however the knowledge about assembly intermediates of its peripheral stalk and of subunit c are still not sufficient. We focused precisely on those unsolved questions in the fields of ATP synthase biogenesis and its secondary functions, by the production of a KO model of catalytic β subunit of mammalian ATP synthase F1 domain (βKO). This model was successfully prepared on the background of HEK293 cell line. Its characterisation revealed that disruption of the F1 structure resulted in the inability to assemble functional monomer and resulted in a decay of individual subunits. The only...
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Molecular characterization of the factors participating in the formation of Giardia intestinalis cyst wall.
Pastyříková, Aneta ; Doležal, Pavel (advisor) ; Hrdý, Ivan (referee)
1 Abstract The surface of Giardia intestinalis cysts is covered by a thick fibrillar wall providing the parasite with the necessary protection against the external environment. The cyst wall consists of proteins and carbohydrates, among which is a unique β-1,3-N-acetylgalactosamine homopolymer (known as Giardan). This carbohydrate is synthesized from glucose by cytosolic enzymes induced during encystation. This thesis focuses on the biosynthesis of Giardan and the functional characterization of UDP-N-acetylglucosamine 4'-epimerase (UA4E), one of the enzymes involved in the synthetic pathway. For these purposes, the corresponding gene was targeted using the CRISPR/Cas9 system. A stable cell line was obtained in which all four copies of the UA4E gene were deleted. The success of gene knock-out was confirmed on the basis of proteomic analysis. Using fluorescence microscopy, we observed that ΔUA4E cells induced to encystation are able to initiate encystation processes and cell differentiation. The presence of ESV vesicles and typical morphological changes corresponding to cysts were observed in the cells. However, these stages had a thin surface layer and were unable to form typical thick wall with fibrillar appearance at their surface leading to the subsequent production of non-viable cysts. Key words Giardia...
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Development of new molecular tools for altering gene expression in Giardia intestinalis .
Horáčková, Vendula ; Doležal, Pavel (advisor) ; Horváthová, Lenka (referee)
Giardia intestinalis is a widespread intestinal parasite that causes diarrhea in human and other vertebrate hosts. Although, fully sequenced genome of G. intestinalis has been published, very little is known about the regulation of gene expression. Together with the tetraploid genome, this complicates the use of many common reverse genetics methods. The aim of this thesis was to develop new molecular tools that can be used to alter gene expression in G. intestinalis. For the purposes of this work, new vectors for tetracycline-inducible gene expression including T7 promoter and endogenous oct promoter were designed. Furthermore, cwp1 gene knock-out was created using CRISPR-Cas9 technology. In order to modify mechanisms of double strand break repair, expression of two key components of bacterial NHEJ pathway - LigD and Ku - was introduced into cells of G. intestinalis.
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Transdermal Permeation in Vitro Study of Creams Containing Transkarbam 12
Dvořáková, Pavla ; Doležal, Pavel (advisor) ; Severa, Jan (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Pavla Dvořáková Consultant Doc. RNDr. Pavel Doležal, CSc. Title of Thesis Transdermal permeation in vitro study of creams containing transkarbam 12 The topics of skin barrier function and transdermal permeation enhancers, particularly their structure - action relationship, are covered in theoretical part of this work. It also deals with transkarbam 12 and its mechanism of action, transdermal application of active substances and electrical resistance as a physical unit. Experimental part of this work describes setup used for permeation in vitro experiments on pig ear skin of full thickness. The aim of this work was to find experimental evidence and interpretation of relation between electrical resistance of the skin and flux of caffeine from donor suspension or creams containing transkarbam 12 and also evaluate feasibility of the correction of the variability of the permeation experiments results using preceding infinite dose experiment. It was found that the higher the skin electrical conductivity, the higher the permeation of caffeine from donor samples. For the logarithmic approximation of the relation between caffeine fluxes from creams and skin electrical...
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